CN102985561A

CN102985561A - Normalizing chromosomes for the determination and verification of common and rare chromosomal aneuploidies

Info

Publication number: CN102985561A
Application number: CN2011800229710A
Authority: CN
Inventors: 里查德·P·拉瓦
Original assignee: Verinata Health Inc
Current assignee: Verinata Health Inc
Priority date: 2011-04-14
Filing date: 2011-04-14
Publication date: 2013-03-20
Anticipated expiration: 2031-04-14
Also published as: AU2011365507A1; WO2012141712A1; CN102985561B

Abstract

The present invention provides a method capable of detecting single or multiple fetal chromosomal aneuploidies in a maternal sample comprising fetal and maternal nucleic acids, and verifying that the correct determination has been made. The method is applicable to determining copy number variations (CNV) of any sequence of interest in samples comprising mixtures of genomic nucleic acids derived from two different genomes, and which are known or are suspected to differ in the amount of one or more sequence of interest. The method is applicable at least to the practice of noninvasive prenatal diagnostics, and to the diagnosis and monitoring of conditions associated with a difference in sequence representation in healthy versus diseased individuals.

Description

Be used for determining and verifying normalization method karyomit(e) common and rare chromosomal aneuploidy

Invention field

The invention provides and a kind ofly can determine in the maternal sample that comprises fetus and parent nucleic acid that single or multiple fetal chromosomal aneuploidies and checking made the correct method of determining.The method is applicable to the enforcement of Non-invasive Prenatal Diagnosis at least, and is applicable to contrast with healthy individual diagnosis and the monitoring of the symptom that the sequence differential expression is associated in the ill individuality.

Background of invention

(the American College of Obstetrics andGynecology of U.S. Obstetric and Gynecologic Department association that announced in 2007; ACOG) implementing notification number 77 supports the first three months of all pregnant women's is carried out the dysploidy risk assessment, this assessment is based on nuchal translucency and measures and substitute biochemical marker, in order to examination mongolism (U.S. Obstetric and Gynecologic Department association implements notification number 77 (ACOG Practice Bulletin No.77), Obstetric and Gynecologic Department (Obstet Gynecol) 109:217-227[2007]).These examination tests only can provide risk to determine, this risk is determined indecisive and had determining and high false positive rate of non-the best.Nowadays, only there is the traumatic method that comprises chorionic villus sampling (CVS), amniocentesis or umbilical cord puncture that clear and definite genetic information about fetus just is provided, but these programs to mother and fetus all risky (people such as Odie ripple (Odibo), Obstetric and Gynecologic Department (Obstet Gynecol) 112:813-819[2008]; The people such as Odie ripple (Odibo), Obstetric and Gynecologic Department (Obstet Gynecol) 111:589-595[2008]; Ai Wensi (Evans) and Wa Puna (Wapner), perinatology collection of thesis (Semin Perinatol) 29:215-218[2005]).Therefore, desirable is a kind of non-invasive means that are used for obtaining about the clear and definite information of fetal chromosomal state.

The cfDNA that obtains from Maternal plasma is carried out extensive parallel dna sequencing produce millions of short sequence labels, these short sequence labels can be compared and be mapped to uniquely the site from the reference human genome, and the counting of the label that shines upon can be used for determine chromosomal overexpression or express not enough (people such as model (Fan), periodical (the Proc Natl Acad Sci USA) 105:16266-16271[2008 of institute of NAS]; Wei Erketing (Voelkerding) and Lyons (Lyon), clinical chemistry (Clin Chem) 56:336-338[2010]).Yet the order-checking degree of depth and follow-up counting statistics have determined the sensitivity that the fetus dysploidy is determined.Obviously can not in specimen colony, determine the trisomy of more than one types, this situation has been emphasized for the demand of the algorithm of the optimization that is used in the Maternal plasma sample the determining chromosomal aneuploidy (people such as Zhao (Chiu), BMJ (BMJ) 342, c7401[2011]; The people such as Eric (Ehrich), U.S.'s journal of obstetrics and gynecology (Am J Obstet Gynecol) 2014:205 e1[2011]).

Existing methodical limitation becomes the basis for the demand of the non invasive method of the best, these best non invasive methods with for antenatal diagnosis and with copy number change the diagnosis of relevant medical science symptom and monitoring provide in specificity, susceptibility and the suitability any one or all in order to diagnose reliably chromosomal aneuploidy.

The present invention has realized some in the demand, and especially provide an advantage, a kind of reliable method namely is provided, and the method has enough susceptibilitys in order to determine single or multiple chromosomal aneuploidies, and correct determining made in the method checking.

Summary of the invention

The invention provides and a kind ofly can determine in the maternal sample that comprises fetus and parent nucleic acid that single or multiple fetal chromosomal aneuploidies and checking made the correct method of determining.The method is applicable to determine the copy number variation (CNV) of any interested sequence in a plurality of samples, these samples comprise the mixture of the genomic nucleic acids that derives from two different genes groups, and known or suspect that these two different genes groups are different aspect the amount of one or more interested sequences.The method is applicable to the enforcement of Non-invasive Prenatal Diagnosis at least, and is applicable to diagnosis and the monitoring of the symptom relevant with sequence differential expression in the ill individuality of healthy individual contrast.

In one embodiment, determine there is or does not exist a kind of fetal chromosomal aneuploidy in the method by following steps in the parent specimen that comprises fetus and parent nucleic acid molecule: (a) obtain the sequence information for fetus in maternal sample and parent nucleic acid, so that identification is for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, relatively indication for described interested chromosomal the first normalized value and threshold value exists or does not exist for described interested chromosomal a kind of dysploidy, and has or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of described interested chromosomal the second normalized value and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described normalized karyomit(e) value (NCV).

In above-mentioned and all subsequent implementation schemes, the step that obtains order-checking information comprises order-checking of future generation (NGS).NGS uses a plurality of reversible dyestuff terminators to carry out synthesis method order-checking (sequencing-by-synthesis).Alternately, NGS can be that connection method order-checking (sequencing-by-ligation) is checked order.NGS can also be single-molecule sequencing.

Similarly, in above-mentioned and all subsequent implementation schemes, be to be selected from karyomit(e) 9,11,14 and 1 for the normalization method karyomit(e) of karyomit(e) 21.In some embodiments, the normalization method karyomit(e) for karyomit(e) 18 is to be selected from karyomit(e) 8,3,2 and 6.In some embodiments, the normalization method karyomit(e) for karyomit(e) 13 is group, karyomit(e) 5 and the karyomit(e) 6 that is selected from karyomit(e) 4, karyomit(e) 2-6.In some embodiments, the normalization method karyomit(e) for chromosome x is to be selected from karyomit(e) 6,5,13 and 3.In some embodiments, the normalization method karyomit(e) for karyomit(e) 1 is to be selected from karyomit(e) 10,11,9 and 15.In some embodiments, the normalization method karyomit(e) for karyomit(e) 2 is to be selected from karyomit(e) 8,7,12 and 14.In some embodiments, the normalization method karyomit(e) for karyomit(e) 3 is to be selected from karyomit(e) 6,5,8 and 18.In some embodiments, the normalization method karyomit(e) for karyomit(e) 4 is to be selected from karyomit(e) 3,5,6 and 13.In some embodiments, the normalization method karyomit(e) for karyomit(e) 5 is to be selected from karyomit(e) 6,3,8 and 18.In some embodiments, the normalization method karyomit(e) for karyomit(e) 6 is to be selected from karyomit(e) 5,3,8 and 18.In some embodiments, the normalization method karyomit(e) for karyomit(e) 7 is to be selected from karyomit(e) 12,2,14 and 8.In some embodiments, the normalization method karyomit(e) for karyomit(e) 8 is to be selected from karyomit(e) 2,7,12 and 3.In some embodiments, the normalization method karyomit(e) for karyomit(e) 9 is to be selected from karyomit(e) 11,10,1 and 14.In some embodiments, the normalization method karyomit(e) for karyomit(e) 10 is to be selected from karyomit(e) 1,11,9 and 15.In some embodiments, the normalization method karyomit(e) for karyomit(e) 11 is to be selected from karyomit(e) 1,10,9 and 15.In some embodiments, the normalization method karyomit(e) for karyomit(e) 12 is to be selected from karyomit(e) 7,14,2 and 8.In some embodiments, the normalization method karyomit(e) for karyomit(e) 14 is to be selected from karyomit(e) 12,7,2 and 9.In some embodiments, the normalization method karyomit(e) for karyomit(e) 15 is to be selected from karyomit(e) 1,10,11 and 9.In some embodiments, the normalization method karyomit(e) for karyomit(e) 16 is to be selected from karyomit(e) 20,17,15 and 1.In some embodiments, the normalization method karyomit(e) for karyomit(e) 17 is to be selected from karyomit(e) 16,20,19 and 22.In some embodiments, the normalization method karyomit(e) for karyomit(e) 19 is to be selected from 22,17,16 and 20.In some embodiments, the normalization method karyomit(e) for karyomit(e) 20 is to be selected from karyomit(e) 16,17,15 and 1.In some embodiments, the normalization method karyomit(e) for chromosome 22 is to be selected from karyomit(e) 19,17,16 and 20.

In another embodiment, determine there is or does not exist a kind of fetal chromosomal aneuploidy in the method by following steps in the parent specimen that comprises fetus and parent nucleic acid molecule: (a) obtain the sequence information for fetus in maternal sample and parent nucleic acid, so that identification is for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of described interested chromosomal the second normalized value and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described normalized karyomit(e) value (NCV).Fetal chromosomal aneuploidy can be chromosomal aneuploidy a kind of part or complete.In these embodiments, fetal chromosomal aneuploidy can be selected from trisomy 21 (T21), 18 trisomys (T18), 13 trisomys (T13), X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, for example blood sample or the blood plasma part that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in the maternal sample is the cfDNA molecule.In some other embodiments, the parent specimen is the plasma sample that obtains from a pregnant woman, and nucleic acid molecule is the cfDNA molecule.

In another embodiment, the method determines to exist or do not exist at least two kinds of different chromosomal aneuploidies.In one embodiment, the method is by determining to exist or do not exist at least two kinds of different fetal chromosomal aneuploidies at least two interested karyomit(e) repeating steps (a)-(c) in the parent specimen that comprises fetus and parent nucleic acid molecule, wherein these steps comprise that (a) obtains the sequence information for fetus in maternal sample and parent nucleic acid, so that identification is for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of described interested chromosomal the second normalized value and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).In some embodiments, the method comprises for all karyomit(e) repetition the method to determine to exist or do not exist two kinds of different fetal chromosomal aneuploidies at least.

In another embodiment, the method determines to exist or do not exist at least two kinds of different chromosomal aneuploidies.In one embodiment, the method is by determining there are or do not exist at least two kinds of different fetal chromosomal aneuploidies in the parent specimen that comprises fetus and parent nucleic acid molecule at least two interested karyomit(e) repeating steps (a)-(c), wherein these steps comprise that (a) obtains the sequence information for fetus in maternal sample and parent nucleic acid, so that identification is for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of described interested chromosomal the second normalized value and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).In some embodiments, the method comprises for all karyomit(e) repetition the method to determine to exist or do not exist two kinds of different fetal chromosomal aneuploidies at least.At least two kinds of different fetal chromosomal aneuploidies can be selected from T21, T18, T13 and X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, for example blood sample or the blood plasma part that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in the maternal sample is the cfDNA molecule.In some other embodiments, the parent specimen is the plasma sample that obtains from a pregnant woman, and nucleic acid molecule is the cfDNA molecule.

In another embodiment, there be or exist determining for interested chromosomal a kind of dysploidy in the method in the parent specimen that comprises fetus and parent nucleic acid molecule by following steps checkings: (a) obtain the sequence information for fetus in sample and parent nucleic acid, so that identification is for number of the sequence label of interested chromosomal a plurality of mappings and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) use for the number of interested chromosomal label and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of chromosomal the second normalized value of described the first normalization method and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as the normalized karyomit(e) value (NCV) of described calculating.

In another embodiment, the method is verified in the parent specimen that comprises fetus and parent nucleic acid molecule by following steps and is had or do not exist determining for interested chromosomal a kind of dysploidy: (a) obtain the sequence information for fetus in sample and parent nucleic acid, so that identification is for number of the sequence label of interested chromosomal a plurality of mappings and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) use for the number of interested chromosomal label and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of chromosomal the second normalized value of described the first normalization method and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as the normalized karyomit(e) value (NCV) of described calculating.Fetal chromosomal aneuploidy can be chromosomal aneuploidy a kind of part or complete.In these embodiments, fetal chromosomal aneuploidy can be selected from T21, T18, T13 and X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, for example blood sample or the blood plasma part that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in the maternal sample is the cfDNA molecule.In some other embodiments, the parent specimen is the plasma sample that obtains from a pregnant woman, and nucleic acid molecule is the cfDNA molecule.

In another embodiment, the method is by determining to exist or do not exist at least two kinds of different fetal chromosomal aneuploidies at least two interested karyomit(e) repeating steps (a)-(c) in the parent specimen that comprises fetus and parent nucleic acid molecule, wherein obtain sequence information for fetus in sample and parent nucleic acid for the step (a)-(c) comprise (a) of each in these at least two the interested karyomit(e), so that identification is for number of the sequence label of interested chromosomal a plurality of mappings and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) use for the number of interested chromosomal label and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, for in these at least two the interested karyomit(e)s each, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of chromosomal the second normalized value of described the first normalization method and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).In some embodiments, the method comprises for all karyomit(e) repetition the method to determine to exist or do not exist two kinds of different fetal chromosomal aneuploidies at least.

In another embodiment, the method is by determining to exist or do not exist at least two kinds of different fetal chromosomal aneuploidies at least two interested karyomit(e) repeating steps (a)-(c) in the parent specimen that comprises fetus and parent nucleic acid molecule, wherein obtain sequence information for fetus in sample and parent nucleic acid for the step (a)-(c) comprise (a) of each in these at least two the interested karyomit(e), so that identification is for number of the sequence label of interested chromosomal a plurality of mappings and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) use for the number of interested chromosomal label and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, for in these at least two the interested karyomit(e)s each, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of chromosomal the second normalized value of described the first normalization method and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).In some embodiments, the method comprises for all karyomit(e) repetition the method to determine to exist or do not exist two kinds of different fetal chromosomal aneuploidies at least.At least two kinds of different fetal chromosomal aneuploidies can be selected from T21, T18, T13 and X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, for example blood sample or the blood plasma part that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in the maternal sample is the cfDNA molecule.In some other embodiments, the parent specimen is the plasma sample that obtains from a pregnant woman, and nucleic acid molecule is the cfDNA molecule.

In another embodiment, the method is determined to exist or do not exist in the Maternal plasma specimen that comprises fetus and parent nucleic acid molecule (for example cfDNA) to be selected from trisomy 21 by following steps, 18 trisomys, 13 trisomys, and a kind of fetal chromosomal aneuploidy of X monosomy: (a) acquisition is for the sequence information of fetus in maternal sample and parent nucleic acid, so that identification, wherein obtains sequence information at least for number of interested chromosomal a plurality of sequence labels and for a number of two chromosomal a plurality of sequence labels of normalization method and comprises and use a plurality of reversible dyestuff terminators to carry out extensive parallel synthesis method order-checking; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).In some embodiments, the method is by determining to exist or do not exist at least two kinds of different chromosomal aneuploidies that are selected from trisomy 21,18 trisomys, 13 trisomys and X monosomy at least two interested karyomit(e) repeating steps (a)-(c) in the Maternal plasma specimen that comprises fetus and parent nucleic acid molecule (for example cfDNA).The method may further include for all karyomit(e) repeating steps (a)-(c) to determine to exist or do not exist two kinds of fetal chromosomal aneuploidies at least.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, for example blood sample or the blood plasma part that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in the maternal sample is the cfDNA molecule.In some other embodiments, the parent specimen is the plasma sample that obtains from a pregnant woman, and nucleic acid molecule is the cfDNA molecule.

In another embodiment, the method is determined to exist or do not exist in the Maternal plasma specimen that comprises fetus and parent nucleic acid molecule (for example cfDNA) to be selected from trisomy 21 by following steps, 18 trisomys, 13 trisomys, and a kind of fetal chromosomal aneuploidy of X monosomy: (a) acquisition is for the sequence information of fetus in sample and parent nucleic acid, so that identification, wherein obtains sequence information at least for number of the sequence label of interested chromosomal a plurality of mappings and for a number of two chromosomal a plurality of sequence labels of normalization method and comprises and use a plurality of reversible dyestuff terminators to carry out extensive parallel synthesis method order-checking; (b) use for the number of interested chromosomal label and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).In some embodiments, the method is by determining to exist or do not exist at least two kinds of different chromosomal aneuploidies that are selected from trisomy 21,18 trisomys, 13 trisomys and X monosomy at least two interested karyomit(e) repeating steps (a)-(c) in the Maternal plasma specimen that comprises fetus and parent nucleic acid molecule (for example cfDNA).The method may further include for all karyomit(e) repeating steps (a)-(c) to determine to exist or do not exist two kinds of fetal chromosomal aneuploidies at least.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, for example blood sample or the blood plasma part that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in the maternal sample is the cfDNA molecule.In some other embodiments, the parent specimen is the plasma sample that obtains from a pregnant woman, and nucleic acid molecule is the cfDNA molecule.

In some above-mentioned embodiments and some subsequent implementation schemes, obtain sequence information for fetus in sample and parent nucleic acid and comprise the fetus in sample and parent nucleic acid molecule are checked order.

By reference combination

All clearly by reference combinations of all patents mentioned herein, patent application and other publications (comprising all sequences disclosed in these reference), its combination degree just is indicated as being by reference combination definitely and individually as each independent publication, patent or patent application.Yet, should not be understood to admit that to the citation of any document it is about prior art of the present invention.

Brief Description Of Drawings

Novel feature of the present invention in appended claims in addition singularity set forth.By with reference to the following detailed description of the invention of having set forth the schematic embodiment of utilizing the principle of the invention with and accompanying drawing will obtain better understanding to feature and advantage of the present invention.

Fig. 1 provides a schema, shows to determine and there are or do not exist two alternate embodiment of the method for dysploidy in checking.

Fig. 2 shows the normalized karyomit(e) value (example 1) for karyomit(e) 21 (zero), 18 (△) and 13 () of determining in from the sample of training set 1.

Fig. 3 shows the normalized karyomit(e) value (example 1) for karyomit(e) 21 (zero), 18 (△) and 13 () of determining in from the sample of test set 1.

Fig. 4 shows the normalized karyomit(e) value (example 1) for karyomit(e) 21 (zero) and 18 (△) that the method for normalizing that uses the people such as Zhao (Chiu) is determined in from the sample of test set 1.

Fig. 5 shows the figure for the normalized karyomit(e) value of karyomit(e) 9 dosage that uses karyomit(e) 11 to determine in 48 samples of test set 1 (example 1) as normalization method karyomit(e).

Fig. 6 shows the figure for the normalized karyomit(e) value of karyomit(e) 8 dosage that uses karyomit(e) 2 to determine in 48 samples of test set 1 (example 1) as normalization method karyomit(e).

Fig. 7 shows the figure for the normalized karyomit(e) value of karyomit(e) 6 dosage that uses karyomit(e) 5 to determine in 48 samples of test set 1 (example 1) as normalization method karyomit(e).

Fig. 8 shows the figure for the normalized karyomit(e) value of karyomit(e) 21 dosage that uses accordingly that karyomit(e) 9 (A), karyomit(e) 10 (B) and karyomit(e) 14 (C) determines in 48 samples of test set 1, this test set 1 comprises the sample of uninfluenced (zero) and influenced (△) (that is, trisomy 21).

Fig. 9 shows and uses karyomit(e) 2 as normalization method karyomit(e) (A) and the figure for the normalized karyomit(e) value of karyomit(e) 8 dosage that uses karyomit(e) 7 to determine in test set 2 (example 4) as normalization method karyomit(e) (B).

Detailed description of the invention

Except as otherwise noted, otherwise enforcement of the present invention relates to routine techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and dna sequencing and the recombinant DNA field, and these technology are all in the technology category of this area.These technology are known to those of skill in the art, and be described in numerous textbooks and the reference (referring to such as people such as Pehanorm Brookers (Sambrook), " molecular cloning experiment guide (Molecular Cloning:A Laboratory Manual) ", second edition (cold spring port (Cold Spring Harbor)), [1989]); And the people such as Ao Subaier (Ausubel), " up-to-date experimental methods of molecular biology compilation (Current Protocols in Molecular Biology) " [1987])

Numerical range comprises the numerical value that limits this scope.Run through each greatest measure limit that this specification sheets provides being intended that of this and comprise the numerical value limit that each is lower, clearly write out at this as this type of low numerical value limit.Run through each minimum value limit that this specification sheets provides and to comprise the numerical value limit that each is higher, clearly write out at this as this type of high value limit.Run through each numerical range that this specification sheets provides and to comprise each the narrower numerical range that drops in this type of wider numerical range, all write out clearly as this type of narrower numerical range herein.

The title that provides herein is not to different aspect of the present invention or the restriction of embodiment, and it can be to have as an overall specification sheets by reference.Therefore, as above specified, directly the term of definition defines as an overall specification sheets more fully by reference hereinafter.

Unless define separately at this, all technology and term science all have the identical meanings that a those of ordinary skill in the field is understood usually under the present invention as used herein.Comprised that the different science dictionaries at this term that comprises are to know and are obtainable for those skilled in the art.Although similar or be equivalent to any method of those methods described herein and material and material implement or test the present invention in found purposes, some preferred method and materials only have been described.Therefore, the term that directly defines is hereinafter illustrated more completely by this specification sheets is consulted namely as a whole.Should be understood that the present invention is not limited to illustrated concrete grammar, rules and reagent because these can change, they by those skilled in the art according to the use of getting off of its situation.

Definition

As used in this, the term of odd number " ", " a kind of " and " being somebody's turn to do " comprise plural reference, unless context clearly indicates in addition.Except as otherwise noted, nucleic acid be by 5 ' from left to right write and aminoacid sequence is from left to right to write to the carboxyl direction by amino to 3 ' direction.

Term " acquisition sequence information " in this article refers to the sequence information that nucleic acid is checked order to obtain to be sequence reading form, and these sequence readings are identified as sequence label when being mapped to uniquely the reference gene group.

Term " normalized value " in this article refer to determine for interested karyomit(e) and make a numerical value that is associated with number for the chromosomal sequence label of normalization method for the number of interested chromosomal sequence label.For instance, " normalized value " can be a karyomit(e) dosage such as other places description herein, and perhaps it can be the NCV (normalized karyomit(e) value) such as other places description herein.

Term " interested karyomit(e) " in this article refers to a kind of karyomit(e) that exists or do not exist a kind of dysploidy to determine.Interested chromosomal example comprises involved karyomit(e) in the common dysploidy (such as trisomy 21), and involved karyomit(e) in the rare dysploidy (such as 2 trisomys).Among karyomit(e) 1-22, X and the Y any one can be interested karyomit(e).

Term " a plurality of (multiple) and a plurality of (plurality) " in this article refers to two or more dysploidy and/or karyomit(e) when about chromosomal aneuploidy number and/or chromosome number use.

Term " threshold value " in this article refers to and uses training dataset calculating and any numerical value that be used as copy number variation (for example dysploidy) diagnosis threshold in the organism.If surpass threshold value from the result who implements the present invention's acquisition, the experimenter can be diagnosed as copy number variation (for example trisomy 21) so.For the appropriate threshold value of described method can be by analyzing to identify to the normalized value (for example karyomit(e) dosage or NCV (normalized karyomit(e) value)) that calculates for the training sample sets that comprises qualified samples (that is, unaffected sample) herein.Threshold value can be used qualified samples and be identified as having the sample of chromosomal aneuploidy (that is, affected sample) and set (referring to example herein).In some embodiments, the training set for the identification appropriate threshold value comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000 or more qualified samples.It may be favourable coming the diagnostic tool of improvement threshold with larger qualified samples collection.

Term " of future generation order-checking (NGS) " in this article refers to and allows sequence measurement that the nucleic acid molecule that increases in clone's mode and single core acid molecule are carried out extensive parallel order-checking.The limiting examples of NGS comprises synthesis method order-checking and the connection method order-checking of using a plurality of reversible dyestuff terminators to carry out.

Term " reading " refers to the to have sufficient length dna sequence dna of (for example at least about 30bp), it can be used for identification larger sequence or zone, and for example it can be compared and specifically belong to karyomit(e) or genome area or gene.

Term " sequence label " in this article can with term " sequence label of mapping " Alternate with mention by than and definitely ownership (that is, mapping) to a sequence reading of larger sequence (for example, reference gene group).The sequence label of mapping is mapped to a reference gene group uniquely, that is, they are belonged to the single position of reference gene group.Do not comprise the label (that is the label that, does not shine upon uniquely) that can be mapped to an above position on the reference gene group in the analysis.

Term " number of sequence label " is when using about the number for an interested karyomit(e) and/or the chromosomal label of one or more normalization method, in this article refer to and be mapped to this interested karyomit(e) and/or this or these chromosomal sequence label of normalization method, these sequence labels are subsets of a plurality of labels of obtaining for all karyomit(e)s in the sample.The number of tags that obtains for a sample can be at least about 1 * 10 ⁶Individual sequence label, at least about 2 * 10 ⁶Individual sequence label, at least about 3 * 10 ⁶Individual sequence label, at least about 5 * 10 ⁶Individual sequence label, at least about 8 * 10 ⁶Individual sequence label, at least about 10 * 10 ⁶Individual sequence label, at least about 15 * 10 ⁶Individual sequence label, at least about 20 * 10 ⁶Individual sequence label, at least about 30 * 10 ⁶Individual sequence label, at least about 40 * 10 ⁶Individual sequence label, or at least about 50 * 10 ⁶Individual sequence label or at least about 60 * 10 ⁶Individual sequence label, or at least about 70 * 10 ⁶Individual sequence label or at least about 80 * 10 ⁶Individual sequence label is included in the reading of (for example 36bp) between 20bp and the 40bp, is that each sample obtains by reading being mapped to the reference gene group.The number that is mapped to any one chromosomal label will depend on karyomit(e) size and chromosome copies number.For instance, the number that is mapped to the label of the karyomit(e) 21 in the trisomy 21 sample will be different from the number that (that is, greater than) is mapped to the label of the karyomit(e) 21 in the unaffected sample.Similarly, being mapped to the number of the label of karyomit(e) 19 will be less than the number of the label that is mapped to karyomit(e) 1 (its be about karyomit(e) 19 sizes 4 times).The number that is mapped to the label of interested sequence (for example karyomit(e)) is also referred to as " sequence label density ".

Term " sequence label density " in this article refers to the number of the sequence reading that is mapped to reference gene group sequence, for example is numbers of the sequence reading of the karyomit(e) that is mapped to the reference gene group 21 that produces by sequence measurement for the sequence label density of karyomit(e) 21.Can determine sequence label density for whole karyomit(e) or for chromosomal part.

As used herein, term " is compared ", " comparison " or " comparing " refer to be identified as and one or more sequences from the known array coupling of reference gene group with regard to their nucleic acid molecule order aspect.This kind comparison can manually be carried out or be undertaken by computerized algorithm, and example comprises that the efficient few nucleotide of allotting as the part of hundred million sensible genomics analysis stream waterlines (Illumina Genomics Analysis pipeline) is according to Local Alignment (Efficient Local Alignment of Nucleotide Data; ELAND) computer program.The coupling of the sequence reading in the comparison can be 100% sequences match or less than 100% (non-Perfect Matchings).

As used herein, term " reference gene group " refers to any specific known group sequence (no matter be part or complete) of any organism or virus, and it can be used for doing reference to the sequence that identifies from the experimenter.For instance, the reference gene group for human experimenter and many other biological bodies sees the www.ncbi.nlm.nih.gov of American National biotechnology information center (National Center for BiotechnologyInformation).

" genome " refers to the complete genetic information of the organism that represents with the nucleotide sequence form or virus.

Term " maternal sample " in this article refers to the biological sample that the experimenter (for example women) from pregnancy obtains.

Term " biological fluid " in this article refers to the liquid of obtaining from biogenetic derivation, and comprises such as blood, serum, blood plasma, phlegm, irrigating solution, cerebrospinal fluid, urine, seminal fluid, sweat, tears, saliva etc.As used herein, term " blood ", " blood plasma " and " serum " are contained their part or the part through processing clearly.Similarly, when sample is when taking from examination of living tissue, swab, smear etc., " sample " contains fragment or the part through processing that derives from examination of living tissue, swab, smear etc. clearly.

Term " parent nucleic acid " and " fetal nucleic acid " refer to the nucleic acid of conceived female subjects and in this article accordingly by the nucleic acid of the female entrained fetus of pregnancy.

Term " experimenter " in this article refers to human experimenter and non-human experimenter, such as Mammals, invertebrates, vertebrates, fungi, yeast, bacterium and virus.Although example herein relates to the mankind and words mainly are that concept of the present invention is applicable to the genome from any plant or animal, and is applicable to the fields such as veterinary science, animal science and research laboratory for relevant human.

Term " normalization method sequence " in this article refers between a plurality of samples and a plurality of order-checking batch and shows that the number that is mapped to the sequence label on it has the sequence of variability, the variability of the number of this sequence label close to it be used as normalized parameter for the variability of number of sequence label of interested sequence, and can best affected sample and one or more unaffected sample be differentiated." normalization method karyomit(e) " is an example of " normalization method sequence ".

Term " sequence dosage " in this article refers to a parameter of the label density dependent connection of the sequence label density that makes interested sequence and normalization method sequence." karyomit(e) dosage " is the number and the ratio that is mapped to the number of the chromosomal sequence label of normalization method that is mapped to the sequence label of karyomit(e) (for example interested karyomit(e)), and it is an example of sequence dosage." cycle tests dosage " parameter that to be the sequence label density that makes in specimen the interested sequence (for example karyomit(e) 21) of determining join with the sequence label density dependent of normalization method sequence (for example karyomit(e) 9).Similarly, " qualified sequence dosage " parameter that to be the sequence label density that makes in qualified samples the interested sequence of determining join with the sequence label density dependent of normalization method sequence.

Term " karyomit(e) dosage " in this article refers to the number and the ratio that is mapped to the number of the chromosomal sequence label of normalization method of the sequence label that is mapped to karyomit(e) (for example interested karyomit(e)).

Term " normalization method karyomit(e) " in this article refers to the karyomit(e) that the number that shows the sequence label that is mapped to it between a plurality of samples and a plurality of order-checking batch has variability, the variability of the number of this sequence label close to it be used to obtain normalized value for the variability of number of interested chromosomal sequence label, and can best affected sample and one or more unaffected sample be differentiated.

Term " interested sequence " in this article refers to the nucleotide sequence relevant with the sequence differential expression in the ill individuality of healthy individual contrast.Interested sequence can be the sequence on the karyomit(e) of false demonstration in disease or hereditary situation (that is, overexpression or express not enough).Interested sequence can also be a chromosomal part or karyomit(e) (that is, interested karyomit(e)).For instance, interested sequence can be overexpression in the dysploidy symptom karyomit(e) (for example karyomit(e) 13,18,21 and X) or coding is expressed not enough tumor-inhibiting factor in cancer gene.Interested sequence is included in the total group of experimenter's cell or the subgroup overexpression or expresses not enough sequence." interested qualified sequence " is the interested sequence in the qualified samples." interested cycle tests " is the interested sequence in the specimen.

Term " qualified samples " in this article refer to comprise with specimen in the sample of mixture of nucleic acid multiple nucleic acids that compare, that exist with the known copy number, and for interested sequence, it is normally (namely, be not aneuploid) sample, for example being used for identification is the sample of a non-trisomy 21 sample for the chromosomal qualified samples of the normalization method of karyomit(e) 21.

Term " training set " and " training sample " be used in reference in this article comprise with specimen in the sample of nucleic acid nucleic acid that compare, that exist with the known copy number.Unless otherwise indicated, otherwise training set comprises qualified and affected sample.

Term " specimen " in this article refers to and comprises the sample that nucleic acid mixture and these nucleic acid comprise copy number at least one nucleotide sequence that has morphed under a cloud.The nucleic acid that is present in the specimen is called as " test nucleic acid ".

Term " dysploidy " refers to by loss or obtains whole karyomit(e) or a chromosomal part and the imbalance of the genetic material that causes at this.

Term " karyomit(e) dysploidy " refers to by loss or obtains whole karyomit(e) and the imbalance of the genetic material that causes at this, and comprises kind being dysploidy and mosaic dysploidy.

Term " part dysploidy " and " chromosome dyad dysploidy " refer to by losing or (for example obtaining a chromosomal part at this, partial monosomy and partial trisomy) and the imbalance of the genetic material that causes, and contain the imbalance that is caused by transposition, deletion and insertion.

Term " nucleic acid molecule ", " polynucleotide " and " nucleic acid " are used interchangeably, and refer to a covalently bound nucleotide sequence (namely, the ribonucleotide of RNA and the deoxyribonucleotide of DNA), 3 ' position of the pentose of one of them Nucleotide is connected to by a phosphodiester group on the 5 ' position of pentose of next Nucleotide, this comprises the sequence of any type of nucleic acid, including, but not limited to RNA, DNA and cfDNA molecule.Term " polynucleotide " comprises and is not limited to strand and polynucleotide two strands.

Term " copy number variation (CNV) " in this article refers to the nucleotide sequence copy number that is present in the specimen and is present in the variation that the nucleotide sequence copy number in the qualified samples (that is, normal sample) is compared.Copy number variation comprises disappearance (comprising micro-deleted), inserts (comprising little insertion), copies, multiplication, inversion, transposition and complicated multi-position make a variation.CNV has been contained the dysploidy of complete chromosomal aneuploidy and part.

Describe

The invention provides and a kind ofly can determine in the maternal sample that comprises fetus and parent nucleic acid that single or multiple fetal chromosomal aneuploidies and checking made the correct method of determining.The method is applicable to determine the copy number variation (CNV) of any interested sequence in a plurality of samples, these samples comprise the mixture of the genomic nucleic acids that derives from least two different genes groups, and known or suspect that these two different genes groups are different aspect the amount of one or more interested sequences.Interested sequence is included in hundreds of the bases genome sequence interior to the dozens of megabase to whole karyomit(e) scope, and these genome sequences are known or be suspect to be relevant with heredity or disease symptom.The example of interested sequence comprises the karyomit(e) relevant with the dysploidy of knowing (for example trisomy 21) and the chromosome segment (for example 8 trisomys of the part in the acute myelocytic leukemia) of multiplication in disease (such as cancer).

The inventive method is included in and obtains order-checking information in one or more parent specimen, to calculate the karyomit(e) dosage for interested sequence (for example karyomit(e)), thereby determine to exist or do not have single or multiple chromosomal aneuploidies, and comprise that checking makes determining of correct dysploidy.Correctly determine in sample to exist or not exist the required accuracy of CNV (for example dysploidy) to be based on the following: be mapped to the variation (with batch order-checking variation) of number of the sequence label of reference gene group between a plurality of samples in order-checking batch, and the variation (order-checking variation between round) of number that is mapped to the sequence label of reference gene group in the different order-checkings batch, these variations can make the impact of distribution of the sequence label that fetal chromosomal aneuploidy penetrates not obvious.For instance, for the label of the canonical sequence that is mapped to GC enrichment or GC poorness, variation may be especially remarkable.In order to proofread and correct this kind variation, the inventive method uses karyomit(e) dosage from having explained in essence the order-checking variability that occurs based on the knowledge of normalization method karyomit(e) (or normalization method karyomit(e) group).

Normalization method karyomit(e) and karyomit(e) dosage

Use from the sequence information of one group of qualified samples that obtains from the experimenter and identify normalization method karyomit(e), these samples are known to comprise the cell that has for the normal copy number of any one the interested sequence diploid of karyomit(e) 21 (for example for).The sequence information that obtains from qualified samples also is used for determining the identification that statistical significance is arranged (referring to example) at the specimen chromosomal aneuploidy.In one embodiment, qualified samples is to obtain from the mother who nourishes fetus, has used the cytogenetics means to confirm that this fetus has the normal chromosome copies number diploid of karyomit(e) 21 (for example for).The biology qualified samples can be a kind of biological fluid (for example blood plasma) or any suitable sample as mentioned below.In some embodiments, qualified samples comprises the mixture of nucleic acid molecule (for example cfDNA molecule).In some embodiments, qualified samples is the Maternal plasma sample that comprises the mixture of fetus and parent cfDNA molecule.

By using any known sequence measurement that at least a portion of nucleic acid (for example fetus and parent nucleic acid) is checked order to obtain for the chromosomal sequence information of normalization method.Preferably, use described any order-checking (NGS) method of future generation in other places herein to come fetus and the parent nucleic acid of the molecular form that is unit molecule or increases in clone's mode are checked order.Millions of sequence readings with predetermined length (for example 36bp) produce by the NGS technology, and are mapped to the reference gene group to treat in counting as sequence label.At least a portion nucleic acid to each qualified samples checks order, and the number that is mapped to each chromosomal sequence label is counted.In some embodiments, the number that is mapped to chromosomal sequence label can normalize to these interested qualified sequences, shine upon them to top length.As label density with respect to the ratio of interested sequence length and definite sequence label density is known as the label density ratio in this article.It is optional to normalize to interested sequence length, but can be used as to reduce counting the number of words the purpose step and included in the numerical value, understands for the mankind to simplify numerical value.When all the qualified sequence labels in each qualified samples mapped and whens counting all, qualified sequence label density for interested sequence (for example clinically relevant sequence) in the qualified samples is determined, and also has been determined for the sequence label density of the other sequence that is used for subsequently therefrom identifying the normalization method sequence.

Based on the qualified label density of calculating, for the qualified sequence dosage (for example karyomit(e) dosage) of interested sequence (for example karyomit(e) 21) separately as for the sequence label density of interested sequence be determined for the ratio of the qualified sequence label density of the other sequence that is used for subsequently therefrom identifying the normalization method sequence.For instance, for the karyomit(e) dosage of interested karyomit(e) (for example karyomit(e) 21) be as for the sequence label density of karyomit(e) 21 with for the ratio of all the other karyomit(e)s (that is, karyomit(e) 1-20, chromosome 22, chromosome x and karyomit(e) Y) sequence label density separately and definite.Can determine qualified sequence dosage for all karyomit(e).

Subsequently, in qualified samples, identify at least two normalization method sequences for interested sequence (for example karyomit(e) 21) based on the sequence dosage that calculates.For instance, identify close to the sequence of the sequence label Density Variation of karyomit(e) 21 as the sequence label Density Variation that has in the qualified samples for the qualified normalization method sequence of karyomit(e) 21.For instance, qualified normalization method sequence is the sequence with minimum variability.In some embodiments, identify plural normalization method sequence.For instance, determined for each the normalization method karyomit(e) with minimum variability among all karyomit(e) 1-22, chromosome x and the karyomit(e) Y.Table 9 in the example 5 provides four normalization method karyomit(e)s, these normalization method karyomit(e)s be confirmed as among karyomit(e) 1-22, chromosome x and the karyomit(e) Y each have four minimum variability.As shown in example, variability numerically can be expressed as the variation coefficient (CV%).The normalization method sequence can also be to distinguish best the sequence of one or more qualified samples and one or more affected samples, that is, the normalization method sequence is the sequence with maximum differentiability.The differentiability degree can be used as karyomit(e) dosage in the qualified samples colony and the significant difference between the karyomit(e) dosage in one or more specimen and determines.For instance, differentiability numerically can be expressed as the T test value, the karyomit(e) dosage in its expression qualified samples colony and the significant difference between the karyomit(e) dosage in one or more specimen.Alternately, differentiability numerically can be expressed as normalized karyomit(e) value (NCV), and it is the z score value for karyomit(e) dosage when NCV is normal distribution.In definite z score value, can use average and the standard deviation of the karyomit(e) dosage in the combination lattice sample.Alternately, can use average and the standard deviation of the karyomit(e) dosage in the training set that comprises qualified samples and affected sample.In other embodiments, normalization method sequence is the sequence with minimum variability and maximum differentiability.

The method has been identified congenitally to have similar feature and tend to and between sample and order-checking batch the similarly sequence of variation has been occured, and these sequences are applicable to determine the sequence dosage in the specimen.

Based on the identification to this or these normalization method sequence in the qualified samples, use the sequence information that obtains for the nucleic acid in the specimen to determine in the specimen one or more sequence dosage (for example karyomit(e) dosage) for interested sequence (for example karyomit(e) 21).In some embodiments, at least two sequence dosage (for example karyomit(e) dosage) for interested sequence have been determined.For instance, use karyomit(e) 9 as the definite the first chromosome dosage for karyomit(e) 21 of first a normalization method karyomit(e), and use karyomit(e) 11 as the definite second karyomit(e) dosage for karyomit(e) 21 of the second normalization method karyomit(e).Cycle tests dosage can further be expressed as described NCV.In some embodiments, can carry out by following steps the classification of specimen: directly will compare and the second cycle tests dosage and a Second Threshold are compared to determine to exist or do not exist a kind of chromosomal aneuploidy in specimen for interested chromosomal the first cycle tests dosage and first threshold.For the comparatively validate of interested chromosomal two karyomit(e) dosage determining of sample classification.Select threshold value to classify sample as " normally ", " affected " or " " judgement (no call) " sample according to user-defined reliability thresholds.In other embodiments, use first a normalization method karyomit(e) to determine for an interested chromosomal the first chromosome dosage, and use second a normalization method karyomit(e) to determine for the chromosomal second karyomit(e) dosage of the first normalization method.Can carry out by following steps the classification of specimen: the first chromosome dosage and first threshold are compared and the second karyomit(e) dosage and a Second Threshold are compared to determine to exist or do not exist a kind of chromosomal aneuploidy in specimen.For interested chromosomal karyomit(e) dosage and a first threshold relatively determined in specimen exist or do not exist for interested chromosomal dysploidy, and for the comparatively validate of chromosomal the second karyomit(e) dosage of normalization method and a Second Threshold sample classification definite.Test chromosome dosage can further be expressed as described NCV, and wherein the first and second karyomit(e) dose forms are shown the first and second NCV; And the classification of specimen is by comparing a NCV and a first threshold and the 2nd NCV and a Second Threshold are compared to carry out.

Although example herein relates to complete chromosomal aneuploidy, concept of the present invention is applicable to the dysploidy of part.In one embodiment, interested sequence is the chromosome segment relevant with the dysploidy (for example chromosome deletion or insertion or unbalanced chromosome translocation) of part, and at least two normalization method sequences are chromosome segments irrelevant with the dysploidy of part, and the sequence label Density Variation of these two normalization method sequences is close to the sequence label Density Variation of the chromosome segment relevant with the dysploidy of part.The dysploidy of part can use the karyomit(e) dose determination (referring to the U.S. Patent application 12/958 of the International Application Serial No. PCT/US2010/058609 that submitted on December 1st, 2010 and submission on December 1st, 2010,352, the title of these applications all be " method (Method for Determining Copy Number Variations) that be used for to determine the copy number variation " and be combined in full this with it by reference).Can use at least two normalization method sequence checkings to have or do not exist a kind of dysploidy of part according to the inventive method.

Fig. 1 provides the schema of two exemplary of method 100, and the method is determined in the sample that comprises two genomic mixtures (for example maternal sample) and there is or does not exist a kind of chromosomal aneuploidy in checking.

In first embodiment, determine there is or does not exist fetal chromosomal aneuploidy in the method by following steps in the parent specimen that comprises fetus and parent nucleic acid: (a) obtain the sequence information for fetus in maternal sample and parent nucleic acid, so that identification is for the number of an interested chromosomal sequence label and for the number of at least two chromosomal sequence labels of normalization method; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist the fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of described interested chromosomal the second normalized value and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is second a karyomit(e) dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described normalized karyomit(e) value (NCV).

Describe the first embodiment according to the step 110,120,130 and 140 of method as shown in Figure 1.The number (110) that checks order to provide sequence label to the fetus that obtains from maternal sample and parent nucleic acid.The sequence label that is mapped to an interested karyomit(e) (for example karyomit(e) 21) and the sequence label that is mapped to two normalization method karyomit(e)s (for example karyomit(e) 9 and karyomit(e) 11) are counted and be used for calculating for interested chromosomal corresponding the first and second normalized values (for example karyomit(e) dosage).In one embodiment, at least two karyomit(e) dosage are normalized values of determining for each interested karyomit(e).In one embodiment, be a first chromosome dosage for interested chromosomal the first normalized value, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And be second a karyomit(e) dosage for interested chromosomal the second normalized value, it is for the number of interested chromosomal sequence label and ratio (120) for the number of a chromosomal sequence label of the second normalization method.Will be for interested chromosomal the first normalized value (namely, the first chromosome dosage) compare with a first threshold, and will compare (130) with a Second Threshold for interested chromosomal the second normalized value (i.e. the second karyomit(e) dosage), and have or do not exist determining and checking (140) of a kind of chromosomal aneuploidy.Alternately, at least two karyomit(e) dose forms are shown the first and second normalized karyomit(e) values (NCV), the one NCV makes the first chromosome dosage be associated with the average of corresponding the first chromosome dosage in a combination lattice sample, and the 2nd NCV makes the second karyomit(e) dosage be associated with the average of corresponding karyomit(e) dosage in same combination lattice sample, as:

{NCV}_{ij} = \frac{x_{ij} - {\hat{μ}}_{j}}{{\hat{σ}}_{j}}

Wherein

With

Estimation average and the standard deviation for j karyomit(e) dosage in a combination lattice sample accordingly, and x _IjFor viewed j the karyomit(e) dosage of specimen i.The first and second normalized values (that is, NCV) are compared (130) with a first threshold with a Second Threshold accordingly separately, and are had or do not exist determining and checking (140) of a kind of chromosomal aneuploidy.The method can be identified extremely rare (for example 9 trisomys) and more common chromosomal aneuploidy (for example trisomy 21), and can identify a plurality of chromosomal aneuploidies from order-checking information, this order-checking information is the single order-checking batch from the specimen nucleic acid (for example cfDNA) and obtaining.As shown in example, there is not trisomy 21 although determine to exist or do not exist the sequence information of trisomy 21 to disclose for being used for of obtaining of sample, this sample comprises 9 trisomys.In some embodiments, identify chromosomal aneuploidy in each in karyomit(e) 1-22, chromosome x and karyomit(e) Y.Can in interested karyomit(e) and/or the first or second normalization method karyomit(e), identify chromosomal aneuploidy.In some embodiments, the method identifies a plurality of chromosomal aneuploidies that are selected from trisomy 21,13 trisomys, 18 trisomys and X monosomy.

In second embodiment, the method is verified in the parent specimen that comprises fetus and parent nucleic acid molecule by following steps and is had or do not exist determining for interested chromosomal a kind of dysploidy: (a) obtain the sequence information for fetus in sample and parent nucleic acid, so that identification is for the number of the sequence label of an interested chromosomal mapping and for the number of at least two chromosomal sequence labels of normalization method; (b) use for the number of interested chromosomal label and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of chromosomal the second normalized value of described the first normalization method and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, and it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as according to normalized karyomit(e) value following calculating, aforesaid (NCV)

{NCV}_{ij} = \frac{x_{ij} - {\hat{μ}}_{j}}{{\hat{σ}}_{j}}

Describe the second embodiment according to the step 110,150,160 and 140 of method as shown in Figure 1.The number (110) that checks order to provide sequence label to the fetus that obtains from maternal sample and parent nucleic acid.To being mapped to the sequence label of an interested karyomit(e) (for example karyomit(e) 21), and the sequence label that is mapped to a normalization method karyomit(e) (for example karyomit(e) 9) is counted and be used for calculating for interested chromosomal corresponding first normalized value (for example karyomit(e) dosage), and as the ratio of the sequence label that is mapped to the first normalization method karyomit(e) (for example karyomit(e) 9) with the number of the sequence label that is mapped to second a normalization method karyomit(e) (for example karyomit(e) 11), calculate for chromosomal second normalized value of the first normalization method (for example karyomit(e) dosage) (150).The first and second normalized values (that is, karyomit(e) dosage) are compared (160) with the first and second threshold values separately accordingly, and have or do not exist determining and checking (140) of a kind of chromosomal aneuploidy.Alternately, two normalized values (namely, two karyomit(e) dosage) be expressed as the first and second normalized karyomit(e) values (NCV), the one NCV makes the first chromosome dosage be associated with the average of corresponding the first chromosome dosage in a combination lattice sample, and the 2nd NCV makes the second karyomit(e) dosage be associated with the average of corresponding karyomit(e) dosage in same combination lattice sample, as:

{NCV}_{ij} = \frac{x_{ij} - {\hat{μ}}_{j}}{{\hat{σ}}_{j}}

Wherein

With

Estimation average and the standard deviation for j karyomit(e) dosage in a combination lattice sample accordingly, and x _IjFor viewed j the karyomit(e) dosage of specimen i.The first and second normalized values (that is, NCV) are compared separately (160), and are had or do not exist determining and checking (140) of a kind of chromosomal aneuploidy with predetermined threshold.

As discussed previously, the method can be identified rare dysploidy (for example 9 trisomys) and common dysploidy (for example trisomy 21), chromosomal aneuploidy, and can identify a plurality of chromosomal aneuploidies from order-checking information, this order-checking information is from about the single order-checking batch acquisition of specimen nucleic acid (for example cfDNA).In some embodiments, the single or multiple chromosomal aneuploidies of identification in each in karyomit(e) 1-22, chromosome x and karyomit(e) Y.Can in interested karyomit(e) and/or the first or second normalization method karyomit(e), identify chromosomal aneuploidy.In some embodiments, the method identifies the single or multiple chromosomal aneuploidies that are selected from trisomy 21,13 trisomys, 18 trisomys, 9 trisomys and X monosomy.

Can concentrate in one or more independently qualified samples and determine normalization method karyomit(e).In some embodiments, can be concentrated definite for all the chromosomal normalization method karyomit(e)s in the genome in one or more qualified samples.Determine for all the chromosomal normalization method karyomit(e)s in the genome, allow to determine chromosomal aneuploidy in genomic each karyomit(e) with order-checking information, the single order-checking batch acquisition of the nucleic acid that this order-checking information is always self-test sample.

In all embodiments, can following selection normalization method karyomit(e).

Normalization method karyomit(e) for karyomit(e) 1 is to be selected from karyomit(e) 10,11,9 and 15.In one embodiment, the first and second normalization method karyomit(e)s for karyomit(e) 1 are karyomit(e) 10 and karyomit(e) 11.

Normalization method karyomit(e) for karyomit(e) 2 is to be selected from karyomit(e) 8,7,12 and 14.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 2 are karyomit(e) 8 and karyomit(e) 7.

Normalization method karyomit(e) for karyomit(e) 3 is to be selected from karyomit(e) 6,5,8 and 18.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 3 are karyomit(e) 6 and karyomit(e) 5.

Normalization method karyomit(e) for karyomit(e) 4 is to be selected from 3,5,6 and 13.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 4 are karyomit(e) 13 and karyomit(e) 5.

Normalization method karyomit(e) for karyomit(e) 5 is to be selected from 6,3,8 and 18.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 5 are karyomit(e) 6 and karyomit(e) 3.

Normalization method karyomit(e) for karyomit(e) 6 is to be selected from 5,3,8 and 18.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 6 are karyomit(e) 5 and karyomit(e) 3.

Normalization method karyomit(e) for karyomit(e) 7 is to be selected from 12,2,14 and 8.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 7 are karyomit(e) 12 and karyomit(e) 2.

Normalization method karyomit(e) for karyomit(e) 8 is to be selected from 2,7,12 and 3.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 8 are karyomit(e) 2 and karyomit(e) 3.

Normalization method karyomit(e) for karyomit(e) 9 is to be selected from 11,10,1 and 14.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 9 are karyomit(e) 11 and karyomit(e) 10.

Normalization method karyomit(e) for karyomit(e) 10 is to be selected from 1,11,9 and 15.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 10 are karyomit(e) 1 and karyomit(e) 11.

To be selected from 1,10,9 and 15 for the normalization method karyomit(e) as interested chromosomal karyomit(e) 11.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 11 are karyomit(e) 1 and karyomit(e) 10.

Normalization method karyomit(e) for karyomit(e) 12 is to be selected from 7,14,2 and 8.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 12 are karyomit(e) 7 and karyomit(e) 14.

Normalization method karyomit(e) for karyomit(e) 13 is group, karyomit(e) 5 and the karyomit(e) 6 that is selected from karyomit(e) 4, karyomit(e) 2-6.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 13 are the group of karyomit(e) 4 and karyomit(e) 2-6 accordingly.The group of karyomit(e) 2-6 can be used as the first or second normalization method karyomit(e) for interested karyomit(e) 13, and can be used as for the chromosomal normalization method karyomit(e) of the first normalization method that is used for karyomit(e) 13.In some embodiments, can carry out all the chromosomal checkings in the group.Two karyomit(e) groups can be as the first and second normalization method karyomit(e)s for karyomit(e) 13, and wherein the karyomit(e) of the first group is different from the karyomit(e) of the second group.

Normalization method karyomit(e) for karyomit(e) 14 is to be selected from 12,7,2 and 9.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 14 are karyomit(e) 12 and karyomit(e) 7.

Normalization method karyomit(e) for karyomit(e) 15 is to be selected from 1,10,11 and 9.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 2 are karyomit(e) 1 and karyomit(e) 10.

Normalization method karyomit(e) for karyomit(e) 16 is to be selected from 20,17,15 and 1.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 16 are karyomit(e) 20 and karyomit(e) 17.

Normalization method karyomit(e) for karyomit(e) 17 is to be selected from 16,20,19 and 22.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 17 are karyomit(e) 16 and karyomit(e) 20.

Normalization method karyomit(e) for karyomit(e) 18 is to be selected from 8,3,2 and 6.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 18 are karyomit(e) 8 and karyomit(e) 3.

Normalization method karyomit(e) for karyomit(e) 19 is to be selected from 22,17,16 and 20.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 19 are chromosome 22 and karyomit(e) 17.

Normalization method karyomit(e) for karyomit(e) 20 is to be selected from 16,17,15 and 1.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 20 are karyomit(e) 16 and karyomit(e) 17.

Normalization method karyomit(e) for karyomit(e) 21 is to be selected from 9,11,14 and 1.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 21 are karyomit(e) 9 and karyomit(e) 11.

Normalization method karyomit(e) for chromosome 22 is to be selected from 19,17,16 and 20.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for chromosome 22 are karyomit(e) 19 and karyomit(e) 17.

Normalization method karyomit(e) for chromosome x is to be selected from 6,5,13 and 3.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for chromosome x are karyomit(e) 6 and karyomit(e) 5.

Normalization method karyomit(e) for karyomit(e) Y is group, karyomit(e) 3, karyomit(e) 4 and the karyomit(e) 5 that is selected from karyomit(e) 2-6.In another embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) Y are the group of karyomit(e) 3 and karyomit(e) 2-6 accordingly.The group of karyomit(e) 2-6 can be used as the first or second normalization method karyomit(e) for karyomit(e) Y, or as the normalization method karyomit(e) for the first normalization method karyomit(e) (for example karyomit(e) 3) that is used for karyomit(e) Y.In some embodiments, verify out not existing of all chromosomal dysploidy in the group of 2-6.Two karyomit(e) groups can be as the first and second normalization method karyomit(e)s for karyomit(e) 13, and wherein the karyomit(e) of the first group is different from the karyomit(e) of the second group.As for karyomit(e) 13 and karyomit(e) Y institute example, normalization method karyomit(e) can be a karyomit(e) or a karyomit(e) group.

In some embodiments, these methods may relate to the analysis for 3 or 4 chromosomal sequence labels of normalization method except interested karyomit(e).

Therefore, in some embodiments, determine there is or does not exist a kind of fetal chromosomal aneuploidy in the method by following steps in the parent specimen that comprises fetus and parent nucleic acid: (a) obtain the sequence information for fetus in maternal sample and parent nucleic acid, in order to identify for the number of an interested chromosomal sequence label and for the number of three chromosomal sequence labels of normalization method; (b) calculate for interested chromosomal first, second and the 3rd normalized value with the number of sequence label; And (c) will compare for interested chromosomal first, second and the 3rd normalized value and one or more threshold value, to determine in maternal sample, to exist or do not exist a kind of fetus dysploidy.In some embodiments, be a first chromosome dosage for interested chromosomal the first normalized value, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And be second a karyomit(e) dosage for interested chromosomal the second normalized value, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method; And be a trisome dosage for interested chromosomal the 3rd normalized value, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the 3rd normalization method.Randomly, first, second and the 3rd normalized value can be expressed as such as described normalized karyomit(e) values in other places (NCV) herein.

In addition, in some embodiments, the method is verified in the parent specimen that comprises fetus and parent nucleic acid molecule by following steps and is had or do not exist determining for interested chromosomal a kind of dysploidy: (a) obtain the sequence information for fetus in maternal sample and parent nucleic acid, in order to identify for the number of an interested chromosomal sequence label and for the number of three chromosomal sequence labels of normalization method; (b) use for the number of the label of interested chromosomal mapping and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value; (c) use for the number of the chromosomal label of the first normalization method and for the number of a chromosomal label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; (d) use for the number of the chromosomal label of the second normalization method and for the number of a chromosomal label of the 3rd normalization method and determine for chromosomal the 3rd normalized value of the second normalization method; And (e) will compare for interested chromosomal first, second and the 3rd normalized value and one or more threshold value, to determine in maternal sample, to exist or do not exist a kind of fetus dysploidy.In some embodiments, the first normalized value is a first chromosome dosage, and it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method; And the 3rd normalized value is a trisome dosage, and it is for the number of the chromosomal sequence label of the second normalization method and ratio for the number of a chromosomal sequence label of the 3rd normalization method.Randomly, first, second and the 3rd normalized value can be expressed as such as described normalized karyomit(e) values in other places (NCV) herein.

In some embodiments, determine there is or does not exist a kind of fetal chromosomal aneuploidy in the method by following steps in the parent specimen that comprises fetus and parent nucleic acid: (a) obtain the sequence information for fetus in maternal sample and parent nucleic acid, in order to identify for the number of an interested chromosomal sequence label and for the number of four chromosomal sequence labels of normalization method; (b) number with sequence label calculates for the interested chromosomal first, second, third and the 4th normalized value; And (c) will compare for the interested chromosomal first, second, third and the 4th normalized value and one or more threshold value, to determine in maternal sample, to exist or do not exist a kind of fetus dysploidy.In some embodiments, be a first chromosome dosage for interested chromosomal the first normalized value, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And be second a karyomit(e) dosage for interested chromosomal the second normalized value, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method; And be a trisome dosage for interested chromosomal the 3rd normalized value, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the 3rd normalization method; And be a tetrasome dosage for interested chromosomal the 4th normalized value, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the 4th normalization method.Randomly, the first, second, third and the 4th normalized value can be expressed as such as described normalized karyomit(e) values in other places (NCV) herein.

In some embodiments, the method is determined in the parent specimen that comprises fetus and parent nucleic acid molecule by following steps and checking exists or do not exist for interested chromosomal a kind of dysploidy: (a) obtain the sequence information for fetus in maternal sample and parent nucleic acid, in order to identify for the number of an interested chromosomal sequence label and for the number of four chromosomal sequence labels of normalization method; (b) use for the number of tags of interested chromosomal mapping and for a chromosomal number of tags of the first normalization method and determine for interested chromosomal first normalized value; (c) use for the number of the chromosomal label of the first normalization method and for the number of a chromosomal label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (d) use for the number of the chromosomal label of the second normalization method and for the number of a chromosomal label of the 3rd normalization method and determine for chromosomal the 3rd normalized value of the second normalization method; (e) use for the number of the chromosomal label of the 3rd normalization method and for the number of a chromosomal label of the 4th normalization method and determine for chromosomal the 4th normalized value of the 3rd normalization method; And (f) will compare for the interested chromosomal first, second, third and the 4th normalized value and one or more threshold value, to determine in maternal sample, to exist or do not exist a kind of fetus dysploidy.In some embodiments, the first normalized value is a first chromosome dosage, and it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method; And the 3rd normalized value is a trisome dosage, and it is for the number of the chromosomal sequence label of the second normalization method and ratio for the number of a chromosomal sequence label of the 3rd normalization method; And the 4th normalized value is a tetrasome dosage, and it is for the number of the chromosomal sequence label of the 3rd normalization method and ratio for the number of a chromosomal sequence label of the 4th normalization method.Randomly, the first, second, third and the 4th normalized value can be expressed as such as described normalized karyomit(e) values in other places (NCV) herein.

In these embodiments, the first, second, third and the 4th normalization method karyomit(e) can be selected from the normalization method karyomit(e) of above elaboration.For instance, the first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 1 can be selected from karyomit(e) 10,11,9 and 15; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 2 can be selected from karyomit(e) 8,7,12 and 14; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 3 can be selected from karyomit(e) 6,5,8 and 18; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 4 can be selected from karyomit(e) 3,5,6 and 13; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 5 can be selected from karyomit(e) 6,3,8 and 18; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 6 can be selected from karyomit(e) 5,3,8 and 18.The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 7 can be selected from karyomit(e) 12,2,14 and 8; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 8 can be selected from karyomit(e) 2,7,12 and 3; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 9 can be selected from karyomit(e) 11,10,1 and 14; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 10 can be selected from 1,11,9 and 15; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 11 can be selected from karyomit(e) 1,10,9 and 15; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 12 can be selected from karyomit(e) 7,14,2 and 8; Can be selected from the group, 5 and 6 of karyomit(e) 4, karyomit(e) 2-6 for the first, second, third and the 4th normalization method karyomit(e) of karyomit(e) 13; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 14 can be selected from karyomit(e) 12,7,2 and 9; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 15 can be selected from 1,10,11 and 9; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 16 can be selected from karyomit(e) 20,17,15 and 1; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 17 can be selected from karyomit(e) 16,20,19 and 22; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 18 can be selected from karyomit(e) 8,3,2 and 6; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 19 can be selected from chromosome 22,17,16 and 20; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 20 can be selected from karyomit(e) 16,17,15 and 1; The first, second, third and the 4th normalization method karyomit(e) for karyomit(e) 21 can be selected from karyomit(e) 9,11,14 and 1; The first, second, third and the 4th normalization method karyomit(e) for chromosome 22 can be selected from karyomit(e) 19,17,16 and 20; The first, second, third and the 4th normalization method karyomit(e) for chromosome x can be selected from karyomit(e) 6,5,13 and 3; And can be selected from group, the karyomit(e) 3,4 and 5 of karyomit(e) 2-6 for the first, second, third and the 4th normalization method karyomit(e) of karyomit(e) Y.

Sequence measurement

In certain methods of the present invention, obtain to come for the sequence information of fetus in sample and parent nucleic acid the number of recognition sequence label, comprise the fetus in the sample and parent nucleic acid molecule are checked order.

Sequence information is by using any in next generation's order-checking (NGS) method that the dna profiling that increases in clone's mode or single DNA molecules is checked order in extensive parallel mode, genomic dna in maternal sample (such as Cell-free DNA) checked order obtain (such as at the people such as Wo Keerding (Volkerding), clinical chemistry (Clin Chem) 55:641-658[2009]; But maze M (Metzker M), naturally comment (Nature Rev) 11:31-46[2010] described in).Except the high-throughput sequence information, NGS provides quantitative information, and wherein each sequence reading is computable " sequence label ", and these sequence labels represent individual cloned DNA template or single dna molecular.The synthesis method that tetra-sodium order-checking, a plurality of reversible dyestuff terminators of use carry out that includes but not limited to the sequencing technologies of NGS checks order, connects by oligonucleotide probe order-checking and the ionic semiconductor order-checking of carrying out.Can check order individually from the DNA (being the singleplex order-checking) of independent sample, perhaps when single order-checking operation, as index genome molecule, DNA from a plurality of samples can be pooled together and be checked order (namely, multiple order-checking), to produce the reading up to some hundred million dna sequence dna.The example of sequencing technologies below has been described, these technology can be used for obtaining the sequence information of the method according to this invention.

Some sequencing technologies are commercially available, for example from high (the Sani Wei Er (Sunnyvale) of company (Affymetrix Inc.) that flies of the U.S., CA) sequencing by hybridization platform, with from 454 Life Sciences Corp. (454Life Sciences) (Bradford (Bradford), CT), California Hayward hundred million sensible/Suo Liesha company (Illumina/Solexa) (Haywards (Hayward), CA) with spiral Biological Science Co., Ltd (Helicos Biosciences) (Cambridge (Cambridge), MA) synthesis method order-checking platform, and from Applied biosystems (Applied Biosystems) (Foster city (Foster City), CA) connection method order-checking platform, as described below.Except the single-molecule sequencing that the synthesis method order-checking of using spiral Biological Science Co., Ltd (HelicosBiosciences) is carried out, other single-molecule sequencing technology comprise the SMRT of Pacific Ocean Biological Science Co., Ltd (Pacific Biosciences) ^TMTechnology, ion Torrent ^TMTechnology, and the nanoporous of just developing order-checking are for example by Oxford nanoporous technology.Although the Sang Geerfa of automatization (Sanger method) is regarded as ' first-generation ' technology, the inventive method can be applied to use the Sang Geer order-checking biological detection of (comprising the Sang Geer order-checking of automatization).In addition, the inventive method can be applied to use the biological assay of nucleic acid imaging technique (for example atomic force microscope (AFM) or transmission electron microscopy (TEM)).Following illustrated example sequencing technologies.

In one embodiment, method of the present invention comprises use single-molecule sequencing technology, single-molecule sequencing (the Helicos True Single Molecule Sequencing that spiral is real; TSMS) technology obtains the sequence information (such as the people such as Harris T.D. (HarrisT.D.), science (Science) 320:106-109[2008] described in) for genomic dna (such as fetus and parent cfDNA).In the tSMS technology, the DNA sample is cut into the chain of about 100 to 200 Nucleotide, and the polyA sequence is added to 3 ' end of each DNA chain.Come each chain of mark by adding fluorescently-labeled adenylic acid (AMP).These DNA chains are then by the extremely mobile groove of hybridization, and it contains millions of few T (oligo-T) that are fixed to the rooved face that flows and catches the site.Template can be at about 100,000,000 template/cm ²Density.Then the groove that will flow is loaded in the instrument, for example HeliScope ^TMSequenator, and the mobile rooved face of illuminated with laser light, the position of disclosing each template.The CCD camera can be drawn the position of the template on the rooved face that flows.Then cut and wash off the template fluorescent marker.Begin sequencing reaction by introducing archaeal dna polymerase and fluorescently-labeled Nucleotide.Few T nucleic acid is as primer.Polysaccharase is in the mode of template-directed, and the Nucleotide of mark is attached on this primer.Remove polysaccharase and unconjugated Nucleotide.By making the rooved face imaging of flowing, distinguish the template of the guiding combination with fluorescently-labeled Nucleotide.After imaging, cutting step has been removed fluorescent marker, and repeats this process with other fluorescently-labeled Nucleotide, until reach the reading length of hope.Add the collection step sequence information with each Nucleotide.The PCR base of getting rid of in the preparation sequencing library by the whole gene order-checking of single-molecule sequencing technology increases, and the direct measurement of the substantivity of sample preparation permission sample, rather than the measurement of the copy of sample.

In another embodiment, method of the present invention comprises uses 454 order-checkings (Roche Holding Ag (Roche)) to obtain sequence information (for example people's nature (Nature) 437:376-380[2005 such as Margules M (Margulies, M.)] described in) for genomic dna (for example fetus and parent cfDNA).454 order-checkings relate to two steps.In the first step, DNA is cut into the fragment of about 300-800 base pair, and these fragments finish with flush end.Then oligonucleotide aptamer is connected to the end of these fragments.The aptamer primer that acts on amplification and these fragments that check order.For example use aptamer B, it contains 5 ' biotin label, and these fragments can be attached to DNA and catch on the pearl, on the pearl that for example Streptavidin applies.In many oil drippings water emulsion pcr amplification be attached to fragment on the pearl.The result is a plurality of copies of the dna fragmentation of the clonal expansion on each pearl.In second step, in hole (the picoliter size), catch these pearls.The parallel tetra-sodium that carries out of each dna fragmentation is checked order.Add one or more Nucleotide and produce a optical signal by the CCD cameras record in the sequenator.The few nucleotide of strength of signal and combination is proportional.The tetra-sodium order-checking has utilized the pyrophosphate (PPi) that discharges when Nucleotide adds.In the presence of adenylic acid (AMP) 5 ' phosphinylidyne vitriol, PPi is converted into ATP by the ATP sulfurylase.Luciferase uses ATP that fluorescein is changed into oxyluciferin, and this reaction produced light, and this light is distinguished and analyzed.

In another embodiment, the inventive method comprises use SOLiD ^TMTechnology (Applied Biosystems, Inc. (Applied Biosystems)) obtains the sequence information for genomic dna (for example fetus and parent cfDNA).At SOLiD ^TMIn the connection method order-checking, genomic dna is cut into fragment, and aptamer is attached to 5 ' and 3 ' end of these fragments, with generation sheet phase library.Alternately, can distribute these fragments by on 5 ' and the 3 ' end that aptamer is connected to these fragments, digest the fragment of these distributions to produce inner aptamer, and 5 ' and the 3 ' end of fragment that aptamer is attached to generation is upper to produce the pairing storehouse, introduces inner aptamer.Next, preparation clone pearl group in the microreactor that contains pearl, primer, template and PCR component.Behind PCR, sex change template and concentrated pearl have the pearl of extending template with separation.Make the template on the selected pearl stand to allow to be attached to the modification of 3 ' on the slide glass.By order hybridization and the connection portion random oligonucleotide base (or base pair) definite with the central authorities of the identification of rolling into a ball by specific fluorescent, can determine this sequence.After the record color, cut and remove the oligonucleotide of connection, and then repeat this process.

In another embodiment, method of the present invention comprises the real-time (SMRT of the unit molecule that uses Pacific Ocean Biological Science Co., Ltd (Pacific Biosciences) ^TM) sequencing technologies obtains the sequence information for genomic dna (for example fetus and parent cfDNA).In SMRT order-checking, image is carried out in the continuous combination to the Nucleotide of dye marker in the DNA building-up process.Single DNA polysaccharase molecule is attached on the basal surface of independent null mode wavelength recognizer (ZMW recognizer), and these recognizers obtain sequence information when the Nucleotide that connects phosphorus is incorporated in the primer strand in the growth.ZMW is an enclosed construction, and it allows to observe by the archaeal dna polymerase for the background of the fluorescent nucleotide of rapid diffusion turnover ZMW (pressing microsecond meter), the combination of mononucleotide.With some milliseconds Nucleotide is attached in the chain of growing.During this period, fluorescence excitation marker and generation fluorescent signal, and cut away this fluorescence labels.The identification of the corresponding fluorescence of dyestuff has shown which base is combined.Repeat this process.

In another embodiment, method of the present invention comprises uses the nanoporous order-checking to obtain sequence information (for example at (Soni) GV of Sony and Mei Le A. (Meller A.), clinical chemistry (Clin Chem) 53:1996-2001[2007] described in) for genomic dna (for example fetus and parent cfDNA).By the positive nanoporous sequenced dna analytical technology of developing of a plurality of companies industrially, comprise Oxford nanoporous company (Oxford Nanopore Technologies) (Oxford, Britain).The nanoporous order-checking is a kind of single-molecule sequencing technology, and along with it passes through a nanoporous, a monomolecular DNA is by direct Sequencing thus.Nanoporous is an aperture, and its rank is diameter 1 nanometer.Nanoporous is immersed in the conductive fluid, and apply an electromotive force (voltage) across it, produced because ionic conduction is passed a slight electric current of nanoporous.For the size and shape of nanoporous, the amount of mobile electric current is responsive.Along with dna molecular passes nanoporous, each Nucleotide on the dna molecular is to block in various degree nanoporous, to change in various degree the magnitude of the electric current that passes nanoporous.Therefore, pass the reading of change representation DNA sequence of the electric current of nanoporous along with dna molecular.

In another embodiment, method of the present invention comprises and uses chemosensitivity field effect electric crystal (chemFET) array to obtain sequence information (for example as described in the U.S. Patent Application Publication No. 20090026082) for genomic dna (for example fetus and parent cfDNA).In an example of this technology, dna molecular can be placed in the reaction chamber, and template molecule can be hybridized on the sequencing primer that is attached to polysaccharase.Can be attached in the new nucleic acid chain by distinguishing with the change in the electric current of chemFET at the one or more triphosphate in 3 ' end place of sequencing primer.An array can have a plurality of chemFET sensors.In another example, mononucleotide can be attached on the pearl, and these nucleic acid can increase on this pearl, and independent pearl can be transferred in the independent reaction chamber on the chemFET array, wherein each chamber has the chemFET sensor, and nucleic acid can be sequenced.

In another embodiment, method of the present invention comprises that the technology of using kingfisher branch and subsidiaries (HalcyonMolecular) obtains the sequence information for genomic dna (for example fetus and parent cfDNA), this utilization transmission electron microscopy (TEM).The method, be called as individual molecule and place rapid nano transfer (Individual Molecule Placement Rapid Nano Transfer, IMPRNT), comprise the monatomic resolving power transmission electron microscope imaging that utilizes by high molecular (150kb or the larger) DNA of heavy atom marker selected marker, and arrange these molecules with consistent base to the ultrathin membrane of base spacing in ultra dense degree (the 3nm chain is to chain) parallel array.Come molecule on the imaging film with electron microscope, determining the position of heavy atom marker, and extract the base sequence information from DNA.In PCT patent disclosure WO 2009/046445, further illustrate the method.The method allows the complete human genome of order-checking within less than ten minutes time.

In another embodiment, the dna sequencing technology is ion torrent company (Ion Torrent) single-molecule sequencing, it makes semiconductor technology and the pairing of simple order-checking chemistry, on semi-conductor chip the information (A, C, G, T) of chemical code directly is translated as numerical information (0,1).In essence, when Nucleotide being attached in the DNA chain by polysaccharase, discharge a hydrogen ion as by product.Ion torrent company (Ion Torrent) uses the hole array of a highdensity micromachined, carries out these Biochemical processes with extensive parallel mode.Each pore volume is received a different dna molecular.Be an ion-sensitive layer under these holes, and be an ionization sensor under it.At a Nucleotide, for example C when being added to a dna profiling and then being attached in the DNA chain, will discharge a hydrogen ion.To change the pH value of solution from the electric charge of this ion, this variation can be identified by the ionization sensor of ion torrent.This sequenator---in essence in the world minimum solid-state pH meter---is judged base, directly from the chemical information to numerical information.A this ion human genome machine (Ion personal GenomeMachine, PGM ^TM) then sequenator sequentially flood this chip with one by one Nucleotide.Do not mate if flood the next Nucleotide of this chip, will record so the change less than voltage, and will can not judge base.If have two the same bases at the DNA chain, voltage will double so, and this chip will be recorded to two the same bases through judging.Direct Recognition allows by the combination of recording Nucleotide second.

In another embodiment, method of the present invention comprises by the synthesis method order-checking of using hundred million sensible companies (Illumina) and based on the order-checking chemistry of reversible terminator millions of dna fragmentations is carried out extensive parallel order-checking and obtains sequence information (such as at the people such as Bentley (Bentley), Nature (nature) 6:53-59[2009] described in) for genomic dna (such as fetus and parent cfDNA).Template DNA can be genomic dna, for example cfDNA.In some embodiments, be used as template from the genomic dna of isolated cell, and be split into the length of hundreds of base pairs.In other embodiments, cfDNA is used as template, and does not need to cut apart, because cfDNA exists with the short-movie section.For instance, fetus cfDNA circulates with the pieces less than 300bp in blood flow, and according to estimates parent cfDNA be with about 0.5 and 1Kb between pieces circulation (people such as Lee (Li), clinical chemistry (ClinChem), 50:1002-1011[2004]).The sequencing technologies of hundred million sensible companies relies on the attached of genomic dna to a plane (the oligonucleotide anchor point is attached to top randomly transparent surface) cut apart.Template DNA is produced the flush end of 5 ' phosphorylation by end reparation, and the polymerase activity of Ke Lienuo (Klenow) fragment is used to add single A base to 3 ' end of flat phosphorylated cdna fragment.This interpolation has prepared the dna fragmentation that is used for being connected on the oligonucleotide aptamer, and in their 3 ' and end has single T base of a suspension, with the increase joint efficiency.These aptamer oligonucleotide and mobile groove anchor point are complementary.Under the restriction diluting condition, aptamer is modified, single-stranded template DNA is added to mobile groove, and is fixed to anchor point by hybridization.Attached dna fragmentation is extended and the bridge-type amplification, produces the mobile groove of super-high density order-checking with several hundred million bunches, and each all contains about 1000 copies of same template.In one embodiment, the genomic dna of random division, for example cfDNA is front in experience cluster amplification (cluster amplification), uses PCR that it is increased.Alternately, use is without amplification gene group storehouse goods, and increases individually genomic dna (that is, the cfDNA) (people such as Ke Zharewa (Kozarewa) of enrichment random division with cluster, natural method (Nature Methods), 6:291-295[2009]).Use a kind of four color DNA synthesis method sequencing technologies of robust these templates that check order, this technology has adopted has the reversible terminator that can remove fluorescence dye.Obtain the identification of hypersensitivity fluorescence with laser excitation and total internal reflection optics.The short sequence reading that will have about 20-40bp (for example 36bp) is compared with respect to the reference gene group of covering repetition, and judges hereditary difference with specifically developed data analysis streamline software.After the first time, reading was finished, can these templates of in-situ regeneration, so that second reading that can obtain from the end opposite of these fragments.Therefore, the single terminal also or in pairs terminal order-checking that can use these dna fragmentations.Carry out the part order-checking of the dna fragmentation that exists in the sample, and will the sequence label of the reading that comprises predetermined length (for example 36bp) reflect known reference gene group.Can shine upon the label of penetrating counts.

In one embodiment, reference gene group sequence is the NCBI36/hg18 sequence, is it in the World Wide Web, at genome.ucsc.edu/cgi-bin/hgGateway? org=Human﹠amp; Db=hg18﹠amp; Hgsid=166260105 can get.In another embodiment, reference gene group sequence is GRCh37/hg19, and it can get at genome.ucsc.edu/cgi-bin/hgGateway in the World Wide Web.Sequence from other reference gene groups of multiple species can get at ncbi.nlm.nih.gov/genomes/leuks.cgi in the NCBI website.Other sources of open sequence information comprise GenBank, dbEST, dbSTS, EMBL (European Molecular Bioglogy Laboratory) and DDBJ (Japanese DNA database).A plurality of computerized algorithms can be used for aligned sequences, comprise rather than limit: restriction BLAST people such as (, 1990) A Erqiuer (Altschul), BLITZ (MPsrch) (Sturrock (Sturrock); Collins (Collins), 1993), FASTA (gloomy (the Person) ﹠amp of amber; Li Puman (Lipman), 1988), the BOWTIE (people such as glug plum moral (Langmead), genome biology (Genome Biology) 10:R25.1-R25.10[2009]) or ELAND (the San Diego, CA, USA hundred million sensible (Illumina of company, Inc.), San Diego (San Diego), CA, USA).In one embodiment, end to the copy of the clone of blood plasma cfDNA molecule expansion checks order, and it is processed by the information biology compare of analysis that is used for Illumina genome analysis instrument, and this analyser has used effectively extensive comparison (ELAND) software of RiboaptDB.

In some embodiments of the method for this explanation, the sequence label of mapping is included as the sequence reading of about 20bp, about 25bp, about 30bp, about 35bp, about 40bp, about 45bp, about 50bp, about 55bp, about 60bp, about 65bp, about 70bp, about 75bp, about 80bp, about 85bp, about 90bp, about 95bp, about 100bp, about 110bp, about 120bp, about 130, about 140bp, about 150bp, about 200bp, about 250bp, about 300bp, about 350bp, about 400bp, about 450bp or about 500bp.The expectation technical superiority will be so that can carry out single-ended reading greater than 500bp, and when producing pairing end reading, this reading allows to for the reading greater than about 1000bp.In one embodiment, the sequence label of mapping is included as the sequence reading of 36bp by the mapping that relatively realizes sequence label of sequence label and reference sequences, with the karyomit(e) source of definite nucleic acid (for example cfDNA) molecule that checks order, and do not need specificity genetic sequence information.Can allow the mispairing (each sequence label 0-2 mispairing) of little degree, may reside in little polymorphism between the genome in reference gene group and the biased sample with explanation.

Each sample obtains a plurality of sequence labels.In some embodiments, from the reference gene group that reading is mapped to each sample, obtained to be included in 20 and the reading of 40bp between (for example 36bp) at least about 3x10 ⁶Individual sequence label, at least about 5x10 ⁶Individual sequence label, at least about 8x10 ⁶Individual sequence label, at least about 10x10 ⁶Individual sequence label, at least about 15x10 ⁶Individual sequence label, at least about 20x10 ⁶Individual sequence label, at least about 30x10 ⁶Individual sequence label, at least about 40x10 ⁶Individual sequence label or at least about 50x10 ⁶Individual sequence label.In one embodiment, the mapped All Ranges that is mapped to the reference gene group of all sequences reading.In one embodiment, label to the All Ranges (for example all karyomit(e)s) that is mapped to the reference gene group is counted, and determine in the hybrid dna sample, the CNV of interested sequence (for example karyomit(e) or its part) (that is, overexpression or expression are not enough).The method does not need two differentiation between the genome.

In some embodiments, the method determines to exist or do not exist a kind of fetal chromosomal aneuploidy by following steps in the parent specimen that comprises fetus and parent nucleic acid molecule: (a) obtain the sequence information for fetus in maternal sample and parent nucleic acid, so that identification is for the number of an interested chromosomal sequence label and for the number of at least two chromosomal sequence labels of normalization method, wherein sequence information comprises order-checking of future generation (NGS), comprise the synthesis method order-checking of using a plurality of reversible dyestuff terminators to carry out, comprise the connection method order-checking, or comprise single-molecule sequencing; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of described interested chromosomal the second normalized value and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is second a karyomit(e) dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).

In some other embodiments, the method is verified in the parent specimen that comprises fetus and parent nucleic acid molecule by following steps and is had or do not exist determining for interested chromosomal a kind of dysploidy: (a) obtain the sequence information for fetus in sample and parent nucleic acid, so that identification wherein obtains sequence information and comprises order-checking of future generation (NGS) for the number of the sequence label of an interested chromosomal mapping and for the number of at least two chromosomal sequence labels of normalization method, comprise the synthesis method order-checking of using a plurality of reversible dyestuff terminators to carry out, comprise the connection method order-checking, or comprise single-molecule sequencing; (b) use for the number of interested chromosomal label and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of chromosomal the second normalized value of described the first normalization method and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, and it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as the normalized karyomit(e) value (NCV) of calculating as described herein.

In some embodiments, the first normalized value is a first chromosome dosage, and it is for the number of described interested chromosome sequence label and ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).

Biological fluid comprises, as limiting examples, and blood, blood plasma, serum, sweat, tears, phlegm, urine, phlegm, ear effluent (ear flow), lymph liquid, saliva, cerebrospinal fluid, irrigating solution (ravages), marrow suspension (bone marrow suspension), vagina effluent (vaginal flow), through the irrigating solution of uterine neck, brain liquid, ascites, milk, the secretory product of breathing, intestines and genitourinary tract, amniotic fluid and white corpuscle exclusion sample.In some embodiments, this sample is by non-invasive process easily obtainable sample, for example blood, blood plasma, serum, sweat, tears, phlegm, urine, phlegm, ear effluent and saliva.Preferably, this biological sample is peripheral blood sample, or blood plasma or serum part.In other embodiments, this biological sample is cotton swab or smear, examination of living tissue sample, or cell cultures.In another embodiment, this sample is the mixture of two or more biological samples, and the biological example product of imitating can comprise two or more biological fluid samples, tissue sample and cell cultures sample.As used in this, term " blood ", " blood plasma " and " serum " are clearly contained their separated part or the part of processing.Similarly, when a sample be take from a kind of examination of living tissue, cotton swab, smear, etc. the time, should " sample " contain clearly derived from this examination of living tissue, cotton swab, smear, etc. separated part or the part of processing.

In some embodiments, sample can derive from a plurality of sources, include but not limited to, from Different Individual, the different stages of development of identical or different individuality, different diseased individuals (for example suffering from individuality cancer or that suspection has genetic block), the sample of normal individual, the sample that obtains in the different steps of the disease of individuality, derive from experience to the sample of the individuality of the difference treatment of disease, sample from the individuality that experiences the varying environment factor, or to a kind of individuality of state of an illness susceptible, or be exposed to the individuality of a kind of transmissible disease factor (for example HIV), and be donorcells, the sample of the recipient's of tissue and/or organ individuality.In some embodiments, sample is the sample that comprises the mixture of the different sources sample that derives from identical or different experimenter.For instance, sample can comprise the mixture that derives from two or more individual cells, as usually finding in the crime scene.In one embodiment, this sample is the maternal sample that derives from conceived female (for example pregnant woman).In this case, this sample can be analyzed with the method in this explanation, so that the antenatal diagnosis of potential chromosome abnormalty in the fetus to be provided.This maternal sample can be tissue sample, biological fluid sample or cell sample.Biological fluid comprises (as limiting examples): blood, blood plasma, serum, sweat, tears, phlegm, urine, phlegm, the ear effluent, lymph, saliva, cerebrospinal fluid, irrigating solution (ravages), marrow suspension, vagina effluent, through the irrigating solution of uterine neck, brain liquid, ascites, milk, the secretory product of breathing, intestines and genitourinary tract, and white corpuscle exclusion sample.In some embodiments, this sample is by non-invasive process obtainable sample easily, for example, and blood, blood plasma, serum, sweat, tears, phlegm, urine, phlegm, ear effluent and saliva.In some embodiments, this biological sample is peripheral blood sample, or blood plasma or serum separated part.In other embodiments, this biological sample is cotton swab or smear, examination of living tissue sample or cell cultures.In another embodiment, maternal sample is the mixture of two or more biological samples, and for example, a kind of biological sample can comprise two or more biological fluid samples, tissue sample and cell cultures sample.Such as above disclosure, term " blood ", " blood plasma " and " serum " are clearly contained their separated part or the part of processing.Similarly, when a sample take from examination of living tissue, cotton swab, smear, etc. the time, this " sample " clearly contain derived from examination of living tissue, cotton swab, smear, etc. separated part or the part of processing.

Sample can also be that the tissue that derives from vitro culture, cell or other contain the source of polynucleotide.The sample of these cultivations can be taken from a plurality of sources, include but not limited to, maintain the culture (for example tissue or cell) under different culture media and the condition (for example pH value, pressure or temperature), kept the culture (for example tissue or cell) of the period of different lengths, with the different factors or reagent (drug candidate for example, or conditioning agent) culture of processing (for example tissue or cell), or the culture of dissimilar tissues or cell.

Be that people know from the method for biological origin isolating nucleic acid, and the character that depends on the source is with difference.Those of ordinary skill in the art can easily isolate from a source as at the needed nucleic acid of the method for this explanation.In some cases, can be favourable with the fracture of the nucleic acid molecule in the nucleic acid samples.Fracture can be at random, and perhaps it can be special, the situation of for example using digestion with restriction enzyme to reach.The method that is used for random fracture is known in this area, and comprises that for example restricted dnase digestion, alkaline purification and physics are sheared.In one embodiment, sample nucleic acid obtains as cfDNA, and it does not experience fracture.In other embodiments, sample nucleic acid obtains as genomic dna, and its experience is broken into about 500 or the fragment of more base pairs, and can easily use the NGS method to it.

Sample that be used for to determine CNV (for example karyomit(e) and part dysploidy) comprises (the being cell) genomic nucleic acids that is present in cell or the genomic nucleic acids of " acellular ".Genomic nucleic acids comprises DNA and RNA.Preferably, genomic nucleic acids is genomic nucleic acids and/or the cfDNA of cell.In some embodiments, the genomic nucleic acids of sample is cell DNA, and it can obtain by extracting genomic dna with artificial or mechanical system from the intact cell with identical or different genetic composition from intact cell.Cell DNA can be for example from the intact cell with identical genetic composition that derives from an experimenter, obtain from the mixture of different experimenters' intact cell or from the mixture at intact cells different aspect the genetic composition that derives from an experimenter.The method of extracting genomic dna from intact cell is known in the art, and depends on the character in source and different.In some embodiments, may be favourable with the cell genomic dna fracture.Fracture can be at random, or it can be specific, realizes as for example using restriction endonuclease digestion.The method of random segment is known in the art, and comprises that for example restrictive dnase digestion, alkaline purification and physics are sheared.In some embodiments, sample nucleic acid is to obtain with the cell genomic dna form, makes the cell genomic dna fracture, becomes the fragment with about 500 or more base pairs, and these fragments can check order by check order (NGS) of future generation.

In some embodiments, obtain cell genomic dna so that identification comprises the chromosomal aneuploidy of the sample of individual gene group.For instance, cell genomic dna can obtain from only comprise conceived female cell sample, and namely this sample does not contain the Fetal genome sequence.From individual gene group (for example only maternal gene group) identification chromosomal aneuploidy can be used for and the chromosomal aneuploidy of in the mixture of the fetus that is present in Maternal plasma and maternal gene group, identifying and/or polymorphism relatively so that the identification fetal chromosomal aneuploidy.Similarly, cell genomic dna can obtain from the patient's (for example cancer patient) who is in different treatment stages, so that by the possible variation of chromosomal aneuploidy in the sample DNA and/or polymorphism being analyzed to estimate the effect for the treatment of plan.

In some embodiments, obtain acellular nucleic acid, for example Cell-free DNA (cfDNA) is favourable.Acellular nucleic acid (comprising Cell-free DNA) can from the biological sample that includes but not limited to blood plasma, serum and urine, obtain by diverse ways known in the art (people such as model (Fan), periodical (the Proc Natl Acad Sci) 105:16266-16271[2008 of institute of NAS]; Littlely go out people such as (Koide), antenatal diagnosis (Prenatal Diagnosis) 25:604-607[2005]; People such as old (Chen), Natural medicine (Nature Med.) 2:1033-1035[1996]; The people such as Lu (Lo), lancet (Lancet) 350:485-487[1997]; The people such as Bo Taizhatu (Botezatu), clinical chemistry (Clin Chem.) 46:1078-1084,2000; And the people such as (Su) of Soviet Union, molecular diagnostics magazine (J Mol.Diagn.) 6:101-107[2004]).For from cellular segregation cfDNA, can use part to separate (fractionation), centrifugal (for example density gradient centrifugation), DNA specificity precipitation or high-flux cell sorting and/or separation method.Can obtain for commercially available test kit (Indianapolis, the state of Indiana Luo Shi diagnostic companies (Roche Diagnostics, Indianapolis, IN) of manually separating with automatization cfDNA; Markon's welfare Ya Zhou Valencia Kai Jie company (Qiagen, Valencia, CA); Delaware State Du Lun cot Lei-Nei Geer company (Macherey-Nagel, Duren, DE)).During the biological sample that comprises cfDNA has been used to analyze, in order to determine existence or do not have multiple chromosome abnormalty (for example trisomy 21) by the sequencing analysis that can determine chromosomal aneuploidy and/or different polymorphism.

Before the preparation sequencing library, can be specifically or non-specifically enrichment be present in cfDNA in the sample.The unspecific enrichment of sample DNA refers to the whole genome amplification of the genomic DNA fragment of sample, and it can be used for increasing the sample DNA level before preparation cfDNA sequencing library.Unspecific enrichment can be to be present in the selective enrichment that comprises one of two genomes in the genomic sample more than.For instance, unspecific enrichment can be the selective enrichment of the Fetal genome in the maternal sample (it can obtain by currently known methods), in order to improve the relative proportion of fetus and mother body D NA in the sample.Alternately, unspecific enrichment can be two genomic non-selective amplifications that are present in the sample.For instance, non-specific amplification can be the non-specific amplification that comprises from fetus and mother body D NA in the sample of the mixture of the DNA of fetus and maternal gene group.The method of whole genome amplification is known in the art.PCR (DOP), primer extension round pcr (PEP) and multiple displacement amplification (MDA) that the degeneracy oligonucleotide is made primer are the examples of whole genome amplification method.In some embodiments, comprise sample from the mixture of the cfDNA of different genes group not for the genomic cfDNA enrichment that is present in the mixture.In other embodiments, the sample that comprises from the mixture of the cfDNA of different genes group carries out unspecific enrichment for any one genome that is present in the sample.

Use

The acellular foetal DNA that circulates in maternal blood and RNA can be used to the early stage Non-invasive Prenatal Diagnosis (NIPD) of the ever-increasing hereditary situation of number, both can be used for management and also can help the reproduction decision-making.The existence of the Cell-free DNA that circulates in blood flow is known having surpassed 50 years.Recently, in the parent blood flow of gestation time, found to exist the foetal DNA (people such as sieve (Lo), lancet (Lancet) 350:485-487[1997]) of in a small amount circulation.Be considered to be derived from dying placenta cells, acellular foetal DNA (cfDNA) be proved to be by on the length typically the short-movie section less than 200bp form, (people such as old (Chan)), clinical chemistry, 50:88-92[2004]), can be distinguished early that (she draws the people such as Nice (Illanes) when only having for 4 weeks pregnant, early stage human grow (EarlyHuman Dev), 83:563-566[2007]), and knownly within a few hours of childbirth, namely from circulating, parent removed the (people such as sieve (Lo), American Journal of Human Genetics (Am J Hum Genet), 64:218-224[1999]).Except cfDNA, in the parent blood flow, can also distinguish the fragment of (cfRNA) of acellular fetal rna, this is to be derived from the gene of being transcribed in fetus or placenta.Provide new chance for NIPD from the extraction of these fetuses heredity elements of maternal blood sample and analysis subsequently.

The method can be used for determining to exist or do not exist a kind of fetal chromosomal aneuploidy at the maternal sample that comprises fetus and parent nucleic acid molecule (for example cfDNA).Present method is the method that is independent of polymorphism of a kind of NIPD of being applicable to, and does not need fetus cfDNA and parent cfDNA distinguished mutually and can realize determining of a kind of fetus dysploidy.

In some embodiments, sample is a kind of biological fluid sample, for example blood sample or its part.Preferably, biological sample is to be selected from blood plasma, serum and urine.In some embodiments, the maternal source sample is a kind of peripheral blood sample.In other embodiments, the maternal source sample is a plasma sample.The order-checking of fetus and parent nucleic acid can realize by any extensive parallel NGS sequence measurement.In one embodiment, order-checking is to the cfDNA molecule that increases in clone's mode or the extensive parallel order-checking of a plurality of independently cfDNA molecules.In another embodiment, order-checking is the described extensive parallel order-checking of using the extensive parallel synthesis method order-checking that a plurality of reversible dyestuff terminators carry out.In another embodiment, order-checking is the extensive parallel order-checking of using extensive parallel connection method order-checking to carry out.

In some embodiments, the method can be determined or verify existence or not have at least two different chromosomal aneuploidies.In one embodiment, the method is by determining to exist or do not exist at least two kinds of different fetal chromosomal aneuploidies at least two interested karyomit(e) repeating steps (a)-(c), wherein these steps comprise that (a) obtains the sequence information for fetus in maternal sample and parent nucleic acid, so that identification is for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) number with sequence label calculates for interested chromosomal first and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of described interested chromosomal the second normalized value and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is second a karyomit(e) dosage, and it is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).

Alternately, the method is by determining to exist or do not exist at least two kinds of different fetal chromosomal aneuploidies at least two interested karyomit(e) repeating steps (a)-(c), wherein these steps comprise that (a) obtains the sequence information for fetus in sample and parent nucleic acid, so that identification is for number of the sequence label of interested chromosomal a plurality of mappings and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) use for the number of interested chromosomal label and for the number of a chromosomal label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; And (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.The first and second threshold values can be identical, and perhaps they can be different.In the step (c) of this method, exist or do not exist for described interested chromosomal a kind of dysploidy for comparison shows that of described interested chromosomal the first normalized value and threshold value, and have or do not exist determining for interested chromosomal a kind of dysploidy for the comparatively validate of chromosomal the second normalized value of described the first normalization method and threshold value.In some embodiments, the first normalized value is a first chromosome dosage, it is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, light and the second normalized value is second a karyomit(e) dosage, and it is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.Randomly, the first and second normalized values can be expressed as described herein normalized karyomit(e) value (NCV).

In these embodiments, can repeat the method to determine to exist or do not exist a kind of fetal chromosomal aneuploidy for all karyomit(e)s.

Confirmable example a kind of or at least two kinds of different chromosomal aneuploidies comprises T21, T13, T18, T2, T9 and X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, for example blood sample or the blood plasma part that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in the maternal sample is the cfDNA molecule.

The example of fetal chromosomal aneuploidy includes but not limited to complete karyomit(e) trisomy or monosomy or partial trisomy or monosomy.The example of complete fetus trisomy comprises trisomy 21 (T21; Mongolism), 18 trisomy (T18; Edward's syndrome (Edward ' s Syndrome)), trisomy 16 (T16), 22 trisomy (T22; Cat's eye syndrome (Cat Eye Syndrome)), 15 trisomys (T15), 13 trisomy (T13; Handkerchief tower syndrome (Patau Syndrome)), 8 trisomy (T8; Wa Keni syndrome (Warkany Syndrome)), 9 trisomys (T9), 2 trisomys and XXY (Klinefelter syndrome (Kleinefelter Syndrome)), XYY or XXX trisomy.The example of partial trisomy comprises 1q32-44, has the 9p trisomy of trisomy, 4 trisomy mosaics, 17p trisomy, part 4q26-qter trisomy, 9 trisomys, part 2p trisomy, part 1q trisomy and/or part 6p trisomy/6q monosomy.The example of fetus monosomy comprises the chromosome x monosomy; And the partial monosomy of karyomit(e) 13, karyomit(e) 15, karyomit(e) 16, karyomit(e) 18, karyomit(e) 21 and chromosome 22, these monosomy are known relevant with abortion.Can also be by the chromosomal partial monosomy of the definite dysploidy that has typically related to of method of the present invention.Monosomy 18p is rare chromosomal disorders, wherein all or part of galianconism (p) (monosomic) of deletion 18.This disease typically is characterised in that of short and small stature, the mental retardation that degree is variable, and development of speech is slow, the deformity in skull and face (cranium face) zone, and/or extra body abnormality.For different cases, relevant craniofacial defect can alter a great deal in scope and seriousness.The patient's condition that is caused by structure and the variation in the number of karyomit(e) 15 comprises peace lattice Mann syndrome and Pu Ruide-Willie Cotard, and they relate to losing of gene activity in the same part (15q11-q13 zone) at karyomit(e) 15.Should be appreciated that in the father and mother carrier, some transpositions and micro-deleted can be asymptomatic, but still can cause the main genetic diseases among the offspring.For example, carry the micro-deleted healthy mother of 15q11-q13 and can bear the child who suffers from peace lattice Mann syndromes (a kind of serious neurodegenerative disease).Therefore, the present invention can be used for this type of excalation of identification fetus.Partial monosomy 13q is a kind of rare chromosomal disorders, when it occurs in one section of karyomit(e) 13 long-armed (q) disappearance (monomer).The baby who suffers from partial monosomy 13q during birth can show low birthweight, the deformity in head and facial (craniofacial region territory), skeletal abnormality (especially hand and pin), and other body abnormalities.Mental retardation is the feature of this patient's condition.Suffer from the individuality of this disease in birth, the mortality ratio between infancy is very high.The case of nearly all partial monosomy 13q does not all have obvious cause and (sporadic) occurs at random.22q11.2 deletion syndrome is also referred to as DiGeorge syndrome, is the syndrome that the disappearance by a bit of chromosome 22 causes.Disappearance (22q11.2) occurs in this to the karyomit(e) near middle on one of karyomit(e) long-armed.This syndromic feature even also can change very extensively in the member of same family and affects a lot of parts of health.Characteristic sign and symptom can comprise inborn defect, such as congenital heart disease, the defective of jaw, the most commonly with close relevant neuromuscular problem (velopharyngeal insufficiency), learning disorder, the Light Difference in the facial characteristics, and recurrent infection.Micro-deleted among the chromosomal region 22q11.2 is to be associated with the risk of schizoid 20 to 30 times increase.In one embodiment, method of the present invention is used to the determining section monosomy, including, but not limited to: monosomy 18p, the partial monosomy of karyomit(e) 15 (15q11-q13), partial monosomy 13q, and can use method of the present invention to determine the partial monosomy of chromosome 22.

1. in some embodiments, chromosomal aneuploidy is the completely chromosomal aneuploidy that occurs with chimeric state.For instance, in some embodiments, chromosomal aneuploidy is the dysploidy that exists with real chromosomal mosaic form, and wherein fetal cell can comprise two kinds of different caryogram.In other embodiments, chromosomal aneuploidy is relevant with the mosaic that mainly is confined to placenta tissue.Difference between the karyomit(e) of cell forms among cell and the baby in placenta mosaic (CPM) the expression placenta that is limited to.The most commonly the trisomy clone in its expression placenta and the normally diploid karyomit(e) complement among the baby when finding CPM.Yet about 10% case is relevant with fetus.Think that the abnormal cells of the suitable high number that exists in the placenta has disturbed normal placental function.Impaired placenta can't support gestation, and this may cause losing the normal baby of karyomit(e) (Tyson (Tyson) and Ka Laosaike (Kalousek), 1992).For many euchromosome trisomys, only there is chimeric case just to survive to mature.For instance, 2 complete trisomys are obviously impelled the abortion of first three months, occur in 0.16% the gestation of generally acknowledging clinically.As if 2 trisomys only have under chimeric state and be just compatible with life when trisomy mainly is confined in the placenta tissue.Although differentiated the case of the 2 antenatal definite trisomy mosaics of certain number, 2 chimeric trisomys have presented one of more difficult consulting situation.The result is not from normally waiting to neonatal death.Oligohydramnios (low amniotic fluid) and bad intrauterine growth are modal features.Unusual the possibility of result mainly is the high and result that may have low-level trisomy in baby self of trisomy level in placenta.Some uncommon trisomys (for example 9 trisomys) can occur under chimeric or non-chimeric state, and present different clinical manifestations.When sampling, chorionic villus finds that mosaic 9 trisomys have presented a kind of consulting situation of difficulty.After diagnosing out 9 trisomys during CVS, should get rid of trisomy in the fetus with amniocentesis and a series of ultrasonic wave, this trisomy causes the symptom that comprises skull, neural system abnormity and mental retardation.The abnormity that heart, kidney and musculoskeletal system also may occur.Find when CVS but not during amniocentesis that the result is normal in most of cases of trisomy.Yet, also abnormal results may appear.Although differentiated the case of the 9 antenatal definite trisomy mosaics of certain number, the result is not from normally waiting to neonatal death.Some trisomys are rare and fatal, and other trisomys can be survived when being confined to placenta cells.In rear kind of situation, determine that can get rid of this trisomy with other test (for example amniocentesis) after the trisomy is the fetus trisomy.

2. the method also is applicable to determine any chromosome abnormalty when one of parents are this unusual known carrier.These including, but not limited to: chromosomal chimeric for little additional markers thing; T (11; 14) (p15; P13) transposition; Unbalanced transposition t (8; 11) (p23.2; P15.5); 11q23 is micro-deleted; The lucky syndrome 17p11.2 disappearance of Smith-Ma; 22q13.3 disappearance; Xp22.3 is micro-deleted; The 10p14 disappearance; 20p is micro-deleted; DiGeorge syndrome [del (22) (q11.2q11.23)]; WILLIAMS-DARLING Ton syndrome (7q11.23 and 7q36 disappearance); The 1p36 disappearance; 2p is micro-deleted; Neurofibroma Class1 (17q11.2 is micro-deleted), the Yq disappearance; Wolf-Hirschhorn syndrome (WHS, 4p16.3 is micro-deleted); 1p36.2 micro-deleted; The 11q14 disappearance; 19q13.2 micro-deleted; Rubinstein-Taybi syndrome (16p13.3 is micro-deleted); 7p21 is micro-deleted; Miller-Di Ke syndrome (17p13.3), the 17p11.2 disappearance; And 2q37 is micro-deleted.

The method can also make up with the analysis of the antenatal symptom that is used for determining that other and mother and/or fetus are relevant.The method also is applicable to determine the copy number variation (CNV) of any interested sequence in a plurality of samples, these samples comprise the mixture of the genomic nucleic acids that derives from least two different genes groups, and known or suspect that these two different genes groups are different aspect the amount of one or more interested sequences.In some embodiments, the method can be used for determining to have or do not exist a kind of chromosomal aneuploidy (referring to example 1) in twin fetus gestation.In hetero-ovular twins gestation, the method can determine in twin pregnancy to exist or not exist a kind of chromosomal aneuploidy, and compares to determine that by setting up for twins fetus mark and the fetus mark that it is relevant with dysploidy separately one or two twin fetuses carry dysploidy.Can determine accordingly by polymorphic sequence (for example SNP among the Maternal plasma cfDNA) is checked order the first and second fetus marks for the first and second twin fetuses.Each fetus mark can be used as by the main allelotrope part of mother's contribution and calculates with the ratio of the inferior equipotential Gene Partial of being contributed by fetus.Be used for determining that the method for Maternal plasma cfDNA fetus mark is described in the following: unsettled U.S. Patent application 12/958,347 (name is called " method (Methods for Determining Fraction of Fetal Nucleic Acids inMaternal Samples) that is used for determining at maternal sample the mark of fetal nucleic acid "), 12/958,356 (name is called " determining simultaneously dysploidy and fetus mark (Simultaneous determination of Aneuploidy and Fetal Fraction) ") (two all are filed on December 1st, 2010), and 13/009,718 (name is called " being identified in the polymorphic sequence (Identification of polymorphic sequences inmixtures of genomic DNA by whole genome sequencing) in the mixture of genomic dna by genome sequencing ", be filed on January 19th, 2011), these patents all are combined in this with it by reference in full.Because hetero-ovular twins will be different in some SNP site at least, so can determine two independently fetus marks (first and second).The NCV for karyomit(e) 21 of known sample for having twin pregnancy, the fetus mark that then is associated with dysploidy can be used as for the difference percentage between the mean value of the karyomit(e) dosage of aneuploid twins sample and 21 dosage of the karyomit(e) in the qualified samples of training set to be estimated, i.e. NCV karyomit(e) 21 dosage of average karyomit(e) 21 dosage of the NCV of NCV karyomit(e) 21 dosage in qualified samples in specimen/in specimen.One first or the second fetus mark being associated with dysploidy and using mark that the NCV for karyomit(e) 21 calculates to determine corresponding to the difference of using in the SNP sequence, identification is that one or two twin fetuses are carried dysploidy thus.

Except the method is used for fetus determines to show the suitability of chromosomal aneuploidy of hereditary situation, can uses the method and determine to exist or do not exist the chromosome abnormalty, the nucleic acid of determining to exist or do not exist pathogenic agent (for example virus) that show disease (for example cancer) and/or morbid state, determine the chromosome abnormalty relevant with graft versus host disease (GVHD) and the contribution of definite individuality in forensic analysis.

CNV in the human genome obviously affect human diversity and vulnerability (strangle the people such as east (Redon), nature (Nature) 23:444-454[2006]; The human genomes such as Sha Yihe (Shaikh) research (Genome Res) 19:1682-1690[2009]).Known CNV facilitates genetic diseases by different mechanisms, causes gene dosage imbalance or gene disruption in most of cases.Except CNV with genetic block is directly related, known they mediated the phenotype that may be harmful to and changed.Recently, several researchs have been reported, compare with normal control, the load of rare or newborn CNV increases in complicated illness (such as autism, ADHD and schizophrenia), emphasized rare or unique CNV potential pathogenicity bo (people such as Sai Bate (Sebat), 316:445-449[2007]; The people such as Walsh (Walsh), science (Science) 320:539-543[2008]).CNV is produced by genome rearrangement, mainly owing to lacking, copy, insert and unbalanced transposition event.

A plurality of embodiment of the present invention provides a kind of method, be used for evaluation in the copy number variation of the interested sequence (sequence of for example being correlated with clinically) of a specimen, this specimen comprises the mixture derived from the nucleic acid of two different genes groups, and these nucleic acid amount in one or more interested sequences known or under a cloud is different.The mixture of nucleic acid is the cell derived from two or more types.In one embodiment, this nucleic acid mixture is the cell derived from normal and cancer, these cell-derived experimenters from suffering from a kind of medical condition (for example cancer).

It is believed that a lot of solid tumors, such as breast cancer, begin to proceed to transfer by the accumulation of some heredity distortion from opening.[helping the people such as rattan (Sato), cancer research (Cancer Res.), 50:7184-7189[1990]; The people such as Jones's agate (Jongsma), clinical pathology magazine (J Clin Pathol): molecular pathology (Mol Path) 55:305-309[2002])].This type of heredity distortion along with their accumulations can cause breeding advantage, genetic instability and follow drill rapidly the drug-fast ability that bears and the vasculogenesis, proteolysis and the metabolism that strengthen.These hereditary distortion can or affect recessive " tumor suppressor gene " or affect active oncogene.Cause disappearance and the restructuring of loss of heterozygosity，LOH (LOH) to be considered in tumour progression, play Main Function by the tumor suppression allelotrope that exposes sudden change.

In suffering from patient's the circulation of malignant tumour, diagnosis had been found that cfDNA, these malignant tumours are including, but not limited to the lung cancer (people such as Pa Sake (Pathak), clinical chemistry (Clin Chem), 52:1833-1842[2006]), prostate cancer (is permitted the people such as watt minister Bach (Schwartzenbach), Clinical Cancer Research (ClinCancer Res), 15:1032-8[2009]), and breast cancer (is permitted the people such as watt minister Bach (Schwartzenbach), can get online [2009] at breast-cancer-research.com/content/11/5/R71).The identification of the confirmable genomic instability relevant with cancer is potential diagnosis and forecasting tool in cancer patients's circulation cfDNA.In one embodiment, method of the present invention has been evaluated the CNV of the interested sequence in sample, this sample comprises the mixture derived from an experimenter's nucleic acid, known or suspect that this experimenter suffers from cancer, for example cancer, sarcoma, lymphoma, leukemia, gonioma and blastoma.In one embodiment, this sample is to derive (processing) from the plasma sample of peripheral blood, and it comprises the mixture derived from the cfDNA of the cell of normal and cancer.In another embodiment, the biological sample that need to determine whether to exist CNV is the mixture derived from cancer and non-cancer cells, and these cells are learned fluid from other biological, these biological fluids are including, but not limited to serum, sweat, tears, phlegm, urine, phlegm, the ear effluent, lymph liquid, saliva, cerebrospinal fluid, irrigating solution (ravages), marrow suspension, the vagina effluent is through the irrigating solution of uterine neck, brain liquid, ascites, milk is breathed, the secretory product of intestines and genitourinary tract, and white corpuscle exclusion sample, perhaps at biopsy, cotton swab, or in the smear.

Interested sequence is a kind of nucleotide sequence, known or suspect this sequence the development of cancer and/or the progress in work.The example of interested sequence comprises nucleotide sequence, and as described below, these sequences are amplified in cancer cells or delete.

The dominant acting gene that is associated with the human entity knurl is typically brought into play their effect by crossing the expression of expressing or changing.Gene amplification is a kind of common mechanism that causes genetic expression to be raised.Evidence from cytogenetical study shows, in surpassing people's breast cancer of 50% remarkable amplification has occured.It should be noted that most, the amplification that is positioned at the proto-oncogene human epidermal growth factor receptor 2 (HER2) on the karyomit(e) 17 has caused the crossing of HER2 acceptor on cell surface to express, thereby excessive and signal dysregulation (the Piao people such as (Park) in causing breast cancer and other malignant tumours, clinical breast cancer (Clinical Breast Cancer), 8:392-401[2008]).Had been found that in other human malignancies multiple oncogene is amplified.The example of cellular oncogene amplification comprises the amplification of the following in the human tumor: promyelocytic leukemia clone HL60, and the c-myc in the small cell lung cancer, former neuroblastoma (Phase I and IV), neuroblastoma clone, Retinoblastoma Cells system and primary tumo(u)r, and the N-myc in small cell lung cancer cell system and the tumour, L-myc in small cell lung cancer cell system and the tumour, in the acute myelocytic leukemia and colon carcinoma cell line in c-myb, the epidermoid carcinoma cell, and the c-erbb in the former glioma of going crazy, lung, colon, bladder, and the c-K-ras-2 in the primary carcinoma of rectum, N-ras in the breast cancer cell line (Wa Musi H. (Varmus H.), genetics yearbook (Ann Rev Genetics), 18:553-612 (1984), [quote at fertile people such as gloomy (Watson), the molecular biology of gene (MolecularBiology of the Gene) (the 4th edition; Benjamin/healthy and free from worry publishing company (Benjamin/CummingsPublishing Co.) 1987)].

The chromosome deletion that relates to tumor suppressor gene can play a kind of vital role in the development of solid tumor and progress.Retinoblastoma tumor suppressor gene (Rb-1) (being positioned at chromosome 13q14) is the tumor suppressor gene of the most widely characterization.Rb-1 gene product (nuclear phosphoprotein of a kind of 105kDa) obviously plays an important role in cell cycle regulating, and (person of outstanding talent is according to people such as (Howe), institute of NAS periodical (Proc Natl Acad Sci) (U.S.), 87:5883-5887[1990]).By by a point mutation also or the allelic inactivation of these two genes of chromosome deletion cause expression change or that lose of Rb albumen.Have been found that the Rb-i gene alteration does not exist only in the retinoblastoma, but also be present in other malignant tumours, such as osteosarcoma, the small cell lung cancer (people such as Rui Gede (Rygaard), cancer research (Cancer Res), 50:5312-5317[1990)]) and breast cancer.Restriction fragment length polymorphism (RFLP) research shows, this type of tumor type has been lost heterozygosity through the 13q that is everlasting, this prompting is because total chromosome deletion, one of allelotrope of Rb-1 gene is lost (the people such as Bai Kaoke (Bowcock), American Journal of Human Genetics (Am J Hum Genet), 46:12[1990]).Comprise relate to karyomit(e) 6 and other with x linkedly copy, the karyomit(e) 1 of disappearance and unbalanced translocation shows the zone of karyomit(e) 1 unusually, particularly q21-1q32 and 1p11-13, may hold and the upper relevant oncogene of morbidity chronic and advanced stage of myeloproliferative tumour or the tumor suppressor gene (people such as OK a karaoke club horse Sa (Caramazza), Europe hematology magazine (Eur J Hematol), 84:191-200[2010]).The myeloproliferative tumour also is associated with the disappearance of karyomit(e) 5.Karyomit(e) 5 complete lost or intercalary deletion is modal chromosome abnormalities in the myelodysplastic syndrome (MDS).The del (5q) that separates/5q-MDS patient has than the more favourable prognosis of those patients of suffering from extra caryogram defective, and they tend to develop myeloproliferative tumour (MPN) and acute myelocytic leukemia.The frequency of unbalanced karyomit(e) 5 disappearances has been drawn an idea, that is: 5q holds one or more tumor suppressor genes, and these genes play basic effect in the growth control of hemopoietic stem cell/hemopoietic progenitor cell (HSCsHPC).Usually the mapping of the cytogenetics in the zone (CDR) of disappearance concentrates on candidate's tumor suppressor gene of 5q31 and 5q32 identification, comprise that ribosomal subunit RPS14, transcription factor Egr1/Krox20 and cytoskeleton reinvent albumen, α-Lian albumen (Ai Siman (Eisenmann), oncogene (Oncogene), 28:3429-3441[2009]).The cytogenetics of fresh tumour and tumor cell line and allelotype research are verified, from the some clear and definite zone on the chromosome 3p (comprising 3p25,3p21-22,3p21.3,3p12-13 and 3p14) allelic lose be in the main epithelial cancer of the wide spectrum of the cancer of lung cancer, breast cancer, kidney, head and neck cancer, ovarian cancer, cervical cancer, colorectal carcinoma, carcinoma of the pancreas, esophagus cancer, bladder cancer and other organs related the earliest with modal genomic abnormality.Some tumor suppressor genes have been mapped to the chromosome 3p zone, and think that intercalary deletion or promotor high methylation are prior to ((the An Geluoni D. (Angeloni D.) that loses at the developing 3p of cancer or complete karyomit(e) 3, functional genomics bulletin (Briefings FunctionalGenomics), 6:19-39[2007]).

The newborn infant and the children that suffer from mongolism (DS) usually present inborn symptomatic leukemia and have acute myelocytic leukemia and the risk of the leukemic increase of acute lymphocytoblast.Karyomit(e) 21 (holding about 300 genes) can involve the various structures distortion, for example transposition in leukemia, lymphoma and solid tumor, disappearance and amplification.In addition, identified the gene that is arranged on the karyomit(e) 21 vital role that risen has occured in tumour.The isostructural distortion of the company of the number of entities of karyomit(e) 21 is associated with leukemia, and specific gene comprises RUNX1, TMPRSS2 and TFF, they are positioned at 21q, (Feng Nacike C (Fonatsch C) gene, karyomit(e) and cancer C work in tumour occurs, (GeneChromosomes Cancer), 49:497-508[2010]).

In one embodiment, the method provides a means to evaluate cognation between the degree that gene amplification and tumour develop.Association between amplification and/or disappearance and carcinoma stage or the grade can be important for prognosis, because this type of information can consist of the definition of hereditary tumor grade, this can predict the following course of disease of the more late tumor with the worst prognosis better.In addition, can be useful with these events about the information of early stage amplification and/or disappearance event when carrying out association aspect the predictive factors of progression of disease subsequently.Can and lack the gene amplification by present method identification carries out related with other known parameters (such as tumour grade, medical history, Brd/Urd marker index, Hormonal States, nodus lymphoideus transferring rate, tumor size, survival time with from epidemiology and obtainable other tumor characteristics of biostatistics research).For example, remain to comprise the carcinoma in situ of atypical hyperplasia, conduit, cancer and the lymphnode metastatic of Phase I-III by the tumour DNA that present method is tested, in order to allow to be identified in amplification and disappearance and the cognation between the stage.The association of making can be so that effectively therapeutic intervention becomes possibility.For example, the zone of consistent amplification can be contained one and cross the gene of expressing, and its product perhaps can attached (for example, the growth factor receptor tyrosine kinase p185 of receiving treatment property ^HER2).

By determining the copy number variation of those nucleic acid from primary carcinoma disease to the cell of transferring to other positions, the method can be used for identification amplification and/or the disappearance event relevant with resistance.A kind of performance of the karyotype instability that if gene amplification and/or disappearance are the permission resistance to be developed rapidly, compare with the tumour from the patient of chemosensitivity so, will expect from more amplifications and/or disappearance in the patient's of chemotherapy resistance the primary tumo(u)r.For example, if the amplification of specific gene has caused drug-fast development, so in from the patient's of chemotherapy resistance tumour cell rather than in primary tumo(u)r, will expect to have obtained consistent amplification around the zone of those genes.The discovery of the cognation between gene amplification and/or disappearance and development of drug resistance can allow to identify the patient that can or can not benefit from adjuvant therapy.

Be used for determining device and the system of CNV

The analysis of sequencing data and definite different computer hardwares, computerized algorithm and the computer program of typically using that draws are thus carried out.Therefore, the inventive method computer-implemented or computer assisted method typically.

In one embodiment, the invention provides a kind of computer program for generation of an output, this output shows in a specimen and to have or not exist a kind of fetus dysploidy.This computer product comprises a computer-readable medium, this medium has the executable logic of a kind of record computer thereon, be used for making treater can determine to exist or do not exist the fetus dysploidy, this logic comprises: a kind of reception program, be used for receiving the sequencing data from least a portion nucleic acid molecule of parent biological sample, wherein said sequencing data comprises the sequence reading; The logic of computer-aided is used for analyzing the fetus dysploidy from the data of described reception; And a kind of written-out program, for generation of exporting to show existing, not existing or kind of described fetus dysploidy.The computer-readable medium that has stored computer-readable instruction above use has can carry out method of the present invention, in order to carry out a kind of method be used to identifying any CNV (for example chromosomal or dysploidy partly).In one embodiment, the invention provides a kind of computer-readable medium, this medium has storage computer-readable instruction thereon and is used for identification suspection at least one karyomit(e) relevant with a kind of chromosomal aneuploidy (for example trisomy 21,13 trisomys, 18 trisomys or X monosomy).

In one embodiment, the invention provides a kind of computer-readable medium, this medium has storage computer-readable instruction thereon and be used for to carry out the method that may further comprise the steps: (a) use the sequence information that obtains from fetus and parent nucleic acid at sample to identify for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will compare and will compare for interested chromosomal the second normalized value and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.Computer-readable medium can have the storage computer-readable instruction that is used for carrying out a kind of method thereon, a first chromosome dosage for interested chromosomal the first normalized value in the method, this the first chromosome dosage is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and be second a karyomit(e) dosage for interested chromosomal the second normalized value in the method, this second karyomit(e) dosage is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.

In one embodiment, the invention provides a kind of computer-readable medium, this medium has storage computer-readable instruction thereon and be used for to carry out the method that may further comprise the steps: (a) use the sequence information that obtains from fetus and parent nucleic acid at sample to identify for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) use for the number of interested chromosomal sequence label and for the number of a chromosomal sequence label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.Computer-readable medium can have the storage computer-readable instruction that is used for carrying out a kind of method thereon, a first chromosome dosage for interested chromosomal the first normalized value in the method, this the first chromosome dosage is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and be second a karyomit(e) dosage for interested chromosomal the second normalized value in the method, this second karyomit(e) dosage is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.

In one embodiment, the invention provides a kind of computer processing system, it is adjusted or disposes executive basis the inventive method.For instance, the invention provides a kind of computer processing system, it is adapted and disposes to carry out the method that may further comprise the steps: (a) use the sequence information that obtains from fetus sample and parent nucleic acid to identify for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) number with sequence label calculates for interested chromosomal first normalized value and second normalized value; And (c) will be for interested chromosomal the first normalized value and first threshold relatively and will be for interested chromosomal the second normalized value and a Second Threshold comparison, to determine in sample, to exist or do not exist a kind of fetus dysploidy.Computer processing system can be adapted and dispose to carry out a kind of method, a first chromosome dosage for interested chromosomal the first normalized value in the method, this the first chromosome dosage is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and be second a karyomit(e) dosage for interested chromosomal the second normalized value in the method, this second karyomit(e) dosage is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.

In one embodiment, the invention provides a kind of computer processing system, it is adapted and disposes to carry out the method that may further comprise the steps: (a) use the sequence information that obtains from the fetus sample and parent nucleic acid to identify for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method; (b) use for the number of interested chromosomal sequence label and for the number of a chromosomal sequence label of the first normalization method and determine for interested chromosomal first normalized value, and use for the number of the chromosomal sequence label of the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of the first normalization method; (c) will compare and will compare for chromosomal the second normalized value of the first normalization method and a Second Threshold for interested chromosomal the first normalized value and a first threshold, to determine in sample, to exist or do not exist a kind of fetus dysploidy.Computer processing system can be adjusted and be disposed to carry out a kind of method, a first chromosome dosage for interested chromosomal the first normalized value in the method, this the first chromosome dosage is for the number of interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and be second a karyomit(e) dosage for interested chromosomal the second normalized value in the method, this second karyomit(e) dosage is for the number of the chromosomal sequence label of the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.

The present invention also provides the device that is adapted or disposes executive basis method of the present invention, and wherein this device randomly comprises being adapted or disposing and comes order-checking device that the fetus in sample and parent nucleic acid molecule are checked order.For instance, the invention provides the device that comprises the following: (a) order-checking device, it is adapted or is disposed for to use as described herein that sequence measurement checks order to the fetus in sample and parent nucleic acid molecule, thus formation sequence information; And (b) computer processing system, it is adapted or is disposed for to use in method as described herein the sequence information by the order-checking device generation, wherein this computer processing system randomly is directly connected to the order-checking device, like this so that sequence information can automatically be transferred to computer processing system from the order-checking device.This device may further include transferring device, and it is adapted or disposes transfers to the order-checking device for order-checking with sample.

To illustrate in greater detail the present invention in following instance, these examples are not intended to as requested scope of the present invention of by any way restriction.Accompanying drawing is the integral part that is intended to be considered to specification sheets of the present invention and explanation.Provide following instance to describe, and also unrestricted desired invention.

Example

Example 1

Use is determined the best that the extensive parallel dna sequencing from the acellular foetal DNA of maternal blood carries out fetal chromosomal abnormalities: the test set 1 that is independent of training set 1

This research is to be undertaken by human experimenter's scientific experimentation plan that qualified fixed point clinical study personnel get permission according to the Ethic review council (IRB) by each mechanism between in April, 2009 and in October, 2010 in 13 clinical areas of the U.S..Participating in having obtained the written consent book from every experimenter before the research.This scientific experimentation plan is designed to provide blood sample and clinical data to support the development of non-invasive PGD method.18 years old or the larger qualified participation of gravid woman of age.Carrying out collecting blood before this program for the chorionic villi sampling (CVS) of experience clinical indication or the patient that amnion pierces through, and collecting equally the result of fetus caryogram.Extract peripheral blood sample (two pipe or altogether about 20mL) from all experimenters and place acid citrate glucose (ACD) pipe (Becton Dickinson Co., Ltd (Becton Dickinson)).All samples all removed identity and specify patient ID number an of anonymity.Blood sample is transported to the laboratory all through the night in the temperature control type conveying containers that provides for institute.Blood drawing and the time that is subject to spending between the sample are registered as the part that sample is ascended the throne.

The case study coordination personnel use anonymous patient ID number will with patient current pregnant situation and history-sensitive clinical data typing research case report form (CRF) in.Sample from non-invasive antenatal program is carried out the cytogenetics analysis of fetus caryogram in each laboratory and the result is recorded in equally among the research CRF.In all data that CRF obtains all in the clinical database in typing laboratory.After 24 to 48 hours venipuncture sampling, utilize two step centrifuging to obtain acellular blood plasma from independent blood tube.Blood plasma from single blood tube enough carries out sequencing analysis.By using the miniature test kit of QIAamp DNA blood (Kai Jie company) (QIAamp DNA Blood Mini kit (Qiagen)) according to the explanation of manufacturers Cell-free DNA to be extracted from cell-free plasma.Because known these acellular dna fragmentations are about 170 base pairs (bp) (Fan et al., Clin Chem 56:1279-1286[2010]) in length, before order-checking, need not to make DNA cracked.

Sample for this training group, cfDNA is delivered to Prognosys Biosciences, Inc. (LaJolla, CA) is used for sequencing library preparation (blunt end and be connected to cfDNA on the common aptamer) and the scientific experimentation of Application standard manufacturers is planned to check order with Illumina Genome Analyzer IIx instrument (http://www.illumina.com/).Obtained the single-ended reading of 36 base pairs.After finishing order-checking, collect all bases and judge files and analyze.For the test group sample, preparation sequencing library and check order at Illumina Genome Analyzer IIx instrument.Being prepared as follows of sequencing library carried out.Illustrated total length scientific experimentation plan mainly is the standard science test plan that Illumina provides, and only different from Illumina scientific experimentation plan on the purifying in the library of increasing.Illumina scientific experimentation plan indication: the library of amplification uses gel electrophoresis to carry out purifying, and uses magnetic bead to carry out identical purification step in the scientific experimentation plan of this explanation.CfDNA with the about 2ng purifying that extracts from Maternal plasma prepares an elementary sequencing library, and this mainly uses

NEBNext ^TMDNA sample preparation DNA reagent collection 1 (NEBNext ^TMDNA Sample Prep DNA Reagent Set 1) (Item Number: E6000L; New England Biolabs, Ipswich, MA) carry out according to the explanation of manufacturers.Except replacing purification column that the product that aptamer connects is carried out the final purifying with Agencourt magnetic bead and reagent, all be to follow NEBNext for the sample preparation of genome dna library according to the scientific experimentation plan in steps ^TMReagent (uses

The GAII order-checking) carries out.NEBNext ^TMStipulations have been followed the stipulations that Illumina provides in essence, and this can obtain at grcf.jhml.edu/hts/protocols/11257047_ChIP_Sample_Prep.pd f place.

The overhang of the cfDNA fragment of about 2ng purifying that will comprise in 40 μ l is by being used in NEBNext with cfDNA in the 1.5ml Eppendorf tube ^TMDNA sample preparation DNA reagent collection 1 (NEBNext ^TMThe diluent of 1: 5 the dna polymerase i of the damping fluid of the phosphorylation of the 5 μ l 10X that provide DNA Sample Prep DNA Reagent Set 1), 2 μ l deoxynucleotide solution mixtures (every part of dNTP has 10mM), 1 μ l, 1 μ l T4 archaeal dna polymerase and 1 μ l T4 polynucleotide kinase were hatched under 20 ℃ 15 minutes, according to Terminal repair module and change into the blunt end of phosphorylation.This sample is cooled to 4 ℃, and use one at QIAQuick PCR purification kit (QIAQuick PCR Purification Kit) (markon's welfare Ya Zhou Valencia (QIAGENInc. of Kai Jie company, Valenicia, CA)) in the quick post of QIA that provides carry out purifying.50 μ l reaction solutions are transferred in the 1.5ml centrifuge tube, and added the Qiagen Buffer PB of 250 μ l.The 300 μ l that obtain are transferred in the quick post of QIA, with its in an Eppendorf centrifuge under 13,000RPM centrifugal 1 minute.With the Qiagen Buffer PE washing of this post with 750 μ l, and centrifugal again.Remaining ethanol by removing under 13,000RPM in centrifugal 5 minutes again.DNA is come wash-out by centrifugal in the triumphant outstanding buffer reagent EB of 39 μ l (QiagenBuffer EB).That uses 16 μ l contains Klenow fragment (3 ' to 5 ' exo minus) (NEBNext ^TMDNA sample preparation DNA reagent collection 1 (NEBNext ^TMDNA SamplePrep DNA Reagent Set 1)) dA tailing master mixed solution is finished the dA tailing of the DNA of 34 μ l blunt ends, and according to manufacturers

DA-Tailing Module was hatched under 37 ℃ 15 minutes.This sample is cooled to 4 ℃, and uses a post that in MinElute PCR Purification Kit (QIAGEN Inc., Valencia, CA), provides to carry out purifying.50 μ l reaction solutions are transferred in the 1.5ml centrifuge tube, and added the Qiagen Buffer PB of 250 μ l.300 μ l are transferred in the MinElute post, with its in an Eppendorf centrifuge under 13,000RPM centrifugal 1 minute.With the Qiagen Buffer PE washing of this post with 750 μ l, and centrifugal again.Remaining ethanol by removing under 13,000RPM in centrifugal 5 minutes again.DNA is come the wash-out basis by centrifugal in the Qiagen Buffer of 15 μ l EB Quick Ligation Module, (Item Number: 1000521) the 2X Quick Ligation Reaction Buffer of diluent, 15 μ l and the quick T4 dna ligase of 4 μ l were hatched under 25 ℃ 15 minutes with 1: 5 the Illumina Genomic Adapter Oligo Mix of 1 μ l with the DNA elutriant of ten microlitres.Sample is cooled to 4 ℃, and uses a following MinElute post.150 microlitre Qiagen Buffer PE are added in the 30 μ l reaction solutions, and whole volumes are transferred in the MinElute post, with its in an Eppendorf centrifuge under 13,000RPM centrifugal 1 minute.With the Qiagen Buffer PE washing of this post with 750 μ l, and centrifugal again.By under 13,000RPM, removing again remaining ethanol in centrifugal 5 minutes.DNA is come wash-out by centrifugal in the Qiagen Buffer of 28 μ l EB.Use Illumina Genomic PCR primer (Item Number: 100537 and 1000538) and at NEBNext ^TMDNA sample preparation DNA reagent collection 1 (NEBNext ^TMDNA SamplePrep DNA Reagent Set 1) the Phusion HF PCR premixed liquid (according to the explanation of manufacturers) that provides in, the DNA elutriant that the aptamer of 23 microlitres is connected stands 18 PCR circulations, and (98 ℃ are lower 30 seconds; 98 ℃ of lower 18 circulation continuous 10 seconds, 65 ℃ lower 30 seconds, and 72 ℃ lower 30 seconds; Finally extend in 72 ℃ lower 5 minutes, and remain under 4 ℃).Use Agencourt AMPure XPPCR purification system (Agencourt Bioscience Corporation, Beverly, MA) according to the explanation (can obtain at www.beckmangenomics.com/products/AMPureXPProtocol_000387 v001.pdf place) of manufacturers the product that increases is carried out purifying.Agencourt AMPure XP PCR purification system has been removed unassembled dNTP, primer, primer dimer, salt and other pollutents, and has reclaimed the amplicon greater than 100bp.With the amplification product behind the purifying at the Qiagen EB of 40 μ l damping fluid wash-out from the Agencourt pearl, and use 2100 Bioanalyzer (Agilent technologies Inc., SantaClara, CA) Agilent DNA 1000 Kit to analysing the distribution of sizes in these libraries.

For training and specimen group the two, the single-ended reading of 36 base pairs is checked order.

Data analysis and sample classification

Be sequence reading and the human genome assembly hg18 that obtains from the UCSC database of 36 bases compare (http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/) with length.Use allows the short gene fragment comparative device (version 0.12.5) of the Bowtie of maximum two base mispairings (Langmead et al., Genome Biol 10:R25[2009]) to compare in comparison process.Only have and know that being mapped to a locational reading of term single gene group just is included.The genomic locus that reading is shone upon has carried out counting and has been included in the calculating of karyomit(e) dosage (referring to following content).Be excluded beyond analysis (exactly, from base 0 to base 2x10 without the zone that any differentiation ground shines upon part at the sequence label from the masculinity and femininity fetus on the Y chromosome ⁶, base 10x10 ⁶To base 13x10 ⁶And base 23x10 ⁶End to Y chromosome.)

The order-checking variation can make the fetus dysploidy not obvious on the impact of the distribution in the sequence site of mapping between during the karyomit(e) of sequence reading distributes same batch and round.In order to proofread and correct this kind variation, in the time will carrying out normalization method for the counting of observing at predetermined normalization method karyomit(e) or one group of normalization method karyomit(e) for the counting in the site of given interested chromosomal mapping, calculate karyomit(e) dosage.At first identify normalization method karyomit(e) or normalization method karyomit(e) collection in the sample subset in the sample training set of unaffected sample (being qualified samples), these samples have for interested karyomit(e) 21,18,13 and the diploid caryogram of X, with each euchromosome be considered as with our interested chromosome counting ratio in potential denominator.Selection makes the minimum denominator karyomit(e) (being normalization method karyomit(e)) of karyomit(e) ratio variation in the order-checking round and between the order-checking round.Each interested karyomit(e) is confirmed as having different denominators (table 1).

In qualified samples, provide for the overall number of the sequence label of each interested chromosomal mapping measuring with respect to the variation of the overall number of the sequence label of all the other karyomit(e)s mapping separately for each interested chromosomal karyomit(e) dosage.Therefore, qualified karyomit(e) dosage can be identified karyomit(e) or karyomit(e) group, be normalization method karyomit(e), it has close to the variation in the sample of interested chromosomal variation, and will as for the ideal sequence of normalized value in order to carry out further statistical evaluation.

In training group (being qualified and affected) for the karyomit(e) dosage of all samples also as be used for the basis of definite threshold during the dysploidy of following illustrated conduct in the specimen of identification.

Table 1

Be used for determining the normalization method chromosome sequence of karyomit(e) dosage

For each interested karyomit(e) in each sample of test group, determined a normalized value and be used to determine to have or do not exist dysploidy.This normalized value calculates as a karyomit(e) dosage, and this karyomit(e) dosage can further be calculated so that a normalized karyomit(e) value (NCV) to be provided.

Karyomit(e) dosage

For test group, calculated a karyomit(e) dosage for each interested karyomit(e) 21,18,13, X and the Y of each sample.As providing in above table 10, the karyomit(e) dosage of karyomit(e) 21 is that the ratio as the number of tags in the number of tags in the specimen that is mapped to the karyomit(e) 21 in the specimen and the specimen that is mapped to the karyomit(e) 9 in the specimen calculates; The karyomit(e) dosage of karyomit(e) 18 is that the ratio as the number of tags in the number of tags and the specimen that is mapped to the karyomit(e) 8 in the specimen that are mapped in the specimen of the karyomit(e) 18 in the specimen calculates; The karyomit(e) dosage of karyomit(e) 13 is that the ratio as the number of tags in the number of tags and the specimen that is mapped to the karyomit(e) 2 to 6 in the specimen that are mapped in the specimen of the karyomit(e) 13 in the specimen calculates; The karyomit(e) dosage of chromosome x is that the ratio as the number of tags in the number of tags and the specimen that is mapped to the karyomit(e) 6 in the specimen that are mapped in the specimen of the chromosome x in the specimen calculates; The karyomit(e) dosage of karyomit(e) Y is that the ratio as the number of tags in the number of tags and the specimen that is mapped to the karyomit(e) 2 to 6 in the specimen that are mapped in the specimen of the karyomit(e) Y in the specimen calculates.

Normalized karyomit(e) value

Use in each specimen for each interested chromosomal karyomit(e) dosage and the corresponding karyomit(e) dosage in the qualified samples of training group, determined, use the normalized karyomit(e) value of following Equation for Calculating (NCV):

{NCV}_{ij} = \frac{x_{ij} - {\hat{μ}}_{j}}{{\hat{σ}}_{j}}

Wherein

With

Estimation training set average and the standard deviation for j karyomit(e) ratio accordingly, and x _IjFor viewed j the karyomit(e) ratio of sample i.When the karyomit(e) ratio was normal distribution, NCV equaled the statistics z score value for ratio.In drawing from the fractile of the NCV of unaffected sample-fractile, do not observe and significantly the departing from of the linear lag.In addition, fail to veto the null hypothesis of normality for the standard testing of the normalizing degree of NCV.For Ke's Er Monuofu-Vladimir Smirnov (Kolmogrov-Smirnov) and Xia Piluo-Weir gram (Shapiro-Wilk) two checks, the significance value is all greater than 0.05.

For test group, calculated a NCV for each interested karyomit(e) 21,18,13, X and the Y of each sample.In order to ensure a safe and efficient classification schemes, for the dysploidy categorizing selection conservative border.For autosomal aneuploid state is classified, need NCV＞4.0 that karyomit(e) is classified as affected (that is being dysploidy for this karyomit(e)); And NCV＜2.5 classify as karyomit(e) unaffected.The sample that euchromosome has the NCV between 2.5 and 4.0 is classified as " not judging ".

In test, heterosomal classification is by all being undertaken by following content sequential use NCV for X and Y:

1. if NCV Y is apart from the mean value of male sex's sample＞-2.0 standard deviations, then this sample is classified as the male sex (XY).

2. if NCV Y is apart from the mean value of male sex's sample＜-2.0 standard deviations, and NCV X is apart from the mean value of women's sample＞-2.0 standard deviations, and then this sample is classified as women (XX).

3. if NCV Y is apart from the mean value of male sex's sample＜-2.0 standard deviations, and NCV X is apart from the mean value of women's sample＜-3.0 standard deviations, and then this sample is classified as monosomy X, i.e. Turner syndrome.

4. if NCV does not meet any above standard, then to be classified as for sex be " judge " to this sample.

The result

The research demography

Between in April, 2009 and in July, 2010, registered altogether 1,014 patient.Patient's demography, invasive Program Type and results of karyotype are summarised in the table 2.Research participant's mean age be 35.6 years old (scope was at 17 to 47 years old) and pregnant age scope be 61 day week to 38 1 day week (15 4 days weeks of average out to).The overall sickness rate of abnormal fetus karyotype is 6.8%, and wherein the T21 sickness rate is 2.5%.In having 946 experimenters of single pregnancy and caryogram, 906 (96%) presents at least a clinical generally acknowledged risk factors for the fetus dysploidy of antenatal process.Only have the high conceived age as the experimenter of its unique indication even remove those, data have still been showed for very high false positive rate of current examination mode.With ultrasonic result of ultrasonography of carrying out be: the nuchal translucency of increase, cystic hygroma or other structural birth defect, these are the strongest abnormal karyotypes of foresight in this age group.

Table 2

Patient demographics

* the result who comprises the fetus of polycyesis, * * is by clinicist's assessment and report

AMA=pregnant woman advanced age, the NT=nuchal translucency

The distribution of various ethnic background of showing in this study population is also shown in the table 2.Generally, 63% patient is the Caucasian in this research, the 17%th, and the Spaniard, the 6%th, the Aisa people, the 5%th, multi-ethnic, and 4% right and wrong descendants American.Notice that race's difference is changed significantly in different places.For example, the three unities has been registered Spain of 60% and 26% Caucasia experimenter, and three clinical points that are positioned at same state are not registered the Spain experimenter.As expected, in our not agnate result, do not observe recognizable difference.

Training data group 1

This training group research has been selected 71 samples 435 samples that collect, that the initial stage accumulates in succession between year December in April, 2009 to 2009.All experimenters that have affected fetus (abnormal karyotype) in the experimenter of this First Series are included for order-checking, and have random choose of suitable sample and data and the unaffected experimenter of random number.Training group patient's Clinical symptoms is demographic consistent with the overall study shown in the table 11.The scope in pregnant age of the sample in the training group is the scopes from 10 0 day week to 23 1 day week.38 people have experienced CVS, 32 people experienced the type that amniocentesis and 1 patient do not have the invasive program of appointment (unaffected caryogram 46, XY).70% patient is the Caucasian, the 8.5%th, and the Spaniard, the 8.5%th, the Aisa people, and 8.5% be multi-ethnic.For the purpose of training, in this group, six samples that checked order have been removed.4 samples are from the experimenter of twin pregnancy (below discuss in detail), and 1 sample has T18, and this sample is contaminated in preparation process, and 1 sample has fetus caryogram 69, XXX, and remaining 65 samples are this training group.

The 13.7M (improvement owing in time passing on sequencing technologies) of the number in unique sequence site (that is, in genome with the label of unique site identification) from the 2.2M of the commitment of this training group research to later stage changes.Surpass any potential change of these 6 times of scopes in order to monitor in the site of uniqueness karyomit(e) ratio, moved different, unaffected sample when finishing in the beginning of research.For the round of front 15 unaffected samples, the average number in unique site is 3.8M and is respectively 0.314 and 0.528 for the average karyomit(e) ratio of karyomit(e) 21 and karyomit(e) 18.For the round of rear 15 unaffected samples, the average number in unique site is 10.7M and is respectively 0.316 and 0.529 for the average karyomit(e) ratio of karyomit(e) 21 and karyomit(e) 18.Between the karyomit(e) ratio of karyomit(e) 21 and karyomit(e) 18, along with the time lapse of training group research, there is not statistical difference.

Illustrate for karyomit(e) 21,18 and 13 training group NCV at Fig. 2.The result is consistent with a kind of hypothesis of normalization method degree shown in figure 2, and this hypothesis is: about 99% diploid NCV will fall into mean value ± 2.5 standard deviations.In 65 samples in this group, the NCV scope that 8 samples of clinical caryogram with indication T21 have is from 6 to 20.The NCV scope that the sample that four clinical caryogram that have indicate fetus T18 has is from 3.3 to 12, and the NCV that two clinical caryogram that have sample of indicating fetus trisomy 13 (T13) has is 2.6 and 4.The distribution of NCV is owing to they dependencys to the per-cent of the fetus cfDNA in the single sample in affected sample.

Similar with euchromosome, in the training group, determined heterosomal mean value and standard deviation.Heterosomal threshold value allows 100% ground to differentiate the masculinity and femininity fetus that the training group is interior.

Test data set 1

Established the karyomit(e) ratio average and with the standard deviation from average of training group after, from the sample of between year June in January, 2010 to 2010, from 575 samples altogether, collecting, selected a test group of 48 samples.One of them is removed from final analysis from the sample of twin pregnancy, so remaining 47 samples in test group.Making for the preparation of the sample of order-checking and the personnel of operating equipment is blind to clinical caryogram information.Pregnant age scope with similar (table 2) in the training group, seen.58% of invasive program is CVS, and is procedural demographic higher than overall, but also with the training category seemingly.50% experimenter is the Caucasian, the 27%th, and Spaniard, the 10.4%th, Aisa people and 6.3% right and wrong descendants American.

In test group, the number of unique sequence label is from about 13M to 26M and difference.For unaffected sample, for karyomit(e) 21 and karyomit(e) 18, the karyomit(e) ratio is respectively 0.313 and 0.527.For karyomit(e) 21, karyomit(e) 18 and karyomit(e) 13, test group NCV is shown in Figure 3 and be sorted in the table 12 and provide.

Table 3

Test group classification data test group categories data

* MX is the monosomy of X chromosome, and Y chromosome does not have sign

In test group, 13/13 experimenter with the caryogram that is designated as fetus T21 is correctly identified as having the NCV of scope from 5 to 14.Eight/eight experimenters with the caryogram that is designated as fetus T18 are correctly identified as having the NCV of scope from 8.5 to 22.In this test group, have the simple sample that classifies as T13 and be classified as wherein that NCV is approximately 3 not judgement.

For test data set, all male sex's samples are correctly identified, and comprise having complex karyotype 46, the sample (table 3) of XY+ marker chromosomes (can not identify by cytogenetics).Have 19 to be correctly validated in 20 women's samples, and women's sample is not classified as judgement.Be three samples of 45, X for caryogram in the test group, have in three two to be correctly validated and to be monosomy X, and 1 be classified as (table 3) do not judged.

Twins

There is one to be from twin pregnancy for having in the initial sample of selecting of training group in four and the test group.Threshold value may be subject to the puzzlement of the different values of the cfDNA that expects in the environment of twin pregnancy as used herein.In the training group, be single chorion 47 from the caryogram of one of them twins sample, XY+21.Second twins sample be different ovum and each fetus carried out separately amniocentesis be.In this twin pregnancy, one of them fetus has the caryogram of 47, XY+21 and another has a normal caryogram 46, XX.In these two cases, based on the acellular classification of method discussed above sample is classified as T21.Two twin pregnancies of in the training group other are correctly classified as for T21 unaffected (all twins all show the diploid caryogram for karyomit(e) 21).For the twin pregnancy in the test group, only to twins B established caryogram (46, XX), and this algorithm correctly to be classified as for T21 be unaffected.

Conclusion

These data show that extensive parallel sequencing can be used to determine a plurality of unusual fetus caryogram from pregnant woman's blood.These data show, can use independently the test group data to identify to 100% correct classification of sample with trisomy 21 and trisomy 18.Even in the situation of the fetus with unusual sex chromosome caryogram, utilize the algorithm of the method not have sample to be sorted out mistakenly.Importantly, this algorithm is determining in the group of two twin pregnancies to exist or not exist aspect the T21 same performance good equally.In addition, this research has checked the many continuous sample from a plurality of centers, not only represented the scope of the abnormal karyotype that people may see in the commercial clinical environment, also show the importance that not accurately sorted out by the sex gestation of common trisome, arrived unacceptable false positive rate with the height of emphasizing in current Prenatal Screening, to exist.These data provide valuable opinion for the great potential of utilizing the method in future.The analytical table of the subset of unique gene locus understands the increase on the consistent Poisson counting statistics value of variance.

These data are set up on the basis of the discovery of Fan and Quake, Fan has confirmed with Quake: use extensive parallel order-checking to determine that without wound ground the sensitivity of fetus dysploidy only is subjected to restriction (Fan and the Quake of counting statistics from Maternal plasma, PLos One 5, e10439[2010]).Because order-checking information spreads all over the collection of whole genome, so this method can be determined any dysploidy or the variation of other copy numbers, comprise and inserting and disappearance.Caryogram from one of them sample has a little disappearance between q21 and q23 in karyomit(e) 11, when sequencing data is analyzed, observe the minimizing of the relative number of regional interior label about 10% of a 25Mb initial at the q21 place in 500k base data box.In addition, in the training group, there are three in the sample owing to the chimerism in the cytogenetics analysis has complicated property caryogram.These caryogram are: i) 47, and XXX[9]/45, X[6]; Ii) 45, X[3]/46, XY[17]; And iii) 47, XXX[13]/45, X[7].The sample ii that shows some cells that contain XY is correctly classified as XY.By cytogenetics analysis (consistent with the mosaic Turner syndrome) all show the sample i (from the CVS process) of the mixture of XXX and X cell and iii (from amniocentesis) be classified as respectively do not judge with monosomy X.

In test during this algorithm, for the karyomit(e) 21 from the sample (Fig. 3) of test group, another interesting data point is observed a NCV who has between-5 and-6.Although this sample is diploid by cytogenetics at karyomit(e) 21, this caryogram has been showed and the triploid chimerism of part: 47, XX+9[9 for karyomit(e) 9]/46, XX[6].Because karyomit(e) 9 is used in the karyomit(e) dosage (table 1) of determining karyomit(e) 21 in the denominator, this has reduced total NCV value.The result has shockingly proved the in the case ability (referring to example 2) of definite fetus trisomy 9 of the method.Determined that a plurality of karyomit(e) ratios are to guarantee for interested chromosomal correct classification.In addition, establish the possibility (referring to example 5) of determining rare dysploidy for all autosomal normalization method karyomit(e)s to improve the leap genome.

At random any or systematic bias that the conclusion of the sensitivity of these methods of relating to persons such as Fan only can be brought sequence measurement at employed algorithm is only correct when taking into account.If this sequencing data is not by suitably normalization method, then the analytical results of gained will be inferior to counting statistics.The people such as Zhao (Chiu) notice in their recent paper, they use karyomit(e) 18 that extensive parallel sequence measurement obtains and 13 measuring result is coarse, and conclusion is need to more study the method is applied to the determining of T18 and T13 (people such as Chiu, BMJ 342:c7401[2011]).The method of using in the people's such as Chiu paper has simply been used the number of interested chromosomal sequence label in their case karyomit(e) 21, this number has carried out normalization method by the overall number of the label in this order-checking round.The challenge part of this approach is: the distribution of label on each karyomit(e) can be from the order-checking round to order-checking round and difference, and has therefore increased the entire change that dysploidy mensuration is measured.For the result of Chiu algorithm is compared with the chromosomal ratio that uses in this example, use the method for people's recommendations such as Chiu to analyze again the test data of karyomit(e) 21 and 18, as shown in Figure 4.Generally, observed compression in the scope of NCV for each of karyomit(e) 21 and 18, and observed reducing of definite rate, wherein utilized the NCV threshold value 4.0 that is used for the dysploidy classification correctly to identify 10/13 T21 and 5/8 T18 sample from our test group.

The people such as Ehrich equally only focus on T21 and have used the algorithm identical with people such as Chiu (Ehrichet al., Am J Obstet Gynecol 204:205 e1-e11[2011]).In addition, after a skew of the test group z score measures of observing them and outside reference data (i.e. training group), they have carried out retraining to establish classification boundaries to test group.Although this method is feasible in principle, in practice with challenging be to need to determine how many samples train and needs how long once carry out retraining guarantee these grouped datas correctly.A kind of method that alleviates this problem is to comprise contrast in each order-checking round, and these are calibrated to the amount of illumination baseline and for quantitative behavior.

The data of using present method to obtain show, when being used for that the chromosome counting data are carried out normalized algorithm when optimised, extensive parallel order-checking can be determined multiple fetal chromosomal abnormalities from pregnant woman's blood plasma.Present method is used for quantitatively not only will check order and reduces to minimum with system change at random between the round, also allows to spread all over whole genome dysploidy is classified, and the most significant is T21 and T18.Need larger sample collection to test for the algorithm of determining T13.For this purpose, carrying out clinical studyes likely, blind, many places with the diagnostic accuracy of further proof present method.

Example 2

Verify determining of dysploidy with the gemini ratio: normalization method karyomit(e) is carried out normalization method

Described in previous example, present method be based on the number that is mapped to interested chromosomal sequence label for be mapped to show with like the interested chromosome races, the normalization method of sample room and the number of the sequence label of the sample of variability between round of checking order.For used normalization method karyomit(e) in the classification of verifying dysploidy and the Exclusion analysis itself is aneuploid karyomit(e) (namely the copy number with deformity exists), following determine the first normalization method karyomit(e) (namely be used for determining karyomit(e) dosage be used for to relate to karyomit(e) 21,18 and the common dysploidy of the X karyomit(e) of classifying) normalization method.

Use described in example 1 from the qualified samples of training set 1 and from the qualified samples of test set 1, analyze order-checking information in order to identify for chromosomal at least one the second normalization method karyomit(e) of the first normalization method, this first normalization method karyomit(e) is used for determining to exist or do not exist T21, T18 or chromosome x dysploidy (accordingly referring to table 4,5 and 6).

A. for the second normalization method karyomit(e) of the first normalization method karyomit(e) 9:

Genotypic definite in order to verify the normal dyeing body 21 of determining such as use the first normalization method karyomit(e) 9 of determining in the example 1, use each other karyomit(e)s calculating for the karyomit(e) dosage of karyomit(e) 9, namely as the label that is mapped to each qualified samples (normal specimens) in training set 1 and the karyomit(e) 9 in each qualified samples in test set and the ratio that is mapped to the label of karyomit(e) 1-8 and 10-22, and calculate CV% (table 4).As discussed previously, be used for the chromosomal CV% of identification normalization method and be the CV value of the karyomit(e) dosage determined at the diploid sample.

Table 4

Determine for the second normalization method of the first normalization method karyomit(e) 9 is chromosomal

Karyomit(e) with minimum variability is confirmed as from the karyomit(e) 11 in the qualified samples of training set and two collection of test set.

Selective staining body 11 with after being used for checking and using determining of 9 pairs of dysploidy for karyomit(e) 21 of the first normalization method karyomit(e) (being T21), calculates karyomit(e) dosage for karyomit(e) 9/ karyomit(e) 11 for each specimen as the second normalization method karyomit(e).Described in example 1, the average karyomit(e) dosage 0.834054 ± 0.005213 (mean value ± S.D) for karyomit(e) 9/ karyomit(e) 11 that uses as determine in the qualified samples of training set is determined the NCV (Fig. 5) for each specimen.

Data show uses karyomit(e) 9 (to be lower than the average 5-6 NCV of all the other specimen for the unusual low NCV that karyomit(e) 21 calculates; Fig. 3) corresponding to when using karyomit(e) 11 as the second normalization method karyomit(e) for the unusual high NCV (the average 5-6 NCV that is higher than all the other specimen) of karyomit(e) 9.Data show that sample has karyomit(e) 9 dysploidy, and have verified determining of diploid karyomit(e) 21 in the sample.This result is consistent with aneuploid caryogram for sample, and this aneuploid caryogram has been shown as trisomy 9 mosaics 47, XX+9[9]/46, XX[6].The caryogram of trisomy 9 is to use amniotic fluid samples to determine.In addition, these data show the method and can identify rare chromosomal aneuploidy (for example trisomy 9).

B. for the second normalization method karyomit(e) of the first normalization method karyomit(e) 8:

For the karyomit(e) dosage (it be the normalization method karyomit(e) that is used to as example 1 described in determine exist or do not exist T18) of each other karyomit(e)s calculating for karyomit(e) 8, i.e. conduct is mapped to the ratio of the label of karyomit(e) 8 and each qualified samples (normal specimens) in training set 1 and the karyomit(e) 1-7 in each qualified samples in test set 1 and 9-22, and calculates CV% (table 5).

Table 5

Determine for the second normalization method of the first normalization method karyomit(e) 8 is chromosomal

Selective staining body 2 with after being used for checking and using determining of 8 pairs of dysploidy for karyomit(e) 18 of the first normalization method karyomit(e) (being T18), calculates karyomit(e) dosage for karyomit(e) 8/ karyomit(e) 2 for each specimen as the second normalization method karyomit(e).The average karyomit(e) dosage 0.60102532 ± 0.00318442 (mean value ± S.D) for karyomit(e) 8/ karyomit(e) 2 that uses as determine in the qualified samples of training set is determined the NCV (Fig. 6) for each specimen.

Fig. 6 shows the dysploidy that does not all exist for the first normalization method karyomit(e) 8 in all specimen, verified thus use karyomit(e) 8 as normalization method karyomit(e) to there being or not existing determining of T18 dysploidy.

C. for the second normalization method karyomit(e) of the first normalization method karyomit(e) 6:

For the karyomit(e) dosage (it be the normalization method karyomit(e) that is used to as example 1 described in determine exist or do not exist the dysploidy of chromosome x) of each other karyomit(e)s calculating for karyomit(e) 6, i.e. conduct is mapped to the ratio of the label of karyomit(e) 6 and each qualified samples (normal specimens) in training set and the karyomit(e) 1-5 in each qualified samples in test set and 7-22, and calculates CV% (table 6).

Table 6

Determine for the second normalization method of the first normalization method karyomit(e) 6 is chromosomal

Karyomit(e) with minimum variability is determined to be in karyomit(e) 5 and the karyomit(e) in the qualified samples of test set 3 in the qualified samples in the training set.

Selective staining body 5 with after being used for checking and using determining of 6 pairs of dysploidy for chromosome x of the first normalization method karyomit(e) (for example monosomy X), calculates karyomit(e) dosage for karyomit(e) 6/ karyomit(e) 5 for each specimen as the second normalization method karyomit(e).The average karyomit(e) dosage 0.954309 ± 0.003149 (mean value ± S.D) for karyomit(e) 6/ karyomit(e) 5 that uses as determine in the qualified samples of training set 1 is determined the NCV for each specimen.

Fig. 7 shows the dysploidy that does not all exist for the second normalization method karyomit(e) 5 in all specimen, verified thus use karyomit(e) 6 as the first normalization method karyomit(e) to there being or not existing determining of chromosome x dysploidy.

These data show that the method can be used for determining rare dysploidy (for example trisomy 9), and the method can be used for verifying the definite result who exists or do not exist for interested chromosomal dysploidy by with the second normalization method karyomit(e) the first normalization method karyomit(e) being carried out normalization method.The chromosomal normalization method of the first normalization method is by alleged occurrence or do not exist for the chromosomal dysploidy of the first normalization method, and determines to exist or do not exist dysploidy to verify the first result in the first or second normalization method karyomit(e).

Example 3

Use for interested chromosomal at least two normalization method karyomit(e)s and determine and verify chromosomal aneuploidy

In order to prove determining and to verify by using for interested chromosomal the first and second normalization method karyomit(e)s of chromosomal aneuploidy, use karyomit(e) 10 and karyomit(e) 14 as the second and the 3rd normalization method karyomit(e) for interested karyomit(e) 21, calculate the karyomit(e) dosage that uses among the example 1A that karyomit(e) 9 calculates as the first normalization method karyomit(e) for karyomit(e) 21.

Fig. 8 A shows the drawing for the NCV of 48 samples in test set 1, and these NCV use the average of the corresponding karyomit(e) dosage in the qualified samples of training set 1 and S.D. to calculate.The average CV% for the karyomit(e) dosage of karyomit(e) 21 in training set 1 is provided in the table 7.

Table 7

Determine for the second normalization method of interested chromosome dyeing body 21 is chromosomal

Show that with arrow the specimen of identifying has the unusual low NCV between-5 and-6 NCV in Fig. 3 among Fig. 8 A, and when using karyomit(e) 9 as the first normalization method karyomit(e), correctly classified as the diploid for karyomit(e) 21.Except using karyomit(e) 9 as the first normalization method karyomit(e), use karyomit(e) 10 and use karyomit(e) 14 as other normalization method karyomit(e), in all specimen of test set 1, determine to exist or do not exist trisomy 21.Use mean values 0.259070 ± 0.002823 S.D. for the second normalization method karyomit(e) 10, and use mean values 0.409420 ± 00.4965 S.D. to come the accordingly NCV shown in the scaling system 8B and 8C for the second normalization method karyomit(e) 14.

Data shown in Fig. 8 B and the C show before to be classified as when karyomit(e) 9 is used as the first normalization method karyomit(e) (Fig. 3 and 8A) for the diplontic sample of karyomit(e) 21 and are proved to be diploid for karyomit(e) 21 when being used as normalization method karyomit(e) at karyomit(e) 10 (Fig. 8 B) or karyomit(e) 14 (Fig. 8 C).

Therefore, determine to exist or do not exist the chromosomal aneuploidy can be by verifying for interested chromosomal normalization method karyomit(e) with at least two coloured differently bodies conducts.

Example 4

In for the second normalization method karyomit(e) of the first normalization method karyomit(e) 8, determine chromosomal aneuploidy

In order to prove except the existence of determining such as the rare chromosome abnormalty that is different from trisomy 9 definite in example 1 and 2, obtain sequence information from the second training set and the second test set, and calculate as mentioned above the NCV for all karyomit(e) dosage separately for karyomit(e) 1-22.

Use karyomit(e) 8 as the first normalization method karyomit(e), carry out in from the sample of test set 2, existing or do not exist determining of the dysploidy that relates to karyomit(e) 18.In specimen, there be or do not exist determining of trisomy 18 in order to verify, for the karyomit(e) dosage of each other karyomit(e)s calculating for karyomit(e) 8, i.e. conduct is mapped to the ratio of the label of karyomit(e) 8 and each qualified samples (normal specimens) in training set 2 and the karyomit(e) 1-7 in each qualified samples in test set 2 and 9-22, and calculates CV% (table 8).

Table 8

Karyomit(e) with minimum variability is determined to be in from the karyomit(e) 2 in the qualified samples of training set and two collection of test set, and is used as the second normalization method karyomit(e) and is used for the determining of dysploidy that there is or does not exist karyomit(e) 18 in checking.Use the first normalization method karyomit(e) 8, for the karyomit(e) dosage of each specimen calculating for karyomit(e) 8/ karyomit(e) 2.The average karyomit(e) dosage 0.601163 ± 0.002408 (mean value ± S.D) for karyomit(e) 8/ karyomit(e) 2 that uses as determine in the qualified samples of training set 2 is determined the NCV (Fig. 9 A) for each specimen.Fig. 9 A shows the dysploidy in specimen of using the first normalization method karyomit(e) 8 to analyze for T18.NCV for karyomit(e) 8 dosage when using karyomit(e) 2 as the second normalization method karyomit(e) unusually low (about-10) shows the dysploidy that exists for karyomit(e) 2 in specimen.In order to verify that dysploidy is karyomit(e) 2 and non-chromosome 8, the average karyomit(e) dosage 0.953953 ± 0.006302 (mean value ± S.D) for karyomit(e) 8/ karyomit(e) 7 that uses as determine in the qualified samples of training set 2 is determined the NCV (Fig. 9 B) for each specimen.Fig. 9 B shows when karyomit(e) 7 is used as the second normalization method karyomit(e) and calculates for the dosage of the first normalization method karyomit(e) 8 and NCV, and specimen does not comprise aneuploid karyomit(e) 8.

These data acknowledgements the method can be used for determine rare dysploidy, and the method can be used for definite result that there is or does not exist dysploidy in checking, namely by determining that the first normalization method karyomit(e) (it is used as be used to the molecule that calculates interested karyomit(e) dosage) does not exist with the copy number of deformity self, namely it is not aneuploid karyomit(e).As shown in example 2 and 3, there be or do not exist determining and to be undertaken by using at least two different normalization method karyomit(e)s of dysploidy.Calculating for interested chromosomal karyomit(e) dosage and NCV, and comparative result to be when determining identical result that different normalization method karyomit(e)s can be used as independently molecule.Alternately, in two different normalization method karyomit(e) first can be used for calculating for interested chromosomal dosage and NCV, and the second normalization method karyomit(e) can be used for calculating the chromosomal dosage of the first normalization method and NCV, does not have dysploidy to verify the first normalization method karyomit(e).

Example 5

Determine that the first and second normalization method karyomit(e)s are used for determining chromosomal aneuploidy

In order to identify for karyomit(e) 1-2, X and Y normalization method karyomit(e) separately, be used to use all karyomit(e)s calculating described in previous example for each chromosomal NCV per-cent by the order-checking information that obtains checking order from each all (the being qualified and affected) samples in training set 1, test set 1 and the training set 2.

The data of showing in the table 9 provide for each four the normalization method karyomit(e)s in all 1-22, X and the Y chromosome, and these four normalization method karyomit(e)s are determined to be in 3 sample sets that provide to have for the minimum CV of matched doses.

Normalization method karyomit(e) with four minimum CV% is provided.Be confirmed as being produced by the mean value for the summation of the karyomit(e) dosage of karyomit(e) 2-6 for the second minimum variability of karyomit(e) 13.When the mean value that uses for the summation of the karyomit(e) dosage of karyomit(e) 2-6, be minimum for the karyomit(e) dosage variability of karyomit(e) Y.

Table 9

For all chromosomal normalization method karyomit(e)

Based on these results, no matter the second normalization method karyomit(e) is for one in interested chromosomal two selected normalization method karyomit(e)s, or second normalization method karyomit(e) be for the normalization method karyomit(e) of the first normalization method karyomit(e) (it is for interested chromosomal the first normalization method karyomit(e)), can select normalization method karyomit(e).

Although in this displaying with described the preferred embodiments of the invention, it is evident that for those of ordinary skills this type of embodiment only provides at this by way of example.Those of ordinary skills will expect 1 numerous variants, change this moment and substitute and need not to deviate from the present invention.It should be understood that a plurality of different replacement scheme that when enforcement is of the present invention, can utilize these embodiments of the present invention described here.Define with following claim in method and structure in scope of the present invention and the scope in these claims and their equivalent cover thus being intended that of this.

Claims

1. one kind is used in the definite method that exists or do not have fetal chromosomal aneuploidy of the parent specimen that comprises fetus and parent nucleic acid molecule, and described method comprises:

(a) obtain for the sequence information at fetus described in the described sample and parent nucleic acid, so that identification is for number of interested chromosomal a plurality of sequence labels and for a number of at least two chromosomal a plurality of sequence labels of normalization method;

(b) number with described sequence label calculates for described interested chromosomal first normalized value and second normalized value; And

(c) will compare and will compare for described interested chromosomal described the second normalized value and a Second Threshold for described interested chromosomal described the first normalized value and a first threshold, to determine in described sample, to exist or do not exist a kind of fetus dysploidy.

2. the method for claim 1, a first chromosome dosage for described interested chromosomal described the first normalized value wherein, described the first chromosome dosage is for the number of described interested chromosomal sequence label and the chromosomal ratio of the first normalization method, and be second a karyomit(e) dosage for described interested chromosomal described the second normalized value wherein, described the second karyomit(e) dosage is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the second normalization method.

3. one kind is used in the definite method that exists or do not have fetal chromosomal aneuploidy of the parent specimen that comprises fetus and parent nucleic acid molecule, and described method comprises:

(b) use for the number of described interested chromosomal described sequence label and for the number of a chromosomal sequence label of the first normalization method and determine for described interested chromosomal first normalized value; And use for the number of the chromosomal described sequence label of described the first normalization method and for the number of a chromosomal sequence label of the second normalization method and determine for chromosomal second normalized value of described the first normalization method;

(c) will compare and will compare for chromosomal described the second normalized value of described the first normalization method and a Second Threshold for described interested chromosomal described the first normalized value and a first threshold, to determine in described sample, to exist or do not exist a kind of fetus dysploidy.

4. method as claimed in claim 3, a first chromosome dosage for described interested chromosomal described the first normalized value wherein, described the first chromosome dosage is for the number of described interested chromosomal sequence label and ratio for the number of a chromosomal sequence label of the first normalization method, and be second a karyomit(e) dosage for described interested chromosomal described the second normalized value wherein, described the second karyomit(e) dosage is the number of the chromosomal sequence label of described the first normalization method and ratio for the number of a chromosomal sequence label of the second normalization method.

5. such as any one described method in the claim 1 to 4, further comprise and determine first a normalized karyomit(e) value and second a normalized karyomit(e) value (NCV), a wherein said NCV makes described the first chromosome dosage be associated with the average of corresponding the first chromosome dosage in a combination lattice sample, and wherein said the 2nd NCV makes described the second karyomit(e) dosage be associated with the average of corresponding the second karyomit(e) dosage in a combination lattice sample, as:

{NCV}_{ij} = \frac{χ_{ij} - {\hat{μ}}_{j}}{{\hat{σ}}_{j}}

Wherein With

Estimation average and the standard deviation for j karyomit(e) dosage in a combination lattice sample accordingly, and x _IjFor viewed j the karyomit(e) dosage of specimen i.

6. such as any one described method in the claim 1 to 5, wherein:

Described normalization method karyomit(e) for karyomit(e) 21 is to be selected from karyomit(e) 9,11,14 and 1;

Described normalization method karyomit(e) for karyomit(e) 18 is to be selected from karyomit(e) 8,3,2 and 6;

Described normalization method karyomit(e) for karyomit(e) 13 is group, karyomit(e) 5 and the karyomit(e) 6 that is selected from karyomit(e) 4, karyomit(e) 2-6;

Described normalization method karyomit(e) for chromosome x is to be selected from karyomit(e) 6,5,13 and 3;

Described normalization method karyomit(e) for karyomit(e) 1 is to be selected from karyomit(e) 10,11,9 and 15;

Described normalization method karyomit(e) for karyomit(e) 2 is to be selected from karyomit(e) 8,7,12 and 14;

Described normalization method karyomit(e) for karyomit(e) 3 is to be selected from karyomit(e) 6,5,8 and 18;

Described normalization method karyomit(e) for karyomit(e) 4 is to be selected from karyomit(e) 3,5,6 and 13;

Described normalization method karyomit(e) for karyomit(e) 5 is to be selected from karyomit(e) 6,3,8 and 18;

Described normalization method karyomit(e) for karyomit(e) 6 is to be selected from karyomit(e) 5,3,8 and 18;

Described normalization method karyomit(e) for karyomit(e) 7 is to be selected from karyomit(e) 12,2,14 and 8;

Described normalization method karyomit(e) for karyomit(e) 8 is to be selected from karyomit(e) 2,7,12 and 3;

Described normalization method karyomit(e) for karyomit(e) 9 is to be selected from karyomit(e) 11,10,1 and 14;

Described normalization method karyomit(e) for karyomit(e) 10 is to be selected from karyomit(e) 1,11,9 and 15;

Described normalization method karyomit(e) for karyomit(e) 11 is to be selected from karyomit(e) 1,10,9 and 15;

Described normalization method karyomit(e) for karyomit(e) 12 is to be selected from karyomit(e) 7,14,2 and 8;

Described normalization method karyomit(e) for karyomit(e) 14 is to be selected from karyomit(e) 12,7,2 and 9;

Described normalization method karyomit(e) for karyomit(e) 15 is to be selected from karyomit(e) 1,10,11 and 9;

Described normalization method karyomit(e) for karyomit(e) 16 is to be selected from karyomit(e) 20,17,15 and 1;

Described normalization method karyomit(e) for karyomit(e) 17 is to be selected from karyomit(e) 16,20,19 and 22;

Described normalization method karyomit(e) for karyomit(e) 19 is to be selected from 22,17,16 and 20;

Described normalization method karyomit(e) for karyomit(e) 20 is to be selected from karyomit(e) 16,17,15 and 1; And

Described normalization method karyomit(e) for chromosome 22 is to be selected from karyomit(e) 19,17,16 and 20.

7. such as any one described method in the claim 1 to 6, wherein determined the existence of at least two kinds of different fetal chromosomal aneuploidies or do not existed.

8. method as claimed in claim 7, wherein:

(i) described method comprise at least two interested karyomit(e)s repeat as claim 1 or method claimed in claim 2 determine existence or do not exist as described in different fetal chromosomal aneuploidies; Or

(ii) described method comprise at least two interested karyomit(e)s repeat as claim 3 or method claimed in claim 4 determine existence or do not exist as described in different fetal chromosomal aneuploidies; Or

(iii) described method comprises at least two interested karyomit(e)s and repeats method as claimed in claim 5.

9. such as claim 7 or method claimed in claim 8, wherein the method comprises for all karyomit(e) and repeating as any one described method in the claim 1 to 5 is determined existence or do not had different fetal chromosomal aneuploidies.

10. such as any one described method in the claim 1 to 6, wherein said fetal chromosomal aneuploidy is to be selected from T21, T13, T18 and monosomy X; Or such as any one described method in the claim 7 to 9, wherein said different fetal chromosomal aneuploidy is to be selected from T21, T13, T18 and monosomy X.

11. such as any one described method in the above claim, wherein:

Described maternal sample obtains from a pregnant woman;

Described maternal sample is a kind of biological fluid sample;

Described maternal sample is a plasma sample; And/or

Described nucleic acid molecule is the cfDNA molecule.

12. as any one described method in the above claim, wherein obtain sequence information for these fetuses in this sample and parent nucleic acid and comprise the fetus in this sample and parent nucleic acid molecule are checked order.

13. method as claimed in claim 12, wherein:

Obtain described sequence information and comprise order-checking of future generation (NGS);

Obtain described sequence information and comprise that using a plurality of reversible dyestuff terminators to carry out synthesis method checks order;

Obtain described sequence information and comprise the connection method order-checking; Or

Obtain described sequence information and comprise single-molecule sequencing.

14. such as any one described method in the above claim, wherein said chromosomal aneuploidy is a kind of part or complete chromosomal aneuploidy.

15. such as any one described method in the above claim, wherein said parent specimen is the plasma sample that obtains from a pregnant woman, and described nucleic acid molecule is the cfDNA molecule.