WO2017045654A1 - 确定受体cfDNA样本中供体来源cfDNA比例的方法 - Google Patents
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Definitions
- the present invention relates to the field of biological information and biological detection, and in particular, the present invention relates to a method for determining the proportion of donor-derived cfDNA in a receptor cfDNA sample, and a device for determining the ratio of donor-derived cfDNA in a receptor cfDNA sample, A method of monitoring immune rejection and a device for monitoring immune rejection.
- Organ and tissue transplantation is one of the most important medical achievements of the 20th century. At present, transplantation has become the most effective treatment for the end stage of tissue and organ failure. Monitoring immune rejection in organ transplant patients is an important means to improve long-term survival after organ transplantation. At present, the diagnosis of acute rejection mainly relies on puncture of transplanted organs for tissue biopsy, such as myocardial and endocardial biopsy after heart transplantation. The method is invasive, costly and prone to complications.
- Non-invasive detection indicators related to rejection such as cytokine detection, lymphocyte detection, complement and adhesion molecule detection, protein and enzyme detection, and MHC molecules
- cytokine detection due to the complexity of the immune response
- lymphocyte detection due to the complexity of the immune response
- complement and adhesion molecule detection due to the complexity of the immune response
- MHC molecules due to the complexity of the immune response
- the methods have their limitations, and the results are easily affected by factors such as bacterial and viral infections, and should not be used as a basis for judging rejection.
- organ transplant rejection involves a wide range of situations and complex conditions, there are no recognized indicators that are non-invasive, sensitive and specific enough for clinical monitoring.
- cfDNA cell-free donor-derived DNA, cfd DNA
- donor-derived donor-derived cfDNA has almost no or very low content, and cfDNA content is up-regulated only in the presence of immunological rejection. Therefore, the proportion of donor-derived cfDNA in the peripheral blood of the recipient can be calculated. Thereby assisting in determining whether the body has a rejection reaction.
- pre-transplant samples are often lacking, especially for donor samples.
- the lack of genetic information of donors also makes it difficult to determine cfdDNA content.
- the present invention is directed to at least one of the above problems or to at least one alternative business means.
- the invention provides a method for determining the proportion of donor-derived cfDNA in a receptor cfDNA sample, the method comprising the steps of: obtaining first sequencing data and second sequencing data, said first sequencing data a result of sequencing of at least a portion of the receptor genomic DNA, comprising a plurality of first reads, the second sequencing data being a sequencing result of at least a portion of the receptor cfDNA, comprising a plurality of second reads; Sequencing data and the second sequencing data are respectively compared with a reference sequence, correspondingly obtaining a first comparison result and a second comparison result; performing SNP detection based on the first comparison result to obtain a first classification result,
- the first typing result comprises a plurality of first homozygous genotype SNPs, indicating that the genotype of the first homozygous genotype SNP in the first sequencing data is AA; based on the second alignment result
- the ratio of the second read of the secondary homozygous genotype SNP is determined, and the ratio
- the invention provides a method for determining the proportion of donor-derived cfDNA in a receptor cfDNA sample, comprising the steps of: obtaining first sequencing data, the first sequencing data being a receptor genomic DNA Sequencing results of at least a portion of the plurality of first readings; comparing the first sequencing data with a reference sequence to obtain a first alignment result; performing SNP detection based on the first alignment result, obtaining a a first typing result, the first typing result comprising a plurality of first homozygous genotype SNPs; obtaining second sequencing data, wherein the second sequencing data is a sequencing result of at least a part of the receptor cfDNA, including a plurality of Reading a second read; comparing the second sequencing data with the reference sequence to obtain a second alignment result; and reading a second read of the second homozygous genotype SNP based on the second alignment result
- the amount of the segment, the ratio of the cfDNA of the donor source is determined, and the second homozyg
- any of the above methods of the present invention is free of reliance on donor genetic samples for the first time, and can be implemented in the form of a flexible, integrated software package that can be deployed independently and efficiently.
- Applying the method to transplant rejection monitoring Because the method is low-invasive or non-invasive, with acceptable cost and intuitive digital results, it can be used as a convenient, early, non-invasive and accurate graft rejection monitoring aid, which can be used as an auxiliary or supplementary means for clinical immune rejection detection. .
- All or part of the steps of the method for determining the ratio of donor-derived cfDNA in a receptor cfDNA sample of any of the above aspects of the invention may be performed using a device/system comprising a detachable corresponding unit function module, or a method program And stored on a machine readable medium, implemented by a machine running the readable medium.
- the invention provides a device for determining the proportion of donor-derived cfDNA in a receptor cfDNA sample for performing donor-derived cfDNA samples in any of the above aspects of the invention All or part of the steps of the method of ratio of cfDNA, the apparatus comprising: a data input unit for inputting data; a data output unit for outputting data; a processor for executing an executable program, the executable program including completion The method of any of the above aspects of the invention; a storage unit coupled to said data input device, said data output device and said memory for storing data, including said executable program.
- executable programs may be stored in a storage medium, which may include: read only memory, random access memory, magnetic or optical disks, and the like.
- a device for monitoring organ transplant rejection comprising: collecting blood at a receptor at different time points to obtain a plurality of blood samples; determining each using the method of any of the above aspects of the invention The ratio of donor-derived cfDNA in the blood sample; the monitoring is performed based on the determined ratio of a plurality of the donor-derived cfDNA.
- a device for monitoring organ transplant rejection the device for performing all or part of the steps of the method for monitoring organ transplant rejection according to one aspect of the invention described above, the device comprising: a sample acquisition unit, The blood is collected from the receptor at different time points to obtain a plurality of blood samples; a donor cfDNA ratio determining unit is coupled to the sample acquiring unit for determining the receptor cfDNA sample by using any of the above aspects of the present invention. Method for determining the ratio of cfDNA in each of the blood samples; the monitoring unit is coupled to the donor cfDNA ratio determining unit for determining cfDNA based on the plurality of said donor sources The ratio is carried out for the monitoring.
- SNPs capable of distinguishing between donors and receptors can be determined based solely on genetic samples of the receptor, and these SNPs can be used as markers for distinguishing cfDNA from donor and recipient sources in cfDNA; With the support of the sequencing reads obtained by these labeling sites, the method and/or device of the present invention can accurately determine the content of cfdDNA in the transplanted receptor cfDNA sample; and apply it to the detection of organ transplant rejection. Because it is a low-invasive or non-invasive test with acceptable cost and intuitive digital results, it can be used as a convenient, early, non-invasive and accurate graft rejection monitoring aid, and is a non-dependent donor genetic sample.
- Technology for clinical Provide advice on the extent of transplant rejection, or as an adjunct or supplement to clinical testing for transplant rejection.
- 1 is a general flow chart of an experiment for obtaining sequencing data in one embodiment of the present invention.
- FIG. 2 is an experimental flow diagram for genotyping the ratio of donor cfDNA in a receptor cfDNA sample in one embodiment of the invention.
- FIG. 3 is an experimental flow diagram of plasma cfDNA detection based on a high throughput sequencing platform in one embodiment of the invention.
- FIG. 4 is a schematic diagram showing the flow of the implementation of the donor-independent organ transplantation immune rejection monitoring software package in one embodiment of the present invention.
- Figure 5 is a graphical representation of a linear relationship between corrected donor cfDNA ratio and true donor cfDNA ratio in one embodiment of the invention.
- first, second, first, secondary and the like are used for descriptive purposes only and are not to be construed as indicating or implying the relative importance or implied indication.
- features defining “first,” “second,” “first,” or “secondary” may include one or more of the features, either explicitly or implicitly.
- a plurality means two or more unless otherwise stated.
- connection shall be understood broadly, and may be, for example, a fixed connection, a detachable connection, or an integral one, unless explicitly stated or defined otherwise.
- Connection it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, which can be the internal connection of the two elements.
- intermediate medium which can be the internal connection of the two elements.
- the so-called donor and recipient are the opposite two individuals and are based on transplantation, for example, based on the donor and recipient sides of the organ or tissue transplant.
- the donor and recipient can be the same species or they can be related Nearly different species that can or may be capable of organ or tissue transplantation.
- a method for determining the proportion of donor-derived cfDNA in a receptor cfDNA sample comprising the steps of:
- S10 acquires first sequencing data and second sequencing data.
- the first sequencing data is a sequencing result of at least a portion of the receptor genomic DNA, comprising a plurality of first reads, the second sequencing data being a sequencing result of at least a portion of the receptor cfDNA, including a plurality of second reads .
- the so-called sequencing data is obtained by sequencing the nucleic acid sequence, and the sequencing can be selected according to the selected sequencing platform, but is not limited to the semiconductor sequencing technology platform such as PGM, Ion Proton, BGISEQ-100 platform, and the technology platform for synthesis and sequencing. Such as Illumina's Hiseq, Miseq sequence platform and single-molecule real-time sequencing platforms such as the PacBio sequence platform.
- the sequencing method can be either single-ended sequencing or double-end sequencing, and the obtained offline data is a segment read out, which is called a read.
- the lengths of the reads in the first or second sequencing data are not the same, and the sequencing data utilizes the BGISEQ-100 sequencing platform of the Huada Gene or the Proton sequencing platform in the Ion Torrent series of Life Technologies.
- the genomic nucleic acid sequence was sequenced.
- the nucleic acid sequence is usually obtained by interrupting the genomic DNA sample of the individual, and then performing the corresponding sequencing library according to the selected sequencing method or sequencing platform, and then sequencing the sequencing library to obtain the offline data. That is, sequencing data. It should be noted that for the cfDNA sample, since it is a fragment itself, it is generally not necessary to interrupt it.
- the obtaining of the first and second sequencing data may be performed sequentially, for example, obtaining the first sequencing data and then acquiring the second sequencing data, or simultaneously.
- S10 acquires first sequencing data and second sequencing data, comprising: S12 acquiring a receptor genomic DNA sample and a receptor cfDNA sample, the genomic DNA sample comprising genomic DNA (gDNA), the cfDNA sample Included in cfDNA; S14 captures the genomic DNA and/or cfDNA to obtain a first target fragment and/or a second target fragment; S16 sequences the first target fragment and/or the second target fragment to obtain the First sequencing data and/or the second sequencing data.
- the order of obtaining the receptor genomic DNA sample and the receptor cfDNA sample in S12 is not limited, and may be acquired sequentially or simultaneously.
- a peripheral blood sample of the receptor is obtained, and the sample is isolated.
- the blood cells in the sample are used as receptor genomic DNA samples, and the remaining plasma samples contain cfDNA, which is a receptor cfDNA sample, and both gDNA samples and cfDNA samples are obtained.
- S14 comprises capturing genomic DNA, comprising: performing the following: S141 fragmenting the genomic DNA to obtain a first DNA fragment, preferably, the size of the first DNA fragment is 150-250 bp; S142 performs end repair on the first DNA fragment to obtain a first repair fragment; S143 performs sequencing joint ligation on the first repair fragment to obtain a first ligation product; S144 performs the first ligation product on the first ligation product Selecting a size to obtain a first ligated product of a predetermined size, preferably, the size of the first ligated product of the selected predetermined size is 210-270 bp; and S145 amplifying the first ligated product of the predetermined size to obtain a An amplification product; and S146 performing the capturing of the first amplification product to obtain the first target fragment.
- S14 comprises capturing cfDNA, comprising: performing a terminal repair on the cfDNA by S114 to obtain a second repair fragment; and S134 performing a sequencing joint connection on the second repair fragment to obtain a a second ligation product; S154 amplifying the second ligation product to obtain a second amplification product; and S174 performing the capturing of the second amplification product to obtain the second target fragment.
- the capture can be performed by using a solid phase chip or by using a liquid phase chip. This embodiment does not limit the capture mode. According to an embodiment of the invention, the capture is performed using a liquid phase chip comprising at least 1000 SNP sites with the last allele frequency of the genome closest to 0.5.
- the captured region comprises the following non-redundant sites combining (i)-(iii) at least two: (i) the heterozygosity in the human population allele frequency database is 0.48 a SNP site of -0.5, (ii) a minor allele frequency of 0.5 in the East Asian population of the Thousand Genome Database, or a SNP with an average minor allele frequency of 0.4-0.5 in all populations in the database Site, (iii) a sub allele frequency greater than 0.4 in the CHB subpool of the HapMap database, or a sub allele frequency of 0.5, or a SNP site with a heterozygosity of 0.48-0.5.
- the donor chip's fdDNA content can be detected using a smaller capture chip, a lower amount of data, ie, a lower cost.
- SNPs are all dimorphic, and genotype refers to a combination of types of a pair of allelic sites on a homologous chromosome.
- the so-called allele frequency of the SNP also known as the minimum allele frequency (MAF) refers to the frequency of the lower frequency allele of the SNP in a given population.
- the MAF of the SNP may be based on information disclosed by the database. In this embodiment, the selected SNP of the MAF that meets the requirements is determined by looking up the information provided in the corresponding database.
- heterozygosity is another frequency parameter of the SNP, and the heterozygosity is 2MAF (1-MAF).
- MAF the higher the heterozygosity or MAF, the closer to 0.5, indicating that the higher the frequency of hybridization of the SNP in the population, the greater the likelihood that the SNP will be determined to distinguish between the acceptor and the donor.
- Optimized design and purposeful screening of the captured target area can reduce the total amount of data, reduce the cost of sequencing, and analyze the cost and time. According to another embodiment of the present invention, by the above screening, the most The resulting captured regions included the SNP sites in Table 1.
- the first sequencing data and the second sequencing data are respectively compared with a reference sequence, and the first alignment result and the second alignment result are obtained correspondingly.
- the alignment of the reads in the sequencing data to the reading alignment means that the sequenced DNA fragments (ie, reads) are localized on the genome. By reading the segmentation, it is convenient to use the genomic location as a bridge to overcome the technical difficulties caused by the short reading caused by sequencing, and integrate the data obtained by sequencing with the annotation results produced by the previous research.
- Read alignment is often used as the first step in the analysis of sequencing data. The quality and speed of the reading will directly affect the subsequent analysis.
- reads can allow up to n base mismatches, n is preferably 1 or 2. If there are more than n bases in the reads, a mismatch is considered.
- the pair of reads cannot be compared to the reference sequence.
- various comparison softwares such as SOAP (Short Oligonucleotide Analysis Package), bwa, Tmap, etc., may be used, which is not limited in this embodiment.
- the reference sequence is a known sequence, and may be any reference template in the biological category to which the target individual belongs, such as a published genome assembly sequence of the same biological category, if the mixed nucleic acid sample is from a human, its genome
- the reference sequence (also referred to as the reference genome) can be selected from the HG19 provided by the NCBI database.
- the comparison result includes an alignment of each read segment with a reference sequence, including whether the read segment can compare the upper reference sequence, the read alignment to the position of the reference sequence, the unique position of the reference to the reference sequence, or multiple locations, How many reads in a certain site are aligned, the base type of the corresponding position of the read of a certain point on the alignment, and the like.
- the average sequencing depth of the target region required to be captured is not less than 200 ⁇ , which corresponds to an average of 200 reading alignments per target site. In this way, the final result is more credible.
- the comparison result and/or the second alignment result after de-emphasis replaces the first alignment result and/or the second alignment result, and then performs subsequent steps, which facilitates accurate determination of the content or ratio.
- S30 performs SNP detection based on the first alignment result.
- Performing SNP detection based on the first alignment result obtaining a first typing result, the first typing result comprising a plurality of first homozygous genotype SNPs, indicating that the first homozygous genotype SNP is in the first sequencing data
- the genotype in is AA.
- homozygous genotype is also called homozygous.
- the homozygous SNP in the first typing result is represented by the letter "AA" here, only for the convenience of referring to the SNP of the type, the non-referring base is A, and does not refer to the specific one or more of the type. SNP.
- SNP detection or SNP identification can utilize various SNP recognition software including, but not limited to, SOAPsnp, SomaticSniper, CaVEMan, SAMtools, MuTect, and TVC.
- SNP recognition software including, but not limited to, SOAPsnp, SomaticSniper, CaVEMan, SAMtools, MuTect, and TVC.
- Dividing reads on the same site into different classes is based on base differences at corresponding positions in the read on the alignment, for example, reading a read to a site whose reference base is A In the middle, the base at this position of a part of the read is A, and the base at the position of the other part of the read is G, and the read to the position is divided into two categories.
- SNP recognition typing software cannot classify homozygous sites that are consistent or inconsistent with the base of the reference sequence.
- SNP identification and typing are first performed using TVC software, and for homozygous sites, TVC software cannot classify it.
- the support of various types of reads of the SNP is used for typing.
- the so-called SNP detection based on the first comparison result is obtained, and the first typing result is obtained, including performing the following a or performing the following a and b: a. according to the proportion of more than 95% a first type of reading, determining the genotype of the locus, b.
- the genotype of the locus is determined, and the difference between the plurality of first reads is that the bases at the corresponding positions of the sites on the common alignment are different.
- Rule a that is, the ratio of the first type of the first reading in the first reading of the upper point is greater than 95%, and the ratio is considered to be homozygous, and the ratio of the base is greater than 95%.
- the base of the corresponding position of the upper read segment; in b, that is, the ratio of two or more types of reads in the read of the upper point is between 25% and 95%, which is considered to be The point is heterozygous, and the base composition is the largest of them, that is, the base at the corresponding position of the two types of reads that are closest to 95%.
- the a and/or b typing rules are applicable to the typing of all types of sites, and those skilled in the art can use the existing genotyping software according to the so-called a and/or b. Directly type the locus.
- the second read is deduplicated to remove duplication due to amplification of the library construction process.
- the first order homozygous genotype SNPs with an allele frequency of 100% and/or a sequencing depth greater than or equal to the average sequencing depth are retained.
- the so-called allele frequency is 100% of the site, and the bases at the corresponding positions of all the first reads of the site are identical. In this way, the rigorous filtering of the first-order homozygous genotype SNP is more accurate and reliable.
- S40 determines the proportion of the donor-derived cfDNA.
- the ratio of donor-derived cfDNA was determined based on the amount of the second read of the second homozygous genotype SNP in the second alignment result.
- the second homozygous genotype SNP is a second alignment in the second alignment that aligns at least a portion of the first homozygous genotype SNP that does not support the second read of allele A.
- Quantity It can be an absolute number, a relative number such as a proportional or a functional relationship.
- the allele is represented by the letter A, but for convenience of explanation, the allele indicating that the site is not the base A.
- the so-called second homozygous genotype SNP which is a homozygote in the recipient, heterozygous in the donor or a homozygous locus, is based on the second reading of the locus.
- the amount of the segment determines the proportion of cfDNA from the donor source.
- the second read of the non-unique alignment in the second alignment result is removed to accurately determine the proportion of donor-derived cfDNA in the receptor cfDNA sample.
- the so-called second homozygous genotype SNP is the second alignment of the second alignment in the second alignment result comprising all of the second reads that do not support allele
- a Grade homozygous genotype SNP calculated according to the following formula, Where N represents the number of second reads, N AB (B) represents the total of the two types of second reads that match the upper homozygous AA and the second read that does not support allele A
- the number, N BB (B) indicates the number of such second reads that match the upper homozygous AA and does not support the second read of allele A, and N AA (A) indicates the alignment.
- the first homozygous AA and supports the number of second reads of allele A.
- the so-called second homozygous genotype SNP is the second alignment of the second alignment in the second alignment result comprising a second read that does not support allele A
- the second homozygous genotype SNP has the following characteristics: in the second alignment result, there is only one type of second reading that does not support the allele A, and the second type of the class
- the alleles supported by the reads are denoted as C, and in this example the genotype of each of the secondary homozygous genotype SNPs in the second sequencing data can be expressed as AC.
- the number of second reads of the support allele C obtained for each secondary homozygous genotype SNP site is proportional to the ratio of the number of reads on that site, and each secondary homozygous genotype
- the frequency of the SNP N C /(N A + N C ), where N C represents the number of second reads of the second homozygous genotype SNP and does not support the allele A, N A represents The second homozygous genotype SNP is aligned and the number of second reads of allele A is supported.
- a large number of such frequencies are available in the second sequencing data, which reflect the ratio of donor cfDNA content to a very small fraction of sequencing or alignment errors.
- the allele different from A is represented by the letter C here, and is only for convenience of explanation to distinguish A, and the base type of the allele is not C.
- the inventors In order to eliminate the influence of sequencing errors or alignment errors on the determination of cfdDNA content, the inventors first proposed the assumption that the data reflecting the donor frequency in the receptor and donor sites can reflect the donor content, and the equipotential is used based on the hypothesis.
- the step includes: clustering the quantity to obtain a clustering result; determining the donor source according to at least one of two types of two-dimensional quantities in the clustering result The ratio of cfDNA.
- the clustering can adopt various clustering algorithms, which is not limited in this embodiment.
- the so-called quantities can be clearly clustered into two categories (clusters), and there are twice the relationship between the two types.
- the proportions of the above-mentioned one or two types of second readings are iteratively aggregated into two clusters according to the mean value, and the chi-square test is performed using the mean value to judge the significance of the double relationship, and two types of mean values are output, wherein the other is the mean value.
- the two-fold mean is the ratio of donor-derived cfDNA.
- a method for determining the proportion of donor-derived cfDNA in a receptor cfDNA sample comprising the steps of: S100 obtaining first sequencing data, the first sequencing data being a receptor genomic DNA Sequencing results of at least a portion of the sample, including a plurality of first reads; S200 comparing the first sequencing data with a reference sequence to obtain a first alignment result; S300 performing SNP detection based on the first alignment result Obtaining a first typing result, the first typing result comprising a plurality of first homozygous genotype SNPs; S400 obtaining second sequencing data, wherein the second sequencing data is a sequencing result of at least a part of the receptor cfDNA, Include a plurality of second reads; comparing the second sequencing data with the reference sequence to obtain a second alignment result; S500 comparing the second homozygous genotypes based on the second alignment result The amount of the second read of the SNP determining the ratio of the donor-derived cfDNA, wherein the second
- the method of any of the above embodiments or embodiments does not depend on the donor genetic sample, and can be implemented in the form of a flexible and integrated software package, which can be independently deployed and operated efficiently.
- the method is applied to transplant rejection monitoring, as any of the methods is low-invasive or non-invasive, has acceptable cost and intuitive digital results, and can be used as a convenient, early, non-invasive, Accurate transplant rejection monitoring assistive technology can be used as an auxiliary or complementary means of clinical immune rejection testing.
- All or part of the steps of the method for determining the ratio of donor-derived cfDNA in a receptor cfDNA sample of any of the above aspects of the invention may be performed using a device/system comprising a detachable corresponding unit functional module, or the method may be programmed Stored on a machine readable medium, implemented by a machine running the readable medium.
- a device for determining a ratio of donor-derived cfDNA in a receptor cfDNA sample the device for performing the above-described receptor cfDNA sample of any one or any of the embodiments of the present invention All or part of the method of the donor source cfDNA ratio, the apparatus comprising: a data input unit for inputting data; a data output unit for outputting data; a processor for executing an executable program, the executable
- the program includes a method of performing any of the above aspects of the present invention; a storage unit coupled to the data input device, the data output device, and the memory for storing data, including the executable program.
- the so-called executable program can be stored in a storage medium, and the storage medium can be: read only memory, random storage Storage, disk or CD.
- a method for monitoring organ transplant rejection comprises: collecting blood at a different time point to obtain a plurality of blood samples; using any of the above embodiments or embodiments of the present invention The method determines a ratio of donor-derived cfDNA in each of the blood samples; the monitoring is performed based on the determined ratio of a plurality of the donor-derived cfDNA.
- An apparatus for monitoring organ transplant rejection according to an embodiment of the present invention, the apparatus for performing all or part of the steps of the method for monitoring organ transplant rejection described above, the apparatus comprising: a sample acquisition unit for respectively different At the time point, blood is collected from the receptor to obtain a plurality of blood samples; a donor cfDNA ratio determining unit is coupled to the sample acquiring unit for determining the receptor cfDNA sample by using any of the above embodiments or examples of the present invention. a method for determining the ratio of cfDNA in each of the blood samples; a monitoring unit coupled to the donor cfDNA ratio determining unit for determining a plurality of the cfDNA based on the donor source Proportion, the monitoring is performed.
- SNPs capable of distinguishing between donors and receptors can be determined based solely on genetic samples of the receptor, and these SNPs can be used as markers for distinguishing cfDNA from donor and recipient sources in cfDNA; With the support of the sequencing reads obtained by these labeling sites, the method and/or device of the present invention can accurately determine the content of cfdDNA in the transplanted receptor cfDNA sample; and apply it to the detection of organ transplant rejection. Because it is a low-invasive or non-invasive test with acceptable cost and intuitive digital results, it can be used as a convenient, early, non-invasive and accurate graft rejection monitoring aid, and is a non-dependent donor genetic sample.
- the technology provides advice for clinically judging the extent of transplant rejection, or as an adjunct or supplement to clinical testing for transplant rejection.
- the experimental method for obtaining the first sequencing data generally includes:
- the example method requires a high sequencing depth of SNP sites, and an average of 200 ⁇ or more, an ordinary chip used in this method causes a large amount of data waste, and the detection cost is greatly improved. Therefore, the inventor follows the sub-allele. The closer the frequency (MAF) value is to 0.5, the independent design and self-synthesis of a small SNP chip for target area capture.
- MAF frequency
- ALFRED allele frequency database filtering according to the heterozygosity range from 0.48 to 0.5, obtaining 946 SNP sites;
- the Chinese (CHB) population sub-library with MAF value greater than 0.4 was selected and filtered according to the condition that the MAF value was equal to 0.5 and the heterozygosity was in the range of 0.48 to 0.5, and a total of 1979 sites were obtained.
- the above SNP loci are combined to remove redundancy, exclude sites located on the X, Y sex chromosomes, and require a unique "rs" number in the dbsnp database. Finally, 3846 target SNP sites were obtained, as shown in Table 1. The target SNP site was extended to 100 bp on both sides for probe design, and finally designed into a small SNP chip suitable for capture in this method.
- Target region capture and high-depth sequencing of plasma cfDNA samples from various blood collection points of the recipient after transplantation The percentage of donor cfDNA in the plasma of each blood collection point was analyzed and analyzed as shown in Figure 3.
- the genomic SNP typing experimental procedure is shown in Figure 2.
- 1 ⁇ g of genomic DNA was broken into the main band as a small fragment DNA.
- the DNA fragment was blunt-ended, and a linker was added to construct a completed library.
- the target region was enriched by the above-mentioned self-designed liquid chip, and then passed through PCR.
- the purified product can be used for sequencing analysis after amplification, and the specific steps are as follows:
- the size of the DNA fragment is selected by agarose gel electrophoresis
- the data analysis method is written into a software package, and the content of the software package generally includes the following:
- the BamDuplicates tool was used to remove PCR repeats from the results of the tmap tool alignment (bam format).
- the BamDuplicates tool is from Ion Torrent Systems, Inc.
- other sequencing platform candidate deduplication tools include samtools rmdup and Picard MarkDuplicates (tool website address: http://broadinstitute.github.io/picard/index.html).
- the recipient blood cells and plasma DNA are expanded according to the target SNP site.
- the first 4 steps are applicable to the recipient blood cell and plasma samples for target region capture sequencing.
- BGISEQ-100 sequencing platform Take the BGISEQ-100 sequencing platform as an example, using the TVC tool (default parameter targetseq_germline_lowstringency_p1_parameters.json file) (Reference: http://ioncommunity.lifetechnologies.com/community/products/torrent-variant-caller) to detect receptor blood cells (or The genetic SNP (Germline SNP) of the tissue) obtained partial genotyping sites. For sites that cannot be typed by TVC tools, the frequency is supported by the proportion of supported reads, maximizing genotyping sites, and simultaneously performing noise reduction processing, retaining only high-quality genotyping results.
- Other sequencing platform candidate genotyping tools include GATK (software acquisition address: https://www.broadinstitute.org/gatk/index.php), etc. The specific operation steps of genotyping are as follows:
- the recipient blood cell DNA data was typed using a TVC tool with a sequencing depth threshold of 6.
- the frequency of different alleles in the recipient plasma DNA is counted in the remaining recipient cell homozygous sites to obtain a list of plasma frequencies.
- a maximum of 2 alleles are required in plasma, and at least 2 reads are supported for different alleles.
- allelic bases that differ from the recipient's blood cells can be considered to be from the donor, a small fraction of which is due to sequencing or alignment errors, and the donor may be heterozygous or homozygous at this site, the specific phenotype is unknown Can be judged by frequency. Take a plasma sample with a 10% donor ratio and a 1000X average sequencing depth. If the reference base is A at a certain site, the recipient blood cells are all A base reads, and the receptor plasma has T base reads, T. Base reads may come from the donor.
- the number of T-supported reads should be 5, the frequency of detecting T is 5%, the number of A-supported reads should be 995, and the frequency ratio of detecting A is 95%; if the donor is homozygous , T supports the number of reads should be 10, the frequency ratio of detecting T is 10%, A supports the number of reads should be 990, and the frequency ratio of detecting A is 90%.
- T supports the number of reads should be 10
- the frequency ratio of detecting T is 10%
- a supports the number of reads should be 990, and the frequency ratio of detecting A is 90%.
- FIG. 4 shows the above data analysis process.
- the currently available high-throughput sequencing methods for detecting the cfDNA content of a relatively small proportion of donor cfDNA samples are either too costly or dependent on donor genetic samples.
- the invention proposes a technical route as follows: 1) performing plasma separation on the blood sample of the recipient after transplantation, and the recipient blood cell (or tissue) DNA is genotyped by target region capture and sequencing, and the homozygous locus is retained; 2) transplantation The post-receptor plasma DNA was captured and sequenced in the target region, and the frequency of different alleles in the homozygous locus of the recipient blood cells was counted.
- the ratio of donor cfDNA was calculated by K-means clustering and chi-square test. .
- the invention encodes the technical route into an integrated operation, independently deployable and efficient operation software package, and one of the purposes is to provide an analysis method for organ transplantation immune rejection monitoring without donor dependence based on high-throughput sequencing method. And software packages.
- the example designs a new chip and corresponding experimental methods as well as data analysis and detection methods, at least the following four aspects of beneficial effects:
- the immune rejection test is performed for the first time by counting the frequencies of different alleles in the homozygous locus of the recipient blood cells. Get rid of The dependence on donor samples is particularly useful for organ transplant patients who have difficulty tracking trace donor samples. Therefore, the experimental method is more widely used, and can assist in detecting various organ transplant immune rejection such as lung transplantation, heart transplantation, liver transplantation, kidney transplantation and the like.
- the data analysis method can be realized by a flexible and integrated software package, which can be independently deployed and operated efficiently.
- the design idea of the example is as follows: Take two normal human blood samples (taken from volunteers), one is the donor and the other is the receptor, and the sample to be tested is mixed and simulated.
- the blood samples were taken to separate blood cells and plasma, and the recipient blood cells (without donor blood cells) were extracted from the genomic DNA, and the DNA was broken and the target region was captured and sequenced for genotyping; after the cfDNA was extracted from the donor and recipient plasmas, Agelint 2100
- the concentration of the cfDNA for the receptor was artificially mixed at a ratio of 3.5%, 5.5%, 8%, and 10%, and then the mixed cfDNA library was captured and sequenced (the sequencer used in the present embodiment was a BGISEQ-100 sequencing platform).
- the steps of the present embodiment are also divided into two steps: 1. capture and sequencing of the target region of the receptor genome; 2. capture and sequence of each mixed cfDNA target region. details as follows:
- the linker and PCR amplification primers were synthesized by Invitrogen, and the COT1 DNA used was purchased from Invitrogen.
- the reagent information used is shown in the following table:
- the supernatant (about 1.5 ml) is dispensed into a 2 ml tube, and the lower layer is a blood cell;
- reaction mixture 100 ⁇ L was gently shaken and mixed uniformly, centrifuged instantaneously, and incubated at 20 ° C for 30 min in a Thermomixe or water bath.
- the Adapter connection reaction system was prepared in a 1.5 ml centrifuge tube, and the system is shown in the following table.
- reaction mixture 100 ⁇ L was gently shaken and mixed uniformly, and after transient centrifugation, it was placed in a Thermomixer at 20 ° C for 15 min.
- step 5 the sample obtained in step 3 of step 1 is run into the glue, and then electrophoresed at 100V for 120 minutes;
- step 5.10.3 was added to a nucleic acid adsorption column (MinElute Spin Column), allowed to stand at room temperature for 2 min, and centrifuged at 17900 g for 1 min.
- a nucleic acid adsorption column MinElute Spin Column
- step 5.10.4 The filtrate of step 5.10.4 was re-added to the adsorption column, allowed to stand at room temperature for 2 min, centrifuged at 17900 g for 1 min, and the filtrate was discarded.
- Purification was carried out by adding 1.5 volumes of Agencourt AMPure beads (150 ⁇ L) and eluting with 32 ⁇ L of Elution Buffer.
- Each library was proportionally Pooled to 750 ng.
- the sample was shaken and mixed, and then centrifuged at full speed for 10 seconds on a centrifuge.
- the sample after centrifugation was transferred to a 95 ° C heatblock for 10 minutes to denature the DNA;
- the PCR instrument hot lid should be set to maintain at 105 ° C;
- the reaction was carried out for 30 min at 20 ° C in a Thermomixer.
- the reaction was carried out at 20 ° C for 20 min in a Thermomixer.
- Reaction procedure 72 ° C for 20 min, 95 ° C for 5 min, 15 cycles of 95 ° C 30 s / 60 ° C 30 s / 70 ° C 30 s / 70 ° C 5 min, 12 ° C ⁇ .
- the target sequence hybridization was captured, eluted, and subjected to PCR amplification, and the next step was sequenced.
- the concentration of the outbound library was as shown in Table 4 below.
- the concentration of the outbound library was in accordance with the normal level of the chip hybridization of 0.3K, and the result of 2100 was normal. Test Sequence analysis.
- the donor was extracted with 6.6 ml of plasma, and the receptor was extracted with 11.4 ml of plasma.
- the results obtained are shown in Table 5.
- the normal human plasma cfDNA concentration was low, and the results showed that the extraction was normal.
- the mixed plasma cfDNA, after end-repair, plus different linkers, after a PCR amplification, the purification concentration is shown in Table 6, the results are normal, and can be used for the next step of sequencing analysis.
- the sequence hybridization is captured, eluted, and subjected to PCR amplification.
- the next step of sequencing can be carried out.
- the concentration of the outbound reservoir is shown in Table 7 below.
- the concentration of the outbound reservoir is in accordance with the normal level of chip hybridization of 0.3K, and the result of 2100 is normal. For sequencing analysis.
- a simulation verification test of the known donor ratio was performed.
- a normal receptor (sample name R) blood cell sample is selected for target region capture sequencing, and the target plasma captured by the donor plasma DNA is also subjected to target region capture sequencing, mixing.
- the proportions were 3.5%, 5.5%, 8%, and 10%, respectively.
- the sample names were named in the mixing ratio.
- the valid data for sequencing was compared by tmap alignment, BamDuplicates de-weighting, quality control (QC), receptor blood cell genotyping, and Body plasma frequency statistics, donor ratio calculation, and finally the donor content test report of 4 blood collection points were obtained to evaluate the degree of organ transplant rejection.
- the process methods of this test system have been integrated into the software Donor_cfDNA.
- the software runs on the Unix/Linux operating system and runs through the Unix/Linux command line.
- the Donor_cfDNA_main.pl command line parameters are shown in the parameter descriptions in Table 8.
- the list represents a simulation experiment named RD, and the ratio of donor DNA to be mixed is 3.5%, 5.5%, 8%, respectively.
- the detection donor ratio and the actual donor content are not completely equal (linear relationship), but the values are close, theoretically increase the plasma target region average sequencing depth to 500X, or even 1000X, detection The scale value is more accurate. It is indicated that the method is technically feasible and can be used or assisted for detecting the cfDNA content of the graft donor, and then dynamically monitoring the acute rejection of the recipient after transplantation.
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Abstract
一种确定受体cfDNA样本中供体来源的cfDNA比例的方法,包括:获取第一和第二测序数据,第一和第二测序数据分别为受体gDNA和受体cfDNA的至少一部分的测序结果;将第一和第二测序数据分别与参考序列比对,获得第一和第二比对结果;基于第一比对结果进行SNP检测,获得第一分型结果,第一分型结果包括多个一级纯合子SNP,表示为AA;基于第二比对结果中比对上二级纯合子SNP的第二读段的量,确定供体来源的cfDNA的比例,二级纯合子SNP为比对上该位点的第二读段中包含不支持等位基因A的第二读段的一级纯合子SNP的至少一部分。
Description
优先权信息
本申请要求申请号为201510599395.6、申请日为2015年9月18日递交至国家知识产权局的中国专利申请的优先权,其通过参考的方式以其全文并入此处并且用于犹如本文所明确并且完全陈述的所有目的。
本发明涉及生物信息和生物检测领域,具体的,本发明涉及一种确定受体cfDNA样本中供体来源cfDNA的比例的方法、一种确定受体cfDNA样本中供体来源cfDNA的比例的装置、一种监测免疫排斥的方法以及一种监测免疫排斥的装置。
器官和组织移植是20世纪最重要的医学成就之一,目前移植术已成为组织、器官功能衰竭终末阶段最有效的治疗措施。对器官移植患者进行免疫排斥反应监测,是提高器官移植患者术后长期存活率的重要手段。目前诊断急性排斥反应主要依靠穿刺移植器官进行组织活检,例如心脏移植后进行心肌、心内膜穿刺活检。该方法创伤性大、费用高且易引发并发症。
而当前与排斥反应有关的非创伤性检测指标如细胞因子的检测、淋巴细胞检测、补体和粘附分子检测、蛋白质和酶类检测及MHC分子等,由于免疫反应的复杂性,上述免疫学检测方法均有其局限性,结果容易受细菌、病毒感染等因素影响,不宜单独作为判断排斥的依据。而且由于器官移植排斥反应涉及范围广泛,情况复杂,现仍未找到公认的、无创伤性的、敏感性和特异性均足以应用于临床监测的指标。
在器官移植患者体内,当移植物受到受体排斥时,细胞从器官脱落,进一步凋亡、裂解,释放供体来源的cfDNA(cell-free donor-derived DNA,cfdDNA)进入受体血液。正常状态下受体血液中供体来源的cfDNA几乎没有或者含量是极低的,只有在发生免疫排斥反应时cfDNA的含量上调,因此,可以通过计算受体外周血中供体来源cfDNA的比例,从而辅助判断机体是否发生排斥反应。
最开始这方面的研究集中在性别不匹配器官移植方面,即女性受体接受男性供体的器官,通过检测Y染色体特异基因从而判断血浆中供体来源cfDNA的含量,这就受到了很多
限制,不能广泛应用于临床。
再者,临床上往往缺乏移植前样本,特别是供体样本,缺乏供体的遗传信息也使得cfdDNA含量的确定困难。
现有的确定受体中供体细胞游离DNA(cfdDNA)含量的方法,以及器官移植排斥检测手段,有待改进或补充。
发明内容
本发明旨在至少解决上述问题至少之一或者提供至少一种可选择的商业手段。
依据本发明的第一方面,本发明提供一种确定受体cfDNA样本中供体来源cfDNA的比例的方法,该方法包括步骤:获取第一测序数据和第二测序数据,所述第一测序数据为受体基因组DNA的至少一部分的测序结果,包括多个第一读段,所述第二测序数据为受体cfDNA的至少一部分的测序结果,包括多个第二读段;将所述第一测序数据和所述第二测序数据分别与参考序列进行比对,对应获得第一比对结果和第二比对结果;基于所述第一比对结果进行SNP检测,获得第一分型结果,所述第一分型结果包括多个一级纯合基因型SNP,表示所述一级纯合基因型SNP在所述第一测序数据中的基因型为AA;基于所述第二比对结果中比对上二级纯合基因型SNP的第二读段的量,确定所述供体来源cfDNA的比例,所述二级纯合基因型SNP为满足以下条件的一级纯合基因型SNP的至少一部分:第二比对结果中比对上该二级纯合基因型SNP的第二读段中包含不支持等位基因A的第二读段。
依据本发明的第二方面,本发明提供一种确定受体cfDNA样本中供体来源的cfDNA的比例的方法,包括以下步骤:获取第一测序数据,所述第一测序数据为受体基因组DNA的至少一部分的测序结果,包括多个第一读段;将所述第一测序数据与参考序列进行比对,获得第一比对结果;基于所述第一比对结果进行SNP检测,获得第一分型结果,所述第一分型结果包括多个一级纯合基因型SNP;获取第二测序数据,所述第二测序数据为受体cfDNA的至少一部分的测序结果,包括多个第二读段;将所述第二测序数据与所述参考序列进行比对,获得第二比对结果;基于所述第二比对结果中比对上二级纯合基因型SNP的第二读段的量,确定所述供体来源的cfDNA的比例,所述二级纯合基因型SNP为所述第二比对结果中比对上该位点的第二读段中包含不支持等位基因A的第二读段的一级纯合基因型SNP的至少一部分。
上述本发明的任一方法,首次摆脱了对供体遗传样本的依赖,而且可以利用灵活的、一体化的软件包的形式实现,能够独立部署、高效运行。将该方法应用于移植排斥监测,
由于该方法为低创或无创检测、具有可接受的成本和直观的数字化结果,可作为一种便捷、早期、无创、准确的移植排斥监测辅助技术,可作为临床免疫排斥检测的辅助或补充手段。
上述本发明任一方面的确定受体cfDNA样本中供体来源的cfDNA的比例的方法的全部或部分步骤,可以利用包含可拆分的相应单元功能模块的装置/系统来施行,或者将方法程序化、存储于机器可读介质,利用机器运行该可读介质来实现。
依据本发明的第三方面,本发明提供一种确定受体cfDNA样本中供体来源cfDNA的比例的装置,该装置用以实施上述本发明任一方面的确定受体cfDNA样本中供体来源的cfDNA的比例的方法的全部或部分步骤,该装置包括:数据输入单元,用于输入数据;数据输出单元,用于输出数据;处理器,用于执行可执行程序,所述可执行程序包括完成上述本发明任一方面的方法;存储单元,与所述数据输入装置、所述数据输出装置和所述存储器相连,用于存储数据,其中包括所述可执行程序。本领域技术人员能够理解,所称的可执行程序可以保存在存储介质中,所称存储介质可以包括:只读存储器、随机存储器、磁盘或光盘等。
依据本发明的第四方面,本发明提供一种监测器官移植排斥的装置,包括:分别于不同时间点对受体进行采血,获得多个血液样本;利用上述本发明任一方面的方法确定每个所述血液样本中供体来源cfDNA的比例;基于确定的多个所述供体来源cfDNA的比例,进行所述监测。
依据本发明的第五方面,提供一种监测器官移植排斥的装置,该装置用以实施上述本发明一方面的监测器官移植排斥的方法的全部或部分步骤,该装置包括:样本获取单元,用以分别于不同时间点对受体进行采血,获得多个血液样本;供体cfDNA比例确定单元,与所述样本获取单元相连,用以利用上述本发明任一方面的确定受体cfDNA样本中供体来源的cfDNA的比例的方法确定每个所述血液样本中供体来源cfDNA的比例;监测单元,与所述供体cfDNA比例确定单元相连,用以基于确定的多个所述供体来源cfDNA的比例,进行所述监测。
利用上述本发明的方法和/或装置系统,能够只依据受体的遗传样本确定出能够区分供体和受体的SNP,将这些SNP作为区分混合cfDNA中供体和受体来源cfDNA的标记;而通过这些标记位点得到的测序读段的支持情况,利用本发明的方法和/或装置,可准确确定移植后的受体cfDNA样本中的cfdDNA的含量;而将其应用于器官移植排斥检测,由于其为低创或无创的检测,且具有可接受的成本、直观的数字化结果展示,能够作为一种便捷、早期、无创和准确的移植排斥监测辅助技术,而且为非依赖供体遗传样本的技术,为临床
判断移植排斥程度提供建议,或者作为临床检测移植排斥的辅助或补充手段。
本发明的上述和/或附加的方面和优点从结合下面附图对实施方式的描述中将变得明显和容易理解,其中:
图1是本发明的一个实施例中的获得测序数据的实验总流程图。
图2是本发明的一个实施例中的确定受体cfDNA样本中的供体cfDNA比例的基因分型的实验流程图。
图3是本发明的一个实施例中的基于高通量测序平台的血浆cfDNA检测的实验流程图。
图4是本发明的一个实施例中的无供体依赖性的器官移植免疫排斥监测软件包实现的流程的示意图。
图5是本发明的一个实施例中的校正供体cfDNA比例与真实供体cfDNA比例的线性关系图。
下面结合附图和具体实施方式对本发明进行详细说明。
所述实施方式在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
在本文中,所使用的术语“第一”、“第二”、“一级”、“二级”等仅用于描述目的,而不能理解为指示或暗示相对重要性、隐含指明所指示的技术特征的数量或者具有顺序关系。由此,限定有“第一”、“第二”、“一级”或“二级”的特征可以明示或者隐含地包括一个或者多个该特征。在本发明的描述中,除非另有说明,“多个”的含义是两个或两个以上。
在本文中,除非另有明确的规定和限定,术语“顺序连接”、“相连”、“连接”等术语应做广义理解,例如,可以是固定连接,也可以是可拆卸连接,或一体地连接;可以是机械连接,也可以是电连接;可以是直接相连,也可以通过中间媒介间接相连,可以是两个元件内部的连通。对于本领域的普通技术人员而言,可以根据具体情况理解上述术语在本发明中的具体含义。
在本文中,所称的供体和受体,是相对的两个个体,是基于移植,例如基于器官或组织移植时的供给一方和接受一方来说的。供体和受体可以是相同物种,也可以是亲缘关系
近的能够或者可能能够进行器官或组织移植的不同物种。
下文的公开了不同的具体实施方式或实施例用来实现本发明的不同方法步骤或装置结构。为了简化本发明的公开,下文中对特定例子的步骤和设置进行描述。当然,它们仅仅为示例,并且目的不在于限制本发明。此外,本发明可以在不同例子中重复参考数字和/或参考字母,这种重复是为了简化和清楚的目的,其本身不指示所讨论各种实施方式和/或设置之间的关系。
根据本发明的一个实施方式提供的一种确定受体cfDNA样本中供体来源cfDNA的比例的方法,包括以下步骤:
S10获取第一测序数据和第二测序数据。
所述第一测序数据为受体基因组DNA的至少一部分的测序结果,包括多个第一读段,所述第二测序数据为受体cfDNA的至少一部分的测序结果,包括多个第二读段。
所称的测序数据通过对核酸序列进行测序得来,测序依据所选的测序平台的不同,可选择但不限于半导体测序技术平台比如PGM、Ion Proton、BGISEQ-100平台,合成边测序的技术平台比如Illumina公司的Hiseq、Miseq序列平台以及单分子实时测序平台比如PacBio序列平台。测序方式可以选择单端测序,也可以选择双末端测序,获得的下机数据是测读出来的片段,称为读段(reads)。根据本发明的一个实施例,所称第一或者第二测序数据中的读段的长度不相同,测序数据利用华大基因的BGISEQ-100测序平台或者LifeTechnologies公司的Ion Torrent系列中的Proton测序平台对基因组核酸序列进行测序获得。所测核酸序列通常是将个体的基因组DNA样本经过打断获得的,接着根据所选用的测序方法或测序平台进行相应的测序文库(library)制备,进而将测序文库上机测序,获得下机数据即测序数据。需要说明的是,对于cfDNA样本,由于其本身就是片段,一般不需再对其进行打断。
第一和第二测序数据的获取,可以先后进行,例如先获取第一测序数据再获取第二测序数据,也可以同时进行。根据本发明的实施例,S10获取第一测序数据和第二测序数据,包括:S12获取受体基因组DNA样本和受体cfDNA样本,所述基因组DNA样本包含基因组DNA(gDNA),所述cfDNA样本包含cfDNA;S14对所述基因组DNA和/或cfDNA进行捕获,获得第一目的片段和/或第二目的片段;S16对所述第一目的片段和/或第二目的片段进行测序,获得所述第一测序数据和/或所述第二测序数据。
该实施例对S12中的受体基因组DNA样本和受体cfDNA样本的获取顺序不作限制,可先后获取或者同时获取,根据本发明的一个实施例,获取受体的外周血样本,分离出其
中的血细胞作为受体基因组DNA样本,剩下的血浆样本包含cfDNA,为受体cfDNA样本,同时获得gDNA样本和cfDNA样本。
根据本发明的一个实施例,S14包括对基因组DNA进行捕获,包括进行以下:S141对所述基因组DNA进行片段化,获得第一DNA片段,较佳的,使所述第一DNA片段的大小为150-250bp;S142对所述第一DNA片段进行末端修复,获得第一修复片段;S143对所述第一修复片段进行测序接头连接,获得第一连接产物;S144对所述第一连接产物进行大小选择,获得预定大小的第一连接产物,较佳的,使选择的预定大小的第一连接产物的大小为210-270bp;S145对所述预定大小的第一连接产物进行扩增,获得第一扩增产物;以及S146对所述第一扩增产物进行所述捕获,以获得所述第一目的片段。
根据本发明的另一个实施例,S14包括对cfDNA进行捕获,包括进行以下:S114对所述cfDNA进行末端修复,获得第二修复片段;S134对所述第二修复片段进行测序接头连接,获得第二连接产物;S154对所述第二连接产物进行扩增,获得第二扩增产物;以及S174对所述第二扩增产物进行所述捕获,以获得所述第二目的片段。
捕获可以利用固相芯片进行,也可以利用液相芯片进行,本实施例对捕获方式不作限制。根据本发明的实施例,利用液相芯片进行所述捕获,捕获的区域包括基因组上次等位基因频率最接近0.5的至少1000个SNP位点。根据本发明的一个较佳实施例,捕获的区域包括以下合并(i)-(iii)至少之二后的非冗余位点:(i)人类群体等位基因频率数据库中的杂合度为0.48-0.5的SNP位点,(ii)在千人基因组数据库的东亚人群中的次等位基因频率为0.5,或者在该数据库中的所有群体中的平均次等位基因频率为0.4-0.5的SNP位点,(iii)在HapMap数据库的CHB子库中的次等位基因频率大于0.4,或者次等位基因频率为0.5,或者杂合度为0.48-0.5的SNP位点。由此,可以使用更小的捕获芯片、更低的数据量,也即更低的成本来检测供体的fdDNA含量。通常所说的SNP都是二态性的,基因型是指同源染色体上一对等位位点的类型的组合。所称的SNP的次等位基因频率,也叫最小等位基因频率(minor allele frequency,MAF)是指该SNP的频率较低的等位基因在给定人群中的频率。SNP的MAF可以依据数据库公开的信息,在该实施例中,挑选的MAF符合要求的SNP是通过查找相应数据库中提供的信息来确定的。所称的杂合度(heterozygosity)是SNP的另一个频率参数,杂合度=2MAF(1-MAF)。杂合度或者MAF越高即越接近0.5,说明该SNP在群体中杂合频率越高,最后被确定为可区分受体和供体的SNP的可能性越大。对捕获的目标区域进行有优化设计、有目的的筛选,能够减少数据总量,降低测序成本、分析成本和时间。根据本发明的另一个实施例,通过上述筛选,最
终获得的捕获的区域包括表1中的SNP位点。
S20比对。
将第一测序数据和第二测序数据分别与参考序列进行比对,对应获得第一比对结果和第二比对结果。
将测序数据中的读段比对到参考序列上(reads mapping或者reads alignment),是指将测序得到的DNA片段(也就是reads)定位在基因组上。通过读段定位,在克服测序产生的reads过短导致的技术困难的同时,也方便利用基因组位置作为桥梁,来将测序得到的数据与前期研究产生的注释结果进行整合。读段比对定位往往被作为测序数据分析的第一步,其质量的好坏以及速度的快慢,都会直接对后续的分析工作产生影响。在比对过程中,根据比对参数的设置,reads最多允许有n个碱基错配(mismatch),n优选为1或2,若reads中有超过n个碱基发生错配,则视为该对reads无法比对到参考序列。具体比对时,可使用各种比对软件,例如SOAP(Short OligonucleotideAnalysis Package),bwa,Tmap等,本实施方式对此不作限定。
所说的参考序列是已知序列,可以是预先获得的目标个体所属生物类别中的任意的参考模板,例如,同一生物类别的已公开的基因组组装序列,若混合核酸样本为来自人类,其基因组参考序列(也称为参考基因组)可选择NCBI数据库提供的HG19。比对结果包含各条读段与参考序列的比对情况,包括读段是否能够比对上参考序列、读段比对到参考序列的位置、比对到参考序列的唯一位置还是多个位置、某一位点多少读段比对上、比对上某位点的读段的相应位置的碱基类型等。
根据本发明的实施例,要求捕获的目标区域的平均测序深度为不小于200×,相当于平均每个目标位点都有200条读段比对上。如此,使最终获得的结果更加可信。
根据本发明的实施例,在获得第一比对结果和/或第二比对结果之后,对第一比对结果和/或第二比对结果进行去重,分别以去重后的第一比对结果和/或去重后的第二比对结果替代第一比对结果和/或第二比对结果,再进行后续步骤,有利于含量或比例的准确确定。
S30基于第一比对结果进行SNP检测。
基于第一比对结果进行SNP检测,获得第一分型结果,第一分型结果包括多个一级纯合基因型SNP,表示所述一级纯合基因型SNP在所述第一测序数据中的基因型为AA。所称的纯合基因型也称为纯合子。需要说明的是,这里以字母“AA”表示第一分型结果中的纯合子SNP,只是为方便指代该类型SNP,非指碱基为A,也非指具体的一个或多个该类型SNP。
SNP检测或者SNP识别可以利用各种SNP识别软件,包括但不限于SOAPsnp、SomaticSniper、CaVEMan、SAMtools、MuTect和TVC。将比对上同一位点的读段分成不同类别,是基于比对上的读段中的对应位置的碱基不同来进行的,例如比对到参考序列碱基为A的位点的读段中,一部分读段的该位置上的碱基为A,另一部分读段的该位置上的碱基为G,则比对到该位点的读段分为两类。
对于与参考序列碱基一致的或者不一致的纯合子位点,常规的SNP识别分型软件不能对其进行分型。根据本发明的一个实施例,先利用TVC软件进行SNP识别及分型,对于纯合子位点,TVC软件没办法对其分型。为最大化位点分型,在本发明的实施例中,利用SNP的各类读段的支持情况来分型。根据本发明的实施例,所称的基于第一比对结果进行SNP检测,获得第一分型结果,包括进行以下a或者进行以下a和b:a.依据所占的比例大于95%的那一类第一读段,确定该位点的基因型,b.依据所占的比例大于等于25%且小于等于95%的多类第一段读段中的所占比例最大的前两类第一读段,确定该位点的基因型,多类第一读段之间的区别在于其共同比对上的位点的相应位置上的碱基不同。规则a,即比对上一位点的第一读段中的某一类第一读段的比例大于95%,则认为该位点为纯合子,组成碱基为比例大于95%的这类比对上的读段的相应位置的碱基;b中,即对比上一位点的读段中有两类或者多于两类的读段的比例介于25%到95%,则认为该位点是杂合子,碱基组成为其中的比例最大,即相对最接近95%的两类读段相应位置上的碱基。需要说明的是,a和/或b分型规则,适用于所有类型的位点的分型,本领域技术人员可以按照所称的a和/或b,不利用现有的基因分型软件,对位点直接分型。
为使上述依据读段的支持比例进行分型的分型结果准确,对后续分析有意义,根据本发明的一个较佳实施例,在进行a和/或b之前,分别对第一读段和第二读段进行去重,去掉由于文库构建过程的扩增带来的重复。
根据本发明的较佳实施例,仅保留等位基因频率为100%、和/或测序深度大于等于平均测序深度的一级纯合基因型SNP。这里,所称的等位基因频率为100%的位点,比对上该位点的所有第一读段的相应位置上的碱基都相同。这样,对一级纯合基因型SNP进行严格过滤,利于确定的结果更加准确可信。
S40确定所述供体来源cfDNA的比例。
基于第二比对结果中比对上二级纯合基因型SNP的第二读段的量,确定供体来源的cfDNA的比例。二级纯合基因型SNP为第二比对结果中比对上该位点的第二读段包含不支持等位基因A的第二读段的一级纯合基因型SNP的至少一部分。需要说明的是,所称的“量”,
可以是绝对数目、相对数目例如比例或者函数关系式。还需要说明的是,以字母A表示等位基因,只是为方便说明,非指示该位点的等位基因为碱基A。所称的二级纯合基因型SNP,亦即为在受体中为纯合子、在供体中为杂合子或者为不同纯合子的位点,依据比对到该类位点的第二读段的量,就能确定供体来源的cfDNA的比例。
根据本发明的实施例,在进行该步骤之前,去除第二比对结果中的非唯一比对的第二读段,以准确确定受体cfDNA样本中供体来源cfDNA的比例。
根据本发明的实施例,所称的二级纯合基因型SNP为第二比对结果中比对上该位点的第二读段包含不支持等位基因A的第二读段的所有一级纯合基因型SNP,依据以下公式计算比例,其中,N表示第二读段的数量,NAB(B)表示比对上一级纯合子AA且不支持等位基因A的第二读段有两类的这两类第二读段的总数量,NBB(B)表示比对上一级纯合子AA且不支持等位基因A的第二读段只有一类的这类第二读段的数量,NAA(A)表示比对上一级纯合子AA且支持等位基因A的第二读段的数量。
根据本发明的实施例,所称的二级纯合基因型SNP为第二比对结果中比对上该位点的第二读段包含不支持等位基因A的第二读段的一级纯合基因型SNP中的一部分,该二级纯合基因型SNP具有以下特点:在第二比对结果中有且只有一类不支持等位基因A的第二读段,将该类第二读段支持的等位基因表示为C,则在该示例中每个二级纯合基因型SNP在第二测序数据中的基因型均可表示为AC。每个二级纯合基因型SNP位点获得的支持等位基因C的第二读段的数目占比对上该位点的所有读段数目的比例称为频率,每个二级纯合基因型SNP的该频率=NC/(NA+NC),其中,NC表示比对上该二级纯合基因型SNP且不支持等位基因A的第二读段的数目,NA表示比对上该二级纯合基因型SNP且支持等位基因A的第二读段的数目。第二测序数据中可得到大量这样的频率,这些频率反映供体cfDNA含量和极小部分的测序测序或比对错误的比例。需要说明的是,这里以字母C表示不同于A的等位基因,只是为方便说明以区分A,非指该等位基因的碱基类型为C。
为排除掉测序错误或比对错误对确定cfdDNA含量的影响,发明人首次提出可区分受体和供体位点中的反映供体频率的数据可反映供体含量的假设,基于该假设运用等位基因频率检测的方法,来计算受体cfDNA中的供体来源cfDNA的比例。根据本发明的实施例,该步骤包括:对所述量进行聚类,获得聚类结果;依据所述聚类结果中呈两倍关系的两类量中的至少一类确定所述供体来源cfDNA的比例。聚类可采用各种聚类算法,本实施例对此不作限定。理论上,所称的量可明显被聚类成两类(簇),且这两类存在两倍关系。根据本发明的一个实施例,所称的量为各个二级纯合基因型SNP(二级纯合子AA)中的不支持等位基因A的一类或各类第二读段所占的比例,使用K-means聚类算法进行聚类,K=2,
将上述一类或各类第二读段所占比例值按均值迭代聚成两个簇,使用均值进行卡方检验,判断两倍关系的显著性,输出两类均值,其中的为另一个均值的两倍的均值即为供体来源cfDNA的比例。
根据本发明的另一个实施方式提供的一种确定受体cfDNA样本中供体来源的cfDNA的比例的方法,包括以下步骤:S100获取第一测序数据,所述第一测序数据为受体基因组DNA样本的至少一部分的测序结果,包括多个第一读段;S200将所述第一测序数据与参考序列进行比对,获得第一比对结果;S300基于所述第一比对结果进行SNP检测,获得第一分型结果,所述第一分型结果包括多个一级纯合基因型SNP;S400获取第二测序数据,所述第二测序数据为受体cfDNA的至少一部分的测序结果,包括多个第二读段;将所述第二测序数据与所述参考序列进行比对,获得第二比对结果;S500基于所述第二比对结果中比对上二级纯合基因型SNP的第二读段的量,确定所述供体来源的cfDNA的比例,所述二级纯合基因型SNP为所述第二比对结果中比对上该位点的第二读段中包含不支持等位基因A的第二读段的一级纯合基因型SNP。前述对本发明实施方式或者任一实施例中的确定受体cfDNA样本中供体来源的cfDNA的比例的方法中的步骤以及特征的解释、以及优点的描述,同样适用本发明这一实施方式的方法,在此不再赘述。
上述任一实施方式或实施例的方法,不依赖于供体遗传样本,可以利用灵活的、一体化的软件包的形式实现,能够独立部署、高效运行。根据本发明的一个实施例,将该方法应用于移植排斥监测,由于任一该方法为低创或无创检测、具有可接受的成本和直观的数字化结果,可作为一种便捷、早期、无创、准确的移植排斥监测辅助技术,可作为临床免疫排斥检测的辅助或补充手段。
上述本发明任一方面的确定受体cfDNA样本中供体来源cfDNA的比例的方法的全部或部分步骤,可以利用包含可拆分的相应单元功能模块的装置/系统来施行,或者将方法程序化、存储于机器可读介质,利用机器运行该可读介质来实现。
根据本发明的再一个实施方式提供的一种确定受体cfDNA样本中供体来源cfDNA的比例的装置,该装置用以实施上述本发明任一实施方式或任一实施例的受体cfDNA样本中供体来源cfDNA的比例的方法的全部或部分步骤,该装置包括:数据输入单元,用于输入数据;数据输出单元,用于输出数据;处理器,用于执行可执行程序,所述可执行程序包括完成上述本发明任一方面的方法;存储单元,与所述数据输入装置、所述数据输出装置和所述存储器相连,用于存储数据,其中包括所述可执行程序。本领域技术人员能够理解,所称的可执行程序可以保存在存储介质中,所称存储介质可以包括:只读存储器、随机存
储器、磁盘或光盘等。
根据本发明的又一个实施方式提供的一种监测器官移植排斥的方法,包括:分别于不同时间点对受体进行采血,获得多个血液样本;利用上述本发明任一实施方式或实施例的方法确定每个所述血液样本中供体来源cfDNA的比例;基于确定的多个所述供体来源cfDNA的比例,进行所述监测。
根据本发明的一个实施方式提供的一种监测器官移植排斥的装置,该装置用以实施上述的监测器官移植排斥的方法的全部或部分步骤,该装置包括:样本获取单元,用以分别于不同时间点对受体进行采血,获得多个血液样本;供体cfDNA比例确定单元,与所述样本获取单元相连,用以利用上述本发明任一实施方式或实施例的确定受体cfDNA样本中供体来源cfDNA的比例的方法确定每个所述血液样本中供体来源cfDNA的比例;监测单元,与所述供体cfDNA比例确定单元相连,用以基于确定的多个所述供体来源cfDNA的比例,进行所述监测。
利用上述本发明的方法和/或装置系统,能够只依据受体的遗传样本确定出能够区分供体和受体的SNP,将这些SNP作为区分混合cfDNA中供体和受体来源cfDNA的标记;而通过这些标记位点得到的测序读段的支持情况,利用本发明的方法和/或装置,可准确确定移植后的受体cfDNA样本中的cfdDNA的含量;而将其应用于器官移植排斥检测,由于其为低创或无创的检测,且具有可接受的成本、直观的数字化结果展示,能够作为一种便捷、早期、无创和准确的移植排斥监测辅助技术,而且为非依赖供体遗传样本的技术,为临床判断移植排斥程度提供建议,或者作为临床检测移植排斥的辅助或补充手段。
以下结合附图和具体实施例对本发明的方法和/或装置进行详细的描述。下面示例,仅用于解释本发明,而不能理解为对本发明的限制。除另有交待,以下实施例中涉及的未特别交待的试剂、序列(接头、标签和引物)、软件及仪器,都是常规市售产品或者开源的,比如购自Life Technologies公司、华大基因等。
实施例一
一、获得第一测序数据的实验方法,一般包括:
(一)目标SNP位点设计
由于该示例方法要求SNP位点的测序深度高,平均需达200×或以上,普通的芯片用于本方法会造成大量数据浪费,使检测成本极大提高,因此,发明人按照次等位基因频率(MAF)值越接近于0.5的思路,自主设计、自主合成了一款小型SNP芯片用于目标区域捕获。
目标SNP位点获取途径主要有以下几种:
1.ALFRED等位基因频率数据库,按照杂合度从0.48至0.5的区间进行过滤,获取946个SNP位点;
2.1000Genomes数据库,在EAS超群子库中按频率为0.5的条件进行过滤,然后按照该库所有群体的平均频率从0.4至0.5的区间再进行过滤,共获取2263个SNP位点;
3.HapMap数据库,选取MAF值大于0.4的中华(CHB)群体子库,按MAF值等于0.5及杂合度在0.48至0.5区间的条件进行过滤,共获取1979个位点。
对以上SNP位点进行合并去冗余,排除位于X、Y性染色体上的位点,同时要求在dbsnp数据库中有唯一的“rs”编号。最终,获得3846个目标SNP位点,如表1所示。对目标SNP位点进行两边延伸至100bp,进行探针设计,最终设计成一款小型、适用该方法中的捕获的SNP芯片。
表1
rs2494624 | rs6683165 | rs11120890 | rs4908616 | rs2640908 | rs707472 | rs9435151 | rs4846000 |
rs730123 | rs4661453 | rs6678469 | rs485897 | rs807455 | rs4920650 | rs2816025 | rs477155 |
rs2235547 | rs6691722 | rs10903111 | rs7513064 | rs2236852 | rs4659370 | rs10794507 | rs7535816 |
rs6698316 | rs4949589 | rs10799103 | rs7519644 | rs404405 | rs6704396 | rs12750693 | rs1149038 |
rs6680621 | rs11590681 | rs114480320 | rs6669395 | rs7546366 | rs4308943 | rs6687753 | rs11208299 |
rs7547176 | rs6664708 | rs6600384 | rs10789403 | rs10890155 | rs3791051 | rs272565 | rs2297812 |
rs1484413 | rs1628297 | rs2297656 | rs12041966 | rs4926985 | rs6697692 | rs6688040 | rs3118036 |
rs494214 | rs684867 | rs1779866 | rs2207789 | rs2886919 | rs3009577 | rs9436678 | rs6588044 |
rs4430373 | rs12140250 | rs4650112 | rs11209397 | rs10489547 | rs1935244 | rs11162136 | rs10747346 |
rs11163300 | rs10493766 | rs17127806 | rs4847196 | rs10874866 | rs12131293 | rs1571049 | rs11166508 |
rs1491646 | rs796544 | rs625926 | rs11185151 | rs10785836 | rs657420 | rs527288 | rs2748680 |
rs4240542 | rs4453038 | rs4272626 | rs12039340 | rs380155 | rs6683142 | rs4950494 | rs11588437 |
rs6587705 | rs6427658 | rs3795741 | rs4661031 | rs10465957 | rs11265289 | rs4656855 | rs2369406 |
rs2502812 | rs2250993 | rs4657130 | rs4657154 | rs4657155 | rs7513132 | rs6700395 | rs10799871 |
rs10799872 | rs2841981 | rs7543224 | rs2205845 | rs2103639 | rs7544590 | rs10919483 | rs6683847 |
rs6425482 | rs10737340 | rs7525372 | rs10912080 | rs2861980 | rs12022368 | rs17574056 | rs10797943 |
rs9425336 | rs6663267 | rs12137257 | rs4651423 | rs1602012 | rs4534385 | rs431889 | rs400172 |
rs516084 | rs815742 | rs845628 | rs1711758 | rs2094025 | rs10733070 | rs9651063 | rs623111 |
rs12118718 | rs10801416 | rs4657806 | rs10919830 | rs585041 | rs9427715 | rs12137900 | rs12125583 |
rs11240672 | rs4951309 | rs6658163 | rs10900447 | rs823114 | rs2357587 | rs61828076 | rs11119948 |
rs12568188 | rs340853 | rs6668907 | rs12738331 | rs2813684 | rs10779288 | rs11117790 | rs6604568 |
rs6604589 | rs4846468 | rs645142 | rs2807845 | rs3010852 | rs35293291 | rs12033057 | rs9943197 |
rs1727029 | rs883583 | rs6541316 | rs7514972 | rs4658890 | rs10752796 | rs6665230 | rs7526011 |
rs9435492 | rs9435539 | rs6586363 | rs10803234 | rs6429466 | rs746997 | rs4659619 | rs12038672 |
rs4406617 | rs10926222 | rs10495474 | rs12144559 | rs2291409 | rs12744297 | rs12119594 | rs2085555 |
rs12143602 | rs12141946 | rs3127469 | rs10802220 | rs6661897 | rs10754558 | rs6688948 | rs9677943 |
rs11674560 | rs4927621 | rs59771193 | rs12476309 | rs11690345 | rs10171401 | rs10929518 | rs16864620 |
rs6756093 | rs6718476 | rs2693818 | rs6741456 | rs12617706 | rs1430289 | rs1367270 | rs1034380 |
rs238629 | rs2356357 | rs12473958 | rs12474828 | rs4669490 | rs16867226 | rs6707887 | rs1054561 |
rs4669492 | rs4669625 | rs11904084 | rs1734341 | rs1734342 | rs1615421 | rs1734358 | rs1734361 |
rs1734365 | rs1387570 | rs2357821 | rs6432266 | rs13033675 | rs72493344 | rs72779862 | rs7574217 |
rs807582 | rs11901571 | rs7577790 | rs12613420 | rs2449623 | rs589842 | rs12999091 | rs11125884 |
rs12466350 | rs1447188 | rs1447190 | rs2593433 | rs212752 | rs6751657 | rs6742602 | rs7565016 |
rs4439939 | rs12475896 | rs2373782 | rs11124965 | rs17032635 | rs12712931 | rs11125128 | rs7602044 |
rs6716567 | rs6545107 | rs10195274 | rs10184260 | rs6733430 | rs4450649 | rs2193690 | rs843646 |
rs7557639 | rs4671358 | rs4672331 | rs7563535 | rs1978404 | rs1472031 | rs11903638 | rs2861632 |
rs12994875 | rs13031428 | rs11687437 | rs10182682 | rs11684466 | rs13420244 | rs6546742 | rs6757131 |
rs13387588 | rs4853065 | rs11675344 | rs2007848 | rs4465789 | rs4853146 | rs2160370 | rs2192922 |
rs2216102 | rs10181895 | rs10205659 | rs925991 | rs11691388 | rs13029122 | rs34767571 | rs62170373 |
rs62170374 | rs10192926 | rs6729714 | rs10165209 | rs12472674 | rs6547306 | rs6758746 | rs6547308 |
rs3100108 | rs35391999 | rs7581471 | rs10203293 | rs17026540 | rs13395344 | rs231556 | rs231558 |
rs4096200 | rs13432811 | rs10202379 | rs12478256 | rs17031229 | rs10185531 | rs12616319 | rs10207608 |
rs12104935 | rs12463442 | rs315952 | rs7594791 | rs17046609 | rs17046615 | rs17046639 | rs6758777 |
rs4849766 | rs11122822 | rs6758991 | rs7587477 | rs12466272 | rs55977322 | rs4848685 | rs10187048 |
rs12619998 | rs3768866 | rs1866460 | rs936131 | rs13424930 | rs13401007 | rs11891214 | rs11891266 |
rs12613726 | rs11695854 | rs6430460 | rs1649570 | rs10204209 | rs7607239 | rs9646663 | rs12472074 |
rs12472110 | rs7561770 | rs6720897 | rs4662303 | rs6722966 | rs11681900 | rs16827230 | rs13419995 |
rs816889 | rs10204829 | rs1482313 | rs4664918 | rs4664919 | rs6716681 | rs12471260 | rs1457235 |
rs7599823 | rs6432740 | rs12052528 | rs3098650 | rs17607603 | rs3791860 | rs16860543 | rs4972516 |
rs11674587 | rs12693237 | rs10185223 | rs17191654 | rs6724378 | rs6737765 | rs10172410 | rs11679657 |
rs13011100 | rs13384417 | rs6747256 | rs7599179 | rs13391723 | rs34997637 | rs7608834 | rs12478266 |
rs16840070 | rs12162325 | rs11692301 | rs7578914 | rs11694792 | rs6738605 | rs12468504 | rs13022344 |
rs6732993 | rs10804115 | rs10497867 | rs7603584 | rs4129010 | rs6717641 | rs7573536 | rs12991600 |
rs6741142 | rs16838023 | rs7574280 | rs4610054 | rs13030205 | rs10192231 | rs1561298 | rs11687587 |
rs828911 | rs705649 | rs12470053 | rs4674066 | rs6728330 | rs667750 | rs1346798 | rs16825437 |
rs2432679 | rs7422445 | rs4099415 | rs4099416 | rs4284822 | rs6757951 | rs10193272 | rs6739418 |
rs10187515 | rs13417255 | rs4355095 | rs10498185 | rs2709423 | rs4455149 | rs10182873 | rs10933235 |
rs10209496 | rs10180608 | rs12615058 | rs12615089 | rs4312487 | rs6436754 | rs6436755 | rs6436758 |
rs4246656 | rs12998075 | rs6437062 | rs2592114 | rs2924814 | rs7569824 | rs6754875 | rs3791424 |
rs10184738 | rs4851996 | rs12469558 | rs749924 | rs11714648 | rs2600029 | rs1178488 | rs11129096 |
rs2320963 | rs9310883 | rs13063069 | rs1396415 | rs1688413 | rs2637546 | rs17709863 | rs9837289 |
rs1872996 | rs2543491 | rs6443222 | rs709641 | rs6795673 | rs1710893 | rs9813290 | rs6765768 |
rs2133132 | rs6774271 | rs4684999 | rs2733534 | rs2257984 | rs2279017 | rs2607755 | rs9844200 |
rs55985119 | rs2278962 | rs4685320 | rs7432238 | rs9859926 | rs9822136 | rs9860391 | rs6797574 |
rs17014597 | rs747313 | rs6794654 | rs7634790 | rs4680763 | rs9849208 | rs13059806 | rs1836181 |
rs13100297 | rs3773652 | rs6550023 | rs12496184 | rs4955278 | rs17279970 | rs6807330 | rs2286939 |
rs2286940 | rs9876116 | rs4678571 | rs6762997 | rs11714912 | rs9968170 | rs2276868 | rs6786191 |
rs499475 | rs12639142 | rs2878628 | rs7426478 | rs7372541 | rs510013 | rs934083 | rs4481124 |
rs12629222 | rs753819 | rs12632824 | rs7613368 | rs1901890 | rs6777784 | rs475747 | rs7636925 |
rs7640062 | rs9837200 | rs7623286 | rs4688495 | rs12495731 | rs9863628 | rs1158924 | rs6549221 |
rs11128143 | rs1367369 | rs1316780 | rs7641488 | rs6785021 | rs12107824 | rs7432324 | rs12487433 |
rs9831375 | rs9873622 | rs4856379 | rs1358606 | rs7625954 | rs12495441 | rs1370209 | rs9850360 |
rs10511133 | rs6806126 | rs9820215 | rs9942007 | rs7627313 | rs9812699 | rs9837006 | rs34310679 |
rs9819239 | rs9852345 | rs6804539 | rs7620246 | rs7620442 | rs1349790 | rs9839497 | rs7618295 |
rs13078920 | rs4928088 | rs1375511 | rs1626282 | rs1164084 | rs13062004 | rs4682251 | rs17202592 |
rs12106967 | rs9840563 | rs9884006 | rs9850446 | rs11714459 | rs1575037 | rs9817322 | rs9869738 |
rs9820381 | rs6810172 | rs1317671 | rs2332690 | rs4527323 | rs7627031 | rs4555544 | rs7650872 |
rs9870482 | rs7653440 | rs11709339 | rs12639254 | rs12488259 | rs10935019 | rs4974500 | rs9858763 |
rs1392920 | rs4894320 | rs3937966 | rs2677421 | rs6780250 | rs7651071 | rs1097939 | rs961929 |
rs11922880 | rs1383609 | rs1481073 | rs7618012 | rs9681852 | rs12631962 | rs35631275 | rs2197749 |
rs11927172 | rs2199940 | rs12636186 | rs10935555 | rs1516545 | rs9653897 | rs6773068 | rs6440467 |
rs4432602 | rs965685 | rs13080561 | rs6765711 | rs9683109 | rs411449 | rs13063739 | rs6440796 |
rs10935984 | rs1448998 | rs7646959 | rs2084507 | rs6787941 | rs13100661 | rs7642473 | rs4955712 |
rs13060964 | rs9880228 | rs6787577 | rs7636253 | rs6444855 | rs6444858 | rs2421772 | rs6766019 |
rs13315469 | rs9824637 | rs6776646 | rs9868184 | rs997369 | rs9854298 | rs1558797 | rs1558798 |
rs710450 | rs1553092 | rs2675416 | rs9858730 | rs9871792 | rs4311173 | rs10755061 | rs55657417 |
rs56946992 | rs6822424 | rs6599390 | rs12644442 | rs10018044 | rs7673523 | rs7663514 | rs6834755 |
rs4696814 | rs3115378 | rs13136584 | rs4697777 | rs7698375 | rs223929 | rs4698249 | rs2159666 |
rs4698374 | rs10002931 | rs10032941 | rs6449138 | rs13129152 | rs976717 | rs2443041 | rs17497475 |
rs11731245 | rs10012903 | rs12501080 | rs4619879 | rs4441745 | rs6849600 | rs4692025 | rs7676237 |
rs13136817 | rs4692066 | rs9990632 | rs7670756 | rs6832648 | rs5029114 | rs17754 | rs2123027 |
rs6829064 | rs3733284 | rs2066788 | rs7693837 | rs6815859 | rs7670903 | rs279844 | rs12644230 |
rs1986648 | rs7694213 | rs904414 | rs3805151 | rs2030364 | rs981963 | rs28708068 | rs28459548 |
rs6819901 | rs1119860 | rs1480323 | rs1480324 | rs1480327 | rs3910148 | rs985954 | rs952003 |
rs7680100 | rs66511235 | rs11930002 | rs9993607 | rs6826243 | rs11736604 | rs11721440 | rs12504877 |
rs12642405 | rs13142709 | rs1565572 | rs9997469 | rs1383624 | rs6814380 | rs10856883 | rs2866046 |
rs4373184 | rs4859487 | rs6858448 | rs10025498 | rs6531779 | rs346502 | rs13130598 | rs9993968 |
rs10033411 | rs10031073 | rs10084787 | rs7694618 | rs10856895 | rs11933557 | rs12645971 | rs902981 |
rs10019142 | rs6835688 | rs10005096 | rs10005450 | rs10027656 | rs6828291 | rs1544390 | rs1544392 |
rs4538488 | rs4254781 | rs4256228 | rs4540052 | rs2013390 | rs7667413 | rs4699251 | rs13130723 |
rs7665905 | rs28701510 | rs4286516 | rs6824318 | rs151394 | rs964503 | rs223345 | rs223344 |
rs223341 | rs223339 | rs223328 | rs223325 | rs223319 | rs223318 | rs223315 | rs223308 |
rs2156505 | rs2672479 | rs28361927 | rs2175985 | rs1399404 | rs2726690 | rs2726688 | rs2672477 |
rs219481 | rs1452697 | rs13114592 | rs7440009 | rs11934129 | rs10155491 | rs6838559 | rs7697632 |
rs9995339 | rs6533787 | rs13108478 | rs1456365 | rs9799664 | rs3775842 | rs13104219 | rs4833769 |
rs11934307 | rs4075065 | rs6842608 | rs11098866 | rs12508861 | rs10033555 | rs10012364 | rs10014509 |
rs1346131 | rs7692730 | rs10024188 | rs1365148 | rs35016117 | rs10222699 | rs12646753 | rs1559782 |
rs1426003 | rs13127440 | rs11099444 | rs4864417 | rs13128168 | rs13129042 | rs12651201 | rs17050379 |
rs11736129 | rs1584783 | rs4956448 | rs2321272 | rs3811787 | rs13127080 | rs951848 | rs1492818 |
rs2353915 | rs10084798 | rs13131791 | rs6834377 | rs10021652 | rs28676537 | rs6838382 | rs2562876 |
rs10005936 | rs2234759 | rs2350467 | rs7688507 | rs7675509 | rs7686898 | rs2314975 | rs7692553 |
rs6833890 | rs6848528 | rs9991207 | rs12331967 | rs6811238 | rs6821998 | rs35281461 | rs11946672 |
rs56286907 | rs55768019 | rs12650554 | rs2200457 | rs4690508 | rs1395477 | rs10866216 | rs7664546 |
rs1585178 | rs7680082 | rs4861830 | rs6822856 | rs57444572 | rs12646548 | rs6822105 | rs6858486 |
rs11726337 | rs13137158 | rs12511186 | rs4861688 | rs13144444 | rs4861741 | rs6826401 | rs12505859 |
rs2019991 | rs4975576 | rs2241602 | rs2962038 | rs11133918 | rs12716127 | rs4975796 | rs11133936 |
rs2897241 | rs16871328 | rs10866554 | rs1019747 | rs7727930 | rs2964164 | rs13182748 | rs412078 |
rs1438301 | rs4391159 | rs7703863 | rs676852 | rs2890356 | rs6866168 | rs6865501 | rs12522645 |
rs12656452 | rs12657605 | rs13158011 | rs4457082 | rs6869914 | rs13155031 | rs7709193 | rs7443190 |
rs7713628 | rs2968253 | rs12187087 | rs6452095 | rs12659069 | rs2877176 | rs4415088 | rs2962787 |
rs6859125 | rs7705367 | rs7725300 | rs10472889 | rs840385 | rs213583 | rs10063005 | rs1353979 |
rs1553575 | rs7448055 | rs1482679 | rs1384447 | rs2128435 | rs4866902 | rs2067980 | rs6882139 |
rs13354106 | rs251331 | rs1593055 | rs381575 | rs256118 | rs417550 | rs4327570 | rs12657711 |
rs2290595 | rs10041616 | rs3804264 | rs160924 | rs4700227 | rs7725687 | rs4353986 | rs12656930 |
rs13159266 | rs6450491 | rs11746657 | rs10075617 | rs4700177 | rs33291 | rs2662398 | rs13179585 |
rs4418079 | rs7728722 | rs7716166 | rs294975 | rs6453133 | rs250757 | rs922538 | rs10514145 |
rs7708478 | rs62364011 | rs245012 | rs33014 | rs12658404 | rs56977687 | rs10942358 | rs2962878 |
rs6897040 | rs13178173 | rs34761 | rs13181336 | rs4642340 | rs419353 | rs12656561 | rs13154870 |
rs10039131 | rs381495 | rs377111 | rs247546 | rs12517486 | rs7729142 | rs10067922 | rs1405472 |
rs3920174 | rs3813308 | rs3797339 | rs10478424 | rs1115482 | rs6886947 | rs34675185 | rs2600398 |
rs2561502 | rs2561503 | rs2561504 | rs2561505 | rs2561506 | rs919306 | rs2974031 | rs153887 |
rs12189340 | rs4835850 | rs6890814 | rs6861554 | rs12515871 | rs10477666 | rs62384958 | rs7736388 |
rs12652706 | rs10520084 | rs7720425 | rs10061925 | rs9790905 | rs11242247 | rs7727766 | rs4380648 |
rs10078040 | rs34022 | rs1444179 | rs720303 | rs12651998 | rs10477360 | rs11948406 | rs13181329 |
rs6895434 | rs4642357 | rs1042713 | rs4705318 | rs4629585 | rs2303022 | rs753459 | rs3776959 |
rs7723312 | rs7723610 | rs9324813 | rs6866674 | rs1820795 | rs4921505 | rs209357 | rs6890136 |
rs10077466 | rs4354068 | rs1422819 | rs2938765 | rs7708885 | rs4868429 | rs4867577 | rs6876868 |
rs4242159 | rs10057145 | rs7728402 | rs2216554 | rs7720043 | rs13175524 | rs28429418 | rs2963675 |
rs10055777 | rs10055826 | rs6866021 | rs13175884 | rs10065976 | rs6871977 | rs6906875 | rs6596810 |
rs2251176 | rs2317965 | rs2745567 | rs2493160 | rs4959620 | rs4959841 | rs1205031 | rs1205033 |
rs7742726 | rs4959935 | rs2798159 | rs6918016 | rs808462 | rs12662634 | rs2070698 | rs12215208 |
rs4434476 | rs9370979 | rs9371004 | rs6918430 | rs4710927 | rs12205236 | rs9465871 | rs6456548 |
rs1087287 | rs6915987 | rs3131042 | rs3131043 | rs3131046 | rs1265064 | rs34907473 | rs9267958 |
rs6457509 | rs113532078 | rs72845752 | rs9272491 | rs9273482 | rs9274529 | rs9275289 | rs6457619 |
rs6457622 | rs7745040 | rs4713607 | rs9469780 | rs9357207 | rs668499 | rs6926781 | rs7739340 |
rs2815020 | rs6925661 | rs56198855 | rs3800282 | rs4607417 | rs1038890 | rs694148 | rs1375701 |
rs4714890 | rs2746323 | rs7750930 | rs9395493 | rs10948570 | rs9463746 | rs6902510 | rs6908786 |
rs1648218 | rs9475319 | rs9370573 | rs12664535 | rs9344213 | rs6901766 | rs1922946 | rs9359969 |
rs2993118 | rs9363853 | rs1671748 | rs9346371 | rs7768796 | rs1898487 | rs1615022 | rs12193133 |
rs1457947 | rs4532411 | rs2753422 | rs9353018 | rs1917065 | rs1572920 | rs6921115 | rs2324788 |
rs1231499 | rs1231498 | rs6941302 | rs12192914 | rs17059246 | rs7773043 | rs2144728 | rs9399702 |
rs1406895 | rs9399703 | rs11155837 | rs4427008 | rs4427009 | rs4240590 | rs1337424 | rs221612 |
rs6934122 | rs12192370 | rs319055 | rs2071945 | rs2817782 | rs9384783 | rs455732 | rs458486 |
rs6912446 | rs7764970 | rs12529501 | rs6907063 | rs9488363 | rs9489275 | rs4946318 | rs4510704 |
rs6937659 | rs9372674 | rs699397 | rs6935700 | rs1543705 | rs12234117 | rs13208666 | rs7775013 |
rs3777435 | rs6931816 | rs1409181 | rs12528474 | rs2205843 | rs962190 | rs3799479 | rs1928277 |
rs9399247 | rs9403134 | rs197508 | rs633802 | rs9403624 | rs12175408 | rs12175421 | rs9321998 |
rs7748978 | rs9377188 | rs1889554 | rs1871859 | rs9384054 | rs6923606 | rs9459828 | rs7749094 |
rs10455887 | rs9347530 | rs9365444 | rs9365446 | rs9347649 | rs9365505 | rs908018 | rs1514331 |
rs13200025 | rs596868 | rs9347924 | rs9364763 | rs12528334 | rs9365775 | rs9347038 | rs9356277 |
rs9366015 | rs6938463 | rs1008457 | rs11754069 | rs1534788 | rs3735108 | rs1713916 | rs6942522 |
rs4722382 | rs12666681 | rs6462094 | rs1991757 | rs11979130 | rs6972061 | rs6959971 | rs6945672 |
rs6462799 | rs4445136 | rs7793828 | rs1709595 | rs10263113 | rs10278301 | rs6959333 | rs10234570 |
rs2189349 | rs2353814 | rs13223466 | rs17574506 | rs1526551 | rs13312424 | rs56054598 | rs2723412 |
rs10264059 | rs7810554 | rs6973638 | rs62438998 | rs6964578 | rs966470 | rs7776567 | rs12540302 |
rs38205 | rs6973890 | rs4721827 | rs73276508 | rs17143272 | rs2270236 | rs7782130 | rs11772635 |
rs6965117 | rs6461625 | rs4722207 | rs10260438 | rs6978480 | rs7797272 | rs2813838 | rs4722456 |
rs10230728 | rs7776731 | rs7793934 | rs4722616 | rs6461957 | rs42089 | rs10951197 | rs4722860 |
rs17160544 | rs2191873 | rs215695 | rs12701556 | rs2735127 | rs6946572 | rs4316059 | rs4314553 |
rs11972350 | rs4720420 | rs2583876 | rs965748 | rs2047915 | rs9784990 | rs4724521 | rs6949085 |
rs6953890 | rs2686827 | rs10237584 | rs10243526 | rs7787700 | rs10230343 | rs2237473 | rs11238155 |
rs1600388 | rs66984970 | rs11978421 | rs1031910 | rs6945649 | rs984196 | rs10260088 | rs7792616 |
rs9969362 | rs4498495 | rs10232043 | rs7776619 | rs4311623 | rs12673895 | rs4870669 | rs12698581 |
rs7783170 | rs11768285 | rs6460672 | rs38584 | rs2074640 | rs4730342 | rs6466331 | rs2302441 |
rs12706023 | rs11760355 | rs1990432 | rs10235211 | rs4730838 | rs2189450 | rs10277514 | rs7778809 |
rs1922060 | rs1558370 | rs10808299 | rs2706896 | rs13247631 | rs11980222 | rs11982431 | rs10954755 |
rs10808307 | rs11761009 | rs10487008 | rs10487009 | rs6465130 | rs2299208 | rs2023744 | rs12667163 |
rs11773773 | rs43007 | rs9641129 | rs388625 | rs2394712 | rs6465604 | rs4729475 | rs4729707 |
rs264373 | rs4730324 | rs7784378 | rs2529491 | rs2056733 | rs55813173 | rs62474759 | rs7810206 |
rs6978560 | rs4731094 | rs4731207 | rs4731214 | rs9886177 | rs4731641 | rs4731679 | rs4731798 |
rs4349926 | rs4731970 | rs2041996 | rs12539993 | rs9649046 | rs17160449 | rs4732334 | rs6962291 |
rs6464471 | rs2240358 | rs11770150 | rs6973174 | rs10261766 | rs454612 | rs7792380 | rs10235552 |
rs10952282 | rs10265794 | rs35573300 | rs10236489 | rs10250599 | rs12703162 | rs7809143 | rs6951959 |
rs10273747 | rs10242760 | rs10238689 | rs10249494 | rs4236179 | rs4909239 | rs6972008 | rs17814323 |
rs4735959 | rs4272406 | rs1033345 | rs10111133 | rs4875865 | rs4876082 | rs4557727 | rs13282229 |
rs4451330 | rs4457357 | rs4875731 | rs4875609 | rs4875743 | rs4875744 | rs12674902 | rs4875773 |
rs4875775 | rs11785517 | rs4875349 | rs12547280 | rs4609213 | rs2616969 | rs7842795 | rs4537322 |
rs4875130 | rs4242499 | rs4875149 | rs1362682 | rs10780170 | rs4875530 | rs10866988 | rs2897813 |
rs7823343 | rs4875535 | rs4875537 | rs2190162 | rs1405246 | rs4875577 | rs4875597 | rs2725685 |
rs4875607 | rs7002659 | rs2979656 | rs4478599 | rs4841005 | rs6994742 | rs2046398 | rs13249013 |
rs2409623 | rs6981758 | rs4392859 | rs4406360 | rs4841386 | rs13255792 | rs4574847 | rs12542170 |
rs9644018 | rs6983961 | rs1528624 | rs430888 | rs4376497 | rs2170397 | rs4298488 | rs17118231 |
rs13257044 | rs6982108 | rs4291272 | rs4831803 | rs4831368 | rs13251172 | rs4521804 | rs10102857 |
rs11777998 | rs7843875 | rs10112502 | rs4443656 | rs7813735 | rs1031882 | rs1031884 | rs4397403 |
rs12674913 | rs4872012 | rs12155880 | rs11135715 | rs11774789 | rs4872154 | rs4543550 | rs4872390 |
rs2976340 | rs7817583 | rs1042992 | rs11135986 | rs4732620 | rs4732863 | rs4732976 | rs4732986 |
rs7017060 | rs4609156 | rs4268090 | rs4452759 | rs10503926 | rs7827391 | rs6986872 | rs6994414 |
rs7014271 | rs4342589 | rs4520153 | rs16884271 | rs11785595 | rs4394356 | rs4427156 | rs16889589 |
rs6983747 | rs1912978 | rs3847183 | rs4554449 | rs10958678 | rs6986504 | rs13271446 | rs4342624 |
rs4873687 | rs454975 | rs2719402 | rs16919978 | rs4236995 | rs4644241 | rs4323454 | rs4738648 |
rs4448263 | rs4237026 | rs56203327 | rs13272076 | rs4237042 | rs1019901 | rs4520200 | rs4273884 |
rs4737773 | rs4737846 | rs4284034 | rs12547594 | rs4737889 | rs4737912 | rs4737915 | rs920530 |
rs4737990 | rs4737991 | rs4738003 | rs4279601 | rs4738017 | rs2380606 | rs6995829 | rs4738201 |
rs4738206 | rs4738223 | rs4738268 | rs4571768 | rs10092623 | rs4385459 | rs4410896 | rs4735683 |
rs4641034 | rs4735749 | rs4507742 | rs13439699 | rs12056492 | rs4526336 | rs4739127 | rs4739180 |
rs1838440 | rs4330731 | rs28522181 | rs10106606 | rs6473354 | rs6473356 | rs28728013 | rs4498534 |
rs4237105 | rs6986074 | rs11783037 | rs12675252 | rs1915011 | rs447618 | rs2205156 | rs4734795 |
rs416829 | rs11994496 | rs58575315 | rs62525163 | rs62525164 | rs4418314 | rs4391396 | rs4579497 |
rs4735302 | rs998980 | rs4624998 | rs4263730 | rs4644202 | rs4735530 | rs4476980 | rs4734407 |
rs11780500 | rs4734443 | rs10095356 | rs4734021 | rs4291239 | rs6985113 | rs1460586 | rs16874595 |
rs4734936 | rs4236795 | rs4506180 | rs11785269 | rs4446710 | rs10955506 | rs7010380 | rs4734201 |
rs12550383 | rs4236804 | rs4735133 | rs4236805 | rs4556111 | rs10755908 | rs7843346 | rs12115151 |
rs7828293 | rs10955657 | rs6469456 | rs6982808 | rs34678450 | rs7831926 | rs7833423 | rs6990320 |
rs4377985 | rs4876357 | rs4876775 | rs13251051 | rs4876841 | rs3134081 | rs4644292 | rs4407920 |
rs16893639 | rs2305599 | rs2326483 | rs4870723 | rs4871178 | rs4871182 | rs4549775 | rs4543545 |
rs4316158 | rs4518674 | rs4870812 | rs4870821 | rs4871323 | rs4870828 | rs4871351 | rs4242341 |
rs4521739 | rs4871593 | rs897147 | rs4871697 | rs4871733 | rs4871750 | rs4143118 | rs1949808 |
rs4407842 | rs10956390 | rs4733789 | rs4733828 | rs4733859 | rs4733687 | rs2579855 | rs4636162 |
rs4285452 | rs298612 | rs4736520 | rs7843127 | rs4736746 | rs4587308 | rs7002377 | rs28415343 |
rs2458753 | rs4736510 | rs4480107 | rs4521737 | rs4736654 | rs11783413 | rs4399592 | rs4897704 |
rs4486240 | rs4897638 | rs4909912 | rs4909629 | rs4909630 | rs4346958 | rs4909851 | rs4385494 |
rs1375250 | rs4398959 | rs4313191 | rs11773949 | rs4909376 | rs4463463 | rs11166744 | rs11166745 |
rs11166746 | rs4412401 | rs10104327 | rs4909694 | rs4422809 | rs4243879 | rs4909767 | rs4736226 |
rs4736081 | rs4736007 | rs4736008 | rs10095006 | rs4736012 | rs4736121 | rs4736123 | rs4736126 |
rs4358783 | rs4374967 | rs4311638 | rs12550497 | rs12548549 | rs7005909 | rs61661433 | rs9657468 |
rs4907408 | rs4917300 | rs4917229 | rs4424237 | rs11775376 | rs13260925 | rs11775811 | rs36090686 |
rs6988480 | rs6982549 | rs4874113 | rs4874122 | rs11775278 | rs2958043 | rs4742225 | rs12378651 |
rs7040899 | rs4741604 | rs1075038 | rs10966860 | rs7033562 | rs7048541 | rs4741729 | rs7869130 |
rs7869275 | rs4741745 | rs3824351 | rs4741769 | rs12353393 | rs4741837 | rs10758554 | rs10974329 |
rs4304371 | rs4741967 | rs10758750 | rs4742263 | rs4620331 | rs2629137 | rs842292 | rs1027583 |
rs10733545 | rs4568657 | rs4563945 | rs4391483 | rs10816255 | rs439467 | rs1322314 | rs10809143 |
rs4483255 | rs10960270 | rs10960277 | rs10756294 | rs4273936 | rs1331346 | rs1411936 | rs10491748 |
rs1952986 | rs2382383 | rs10756454 | rs12554886 | rs9407716 | rs7861593 | rs10810551 | rs10756749 |
rs4442246 | rs4490948 | rs10810918 | rs4610856 | rs4418426 | rs2153416 | rs2506118 | rs7868334 |
rs4636294 | rs4446815 | rs10966560 | rs7853877 | rs1537264 | rs2482525 | rs4461986 | rs480866 |
rs2383759 | rs2167643 | rs4879382 | rs431008 | rs476578 | rs7847234 | rs4878501 | rs4879795 |
rs6476455 | rs10972224 | rs4879848 | rs587118 | rs4879913 | rs4880008 | rs10973803 | rs10973805 |
rs4593645 | rs449851 | rs4745231 | rs4745296 | rs4745466 | rs4745520 | rs4744873 | rs4744604 |
rs4615645 | rs4744610 | rs4526420 | rs4620343 | rs4745073 | rs4606122 | rs4475568 | rs4745114 |
rs4745155 | rs4256646 | rs4255204 | rs4332197 | rs4745601 | rs4745607 | rs4361823 | rs10867213 |
rs4877475 | rs9314677 | rs4877152 | rs7873885 | rs267593 | rs17199987 | rs4501665 | rs7030587 |
rs7035454 | rs11138628 | rs4428744 | rs945162 | rs4877679 | rs2788115 | rs4322086 | rs4877761 |
rs4471123 | rs7020579 | rs4877947 | rs791323 | rs4492454 | rs6559978 | rs4878117 | rs4878121 |
rs11142120 | rs4877127 | rs4876973 | rs4876993 | rs4744106 | rs7849350 | rs4744473 | rs2613326 |
rs4604577 | rs4743855 | rs4744162 | rs10761190 | rs4744227 | rs10821134 | rs4743923 | rs10992756 |
rs10821142 | rs2001994 | rs4744283 | rs4744316 | rs1754435 | rs4744386 | rs3780567 | rs4743288 |
rs589458 | rs4430197 | rs1810711 | rs4743056 | rs11581 | rs10759742 | rs7869116 | rs7872629 |
rs4742727 | rs2295932 | rs4742814 | rs4742820 | rs7849782 | rs4554533 | rs4521823 | rs450382 |
rs410737 | rs2417463 | rs4742669 | rs4743039 | rs10739239 | rs10978839 | rs10759245 | rs7035870 |
rs10816720 | rs491744 | rs481661 | rs10435858 | rs7875113 | rs4302915 | rs4532674 | rs484611 |
rs2691669 | rs2900556 | rs4344146 | rs10817494 | rs10817508 | rs4979275 | rs10759653 | rs4836732 |
rs4836849 | rs4838041 | rs4240422 | rs4837462 | rs4837521 | rs10983982 | rs6478387 | rs4837628 |
rs1333888 | rs12376794 | rs4837820 | rs4837853 | rs4304393 | rs4837949 | rs7390359 | rs12683599 |
rs2282089 | rs4838177 | rs1570581 | rs4838242 | rs4837016 | rs490333 | rs4837043 | rs7860066 |
rs4837096 | rs4322101 | rs11794980 | rs2417065 | rs2996672 | rs4837393 | rs4740383 | rs11243535 |
rs7036013 | rs7870760 | rs10901205 | rs11537116 | rs2427960 | rs2427963 | rs2427966 | rs4917339 |
rs4842031 | rs4240702 | rs4842145 | rs4842168 | rs4842169 | rs4841931 | rs4841934 | rs4842188 |
rs9410013 | rs7865668 | rs4841889 | rs936250 | rs7074221 | rs7092412 | rs17156779 | rs4880961 |
rs7083892 | rs7099247 | rs10903635 | rs10903636 | rs12257452 | rs11251910 | rs4881186 | rs10795097 |
rs4747351 | rs11259272 | rs11255376 | rs920284 | rs11598544 | rs10905646 | rs1876066 | rs7916758 |
rs12217971 | rs10906204 | rs4748006 | rs589345 | rs4747267 | rs11254002 | rs17139010 | rs10904935 |
rs7098901 | rs7079026 | rs1582059 | rs6481881 | rs1475361 | rs7895512 | rs9651356 | rs2484650 |
rs3006263 | rs11015119 | rs1360844 | rs1168579 | rs4749280 | rs7893462 | rs11006779 | rs34717952 |
rs7896951 | rs10826492 | rs1007082 | rs7905328 | rs2488315 | rs7896318 | rs2050503 | rs10159976 |
rs10793544 | rs7069381 | rs10793597 | rs718332 | rs3793784 | rs1180565 | rs4948422 | rs10761504 |
rs10994365 | rs4144119 | rs10509170 | rs2394139 | rs4333908 | rs10740228 | rs10430507 | rs7076617 |
rs7083972 | rs6480237 | rs10822948 | rs7093905 | rs10762138 | rs12220599 | rs10822975 | rs10740269 |
rs10822976 | rs12249046 | rs10997509 | rs7894109 | rs7073570 | rs726872 | rs10762139 | rs6480261 |
rs2812541 | rs827349 | rs827287 | rs11001685 | rs12245799 | rs7898958 | rs12219399 | rs1370562 |
rs2345209 | rs4933780 | rs4933782 | rs11189865 | rs10509435 | rs7074306 | rs677221 | rs616012 |
rs7915460 | rs11199083 | rs10886912 | rs66814819 | rs4568951 | rs6585852 | rs11201031 | rs1338845 |
rs7902588 | rs949809 | rs1933937 | rs1104868 | rs10788415 | rs10749512 | rs10788417 | rs7893154 |
rs7917993 | rs7901447 | rs7913717 | rs7913198 | rs7899048 | rs7914414 | rs7074345 | rs10887419 |
rs11201350 | rs9988691 | rs9988694 | rs7918984 | rs7904036 | rs10887420 | rs4459233 | rs7908982 |
rs7908193 | rs7073421 | rs7093956 | rs2211178 | rs10749553 | rs1234213 | rs10887882 | rs10887883 |
rs12775232 | rs7095466 | rs10785956 | rs10785962 | rs2420000 | rs7907658 | rs7100415 | rs11188256 |
rs4919104 | rs4919235 | rs11193738 | rs10787145 | rs10884734 | rs12220706 | rs7077374 | rs10884951 |
rs10884952 | rs4918581 | rs12254016 | rs1537768 | rs7907069 | rs2185015 | rs585070 | rs2277206 |
rs4918872 | rs4918874 | rs11196514 | rs7911899 | rs4752728 | rs11196799 | rs181637 | rs10749328 |
rs2420642 | rs12251811 | rs11593033 | rs12261081 | rs11248538 | rs1152653 | rs7068628 | rs2154179 |
rs995122 | rs4751122 | rs12253830 | rs10765003 | rs10765034 | rs7075041 | rs10765050 | rs11017816 |
rs10765051 | rs2944477 | rs2944478 | rs11146138 | rs11146419 | rs11146457 | rs2070675 | rs2178798 |
rs747302 | rs12286769 | rs10836510 | rs3750944 | rs11040966 | rs7483922 | rs4758126 | rs12365491 |
rs10769768 | rs7102305 | rs3794018 | rs11041797 | rs3911309 | rs3911310 | rs11042076 | rs2568045 |
rs11042149 | rs4396289 | rs2403264 | rs7947588 | rs10840531 | rs11021780 | rs16910133 | rs1455250 |
rs10831675 | rs11022043 | rs6485257 | rs11606956 | rs7947202 | rs4757965 | rs10766039 | rs7937060 |
rs4757151 | rs11022781 | rs10766106 | rs2351958 | rs9988855 | rs6486303 | rs10832659 | rs7111945 |
rs471465 | rs11500050 | rs7127040 | rs4757045 | rs7117347 | rs10430902 | rs10833407 | rs7103926 |
rs4570579 | rs7480684 | rs11027554 | rs2716542 | rs1159087 | rs7930199 | rs11029955 | rs7933639 |
rs722608 | rs7928133 | rs4923495 | rs9300002 | rs2933163 | rs690444 | rs4923595 | rs10836092 |
rs11032330 | rs11032331 | rs2957497 | rs10836377 | rs659479 | rs11033575 | rs4756593 | rs7950424 |
rs2067048 | rs1353465 | rs11037366 | rs3134909 | rs6485496 | rs4755920 | rs10839408 | rs10897472 |
rs3751120 | rs12281717 | rs12365443 | rs7947409 | rs4944840 | rs7112131 | rs10898977 | rs10899033 |
rs689095 | rs684065 | rs11236683 | rs10899249 | rs11600791 | rs12793830 | rs482893 | rs34359544 |
rs10792603 | rs530158 | rs2040309 | rs556942 | rs10765449 | rs4501950 | rs1542932 | rs9633810 |
rs500558 | rs10831243 | rs7110167 | rs7111231 | rs4354655 | rs606227 | rs6483473 | rs496852 |
rs4754526 | rs11222495 | rs12291672 | rs7931713 | rs10750677 | rs1021290 | rs1483513 | rs11226633 |
rs4754184 | rs326948 | rs2053002 | rs7926635 | rs6589300 | rs4938017 | rs2852435 | rs10891655 |
rs4438073 | rs4938221 | rs948074 | rs10891936 | rs11215864 | rs6589535 | rs2542052 | rs675929 |
rs2508806 | rs513136 | rs10790598 | rs7948511 | rs12806239 | rs11222406 | rs11222496 | rs11223305 |
rs619091 | rs4290247 | rs10894899 | rs4937979 | rs4936258 | rs1316723 | rs722097 | rs7298096 |
rs7303748 | rs7311151 | rs4765687 | rs11062373 | rs7315917 | rs4766100 | rs7308842 | rs7965200 |
rs4765746 | rs4766152 | rs7300031 | rs4766227 | rs4766286 | rs7305525 | rs7308687 | rs10849273 |
rs4930767 | rs3782583 | rs7314204 | rs216885 | rs4764485 | rs4329748 | rs7306824 | rs7958782 |
rs7310448 | rs11043862 | rs2970174 | rs717178 | rs252020 | rs4883241 | rs4763630 | rs4764108 |
rs4764327 | rs7975186 | rs4763707 | rs974728 | rs7300814 | rs6488494 | rs6488518 | rs4763890 |
rs4763305 | rs4763947 | rs730457 | rs4763999 | rs7301500 | rs6488624 | rs7310659 | rs7312042 |
rs7303424 | rs10772914 | rs2975149 | rs7304699 | rs7304811 | rs4619200 | rs4323926 | rs6486835 |
rs10840805 | rs10840837 | rs10431209 | rs4565957 | rs7973335 | rs4609650 | rs7969692 | rs10841141 |
rs7296301 | rs7484891 | rs7975918 | rs4762956 | rs4502032 | rs7300210 | rs7294946 | rs4149022 |
rs7135717 | rs4578491 | rs7302352 | rs1037922 | rs4931075 | rs7310809 | rs10771360 | rs4930945 |
rs723036 | rs7970149 | rs7310157 | rs11050058 | rs4931215 | rs11050438 | rs10843509 | rs10743677 |
rs10771604 | rs10771657 | rs10843652 | rs1485414 | rs11052762 | rs10844643 | rs12320504 | rs2730911 |
rs11168782 | rs2388058 | rs11169407 | rs7312898 | rs10878358 | rs7298930 | rs7307562 | rs7306163 |
rs4768418 | rs11181607 | rs7316154 | rs7308182 | rs7962449 | rs4768560 | rs2218943 | rs4768575 |
rs4601845 | rs7301594 | rs7487075 | rs6582656 | rs4768139 | rs4760648 | rs4760685 | rs4762040 |
rs7305599 | rs10876254 | rs7308751 | rs695003 | rs4761944 | rs1976935 | rs4759062 | rs7310681 |
rs1317996 | rs7297662 | rs1874888 | rs7302420 | rs1800187 | rs1800191 | rs1800165 | rs1140648 |
rs7956957 | rs7977908 | rs11172190 | rs7304290 | rs7961221 | rs7968490 | rs2655886 | rs2655888 |
rs2655879 | rs4758868 | rs4340119 | rs7304109 | rs7297618 | rs7310834 | rs7959278 | rs4762096 |
rs1152908 | rs1152909 | rs1168329 | rs1168292 | rs1168308 | rs1168321 | rs9738014 | rs2920039 |
rs12427352 | rs7398135 | rs4761162 | rs7307739 | rs710681 | rs438847 | rs7958846 | rs12321332 |
rs7305075 | rs1280602 | rs10748196 | rs10784970 | rs10082992 | rs12582138 | rs7316080 | rs7311420 |
rs7314330 | rs11180473 | rs12366348 | rs7300308 | rs7297654 | rs6538115 | rs7304242 | rs4761363 |
rs4761378 | rs7302948 | rs11106845 | rs11106871 | rs1609977 | rs7315214 | rs7960886 | rs4842423 |
rs7309071 | rs7304484 | rs4882436 | rs7296849 | rs4882445 | rs4578433 | rs10862864 | rs4842684 |
rs4842697 | rs4842699 | rs4842509 | rs7138906 | rs4399376 | rs1489895 | rs4567507 | rs2099618 |
rs4761590 | rs7296951 | rs1553596 | rs7138694 | rs12582251 | rs11108609 | rs11108786 | rs6538775 |
rs4762486 | rs934930 | rs12227960 | rs7301622 | rs12371279 | rs11110023 | rs4764997 | rs4764755 |
rs4764776 | rs7294778 | rs7310931 | rs903248 | rs4409923 | rs7311274 | rs12832619 | rs11112810 |
rs7306232 | rs1346173 | rs7313861 | rs246079 | rs4766584 | rs752015 | rs4766522 | rs4509829 |
rs2464288 | rs9989025 | rs4144711 | rs12321350 | rs12422982 | rs7305258 | rs4450210 | rs7311627 |
rs12422915 | rs4767374 | rs4767408 | rs11068055 | rs7314617 | rs4767430 | rs4766807 | rs4492907 |
rs4766834 | rs7295972 | rs4767535 | rs10774972 | rs4767643 | rs4767658 | rs7301936 | rs4767788 |
rs4766939 | rs4384428 | rs7315003 | rs433091 | rs4766950 | rs3809313 | rs1151849 | rs7296507 |
rs10840615 | rs10846616 | rs4144457 | rs917059 | rs4765311 | rs7296549 | rs4761001 | rs7297975 |
rs4882706 | rs7310152 | rs7960920 | rs7310927 | rs470609 | rs7309864 | rs7310081 | rs11060524 |
rs4760040 | rs4759721 | rs4759493 | rs7138800 | rs10848298 | rs7133251 | rs4759856 | rs10848299 |
rs10848300 | rs12809203 | rs7300298 | rs7314138 | rs4759915 | rs11061466 | rs7138697 | rs4883614 |
rs7489112 | rs4883577 | rs7983740 | rs4770114 | rs1579374 | rs942873 | rs17081058 | rs9511657 |
rs9507469 | rs2441067 | rs9512449 | rs1778797 | rs4372556 | rs9547056 | rs1012129 | rs11839815 |
rs589524 | rs2182444 | rs4494418 | rs1924313 | rs7994850 | rs11147782 | rs4943746 | rs9549172 |
rs1885270 | rs9549214 | rs1009823 | rs7333648 | rs2147354 | rs1175196 | rs7317245 | rs9562698 |
rs9567872 | rs2766097 | rs2897689 | rs9596430 | rs7337462 | rs990157 | rs6561751 | rs7320685 |
rs1436703 | rs9569256 | rs9563309 | rs7319488 | rs9597145 | rs9537053 | rs7989059 | rs9537059 |
rs7327180 | rs6561900 | rs4616179 | rs17058611 | rs12585457 | rs9599133 | rs12871277 | rs12323259 |
rs9317762 | rs12429958 | rs2181996 | rs17837307 | rs4885015 | rs9573312 | rs1326072 | rs9600911 |
rs7991594 | rs9574312 | rs563445 | rs507869 | rs7324020 | rs1010392 | rs9545653 | rs9565787 |
rs9575121 | rs4884222 | rs7336097 | rs9588629 | rs11617299 | rs2349955 | rs11069953 | rs2805687 |
rs1907428 | rs4773670 | rs16947580 | rs7322391 | rs12866697 | rs9525158 | rs9590373 | rs9525160 |
rs9516644 | rs9556813 | rs7333055 | rs1058143 | rs7994234 | rs9557494 | rs7995465 | rs4772500 |
rs9585995 | rs4150360 | rs9558055 | rs7991470 | rs7328280 | rs1079772 | rs9519692 | rs4771519 |
rs9558670 | rs353768 | rs11616946 | rs729549 | rs9555612 | rs7324175 | rs2005632 | rs7328030 |
rs9550057 | rs7334530 | rs9562080 | rs4058351 | rs8017588 | rs4982495 | rs12888199 | rs1983518 |
rs928955 | rs17255440 | rs12434279 | rs12886508 | rs10140328 | rs10147992 | rs10129348 | rs8003702 |
rs7158688 | rs10150235 | rs6574836 | rs1950421 | rs962075 | rs4983181 | rs203594 | rs11620936 |
rs11160611 | rs11160612 | rs7160283 | rs2378971 | rs6571509 | rs10483397 | rs10134645 | rs8004791 |
rs4267220 | rs2383437 | rs2383438 | rs8014514 | rs10131549 | rs11623701 | rs11156807 | rs7359105 |
rs4982274 | rs9322953 | rs4904155 | rs2073244 | rs4904210 | rs4901272 | rs969446 | rs10141957 |
rs10162550 | rs11624150 | rs4902768 | rs4902773 | rs1955711 | rs12881979 | rs8011258 | rs7151249 |
rs10134396 | rs10134408 | rs4294728 | rs7157735 | rs11625182 | rs11625089 | rs11625126 | rs4904207 |
rs4899858 | rs1959938 | rs1959941 | rs4144001 | rs10135079 | rs11627668 | rs7146737 | rs17115412 |
rs4494462 | rs7151968 | rs17119821 | rs17120840 | rs4131981 | rs7140155 | rs12879891 | rs11627579 |
rs2761884 | rs2855530 | rs4898820 | rs911621 | rs6573042 | rs10136408 | rs12891489 | rs12889725 |
rs8004472 | rs389596 | rs389413 | rs2348875 | rs6573290 | rs12879559 | rs978511 | rs1387673 |
rs4902147 | rs2355168 | rs10137624 | rs11624884 | rs5010674 | rs10162413 | rs4584750 | rs8022730 |
rs8017368 | rs7157513 | rs3951150 | rs4411434 | rs1255976 | rs12887115 | rs7142757 | rs8005241 |
rs12590176 | rs985956 | rs12433645 | rs17111151 | rs10129919 | rs746629 | rs2210175 | rs7156394 |
rs10133593 | rs727258 | rs9323861 | rs7141239 | rs7142471 | rs2025030 | rs4905157 | rs12434757 |
rs4509952 | rs8009218 | rs1190964 | rs1190966 | rs1190967 | rs1743623 | rs11629252 | rs7149353 |
rs11160634 | rs2400994 | rs6575836 | rs7145753 | rs7148567 | rs10144051 | rs6575986 | rs7144271 |
rs10148970 | rs11160751 | rs10162425 | rs11620897 | rs11627446 | rs55658071 | rs1138400 | rs7147664 |
rs2403192 | rs942862 | rs4906421 | rs10438245 | rs7145300 | rs66798104 | rs7150731 | rs7161740 |
rs4293342 | rs4778368 | rs7164801 | rs7174637 | rs2732035 | rs4344720 | rs8033133 | rs3742952 |
rs7496840 | rs4260026 | rs4467042 | rs487586 | rs486535 | rs6576560 | rs4906908 | rs4283207 |
rs4778189 | rs4778192 | rs12592625 | rs4779952 | rs4780054 | rs4780212 | rs4780082 | rs7172763 |
rs2278313 | rs8027209 | rs8027570 | rs6495681 | rs7175617 | rs7180592 | rs7173992 | rs4923979 |
rs4923980 | rs4299106 | rs8030155 | rs28667981 | rs4506848 | rs4294776 | rs4924223 | rs4924268 |
rs1371962 | rs9806435 | rs8031241 | rs2576932 | rs4924389 | rs1166722 | rs1166723 | rs16970474 |
rs7178233 | rs12593359 | rs1017518 | rs4774770 | rs4774224 | rs4363807 | rs4775256 | rs7164700 |
rs4775777 | rs3098174 | rs7174839 | rs12592778 | rs10459592 | rs4143723 | rs4545756 | rs4775961 |
rs4238383 | rs11070877 | rs3751603 | rs4776110 | rs8025815 | rs4774662 | rs6493661 | rs12441013 |
rs4774706 | rs951048 | rs7163897 | rs1528461 | rs12906506 | rs4310791 | rs8030835 | rs4143225 |
rs4775032 | rs4774308 | rs9646204 | rs17302247 | rs4775162 | rs4775165 | rs4775175 | rs10851660 |
rs4775214 | rs4774349 | rs4519291 | rs4775260 | rs2414686 | rs28579202 | rs10162779 | rs466049 |
rs448458 | rs4775517 | rs4775519 | rs7350775 | rs10519186 | rs4619348 | rs11071863 | rs4776831 |
rs4776862 | rs4777110 | rs745636 | rs488156 | rs4075802 | rs4887161 | rs4886844 | rs4886574 |
rs351156 | rs7182283 | rs6495174 | rs4886704 | rs4886705 | rs4430707 | rs4073214 | rs28607641 |
rs11636251 | rs4886755 | rs11072567 | rs4886485 | rs7179157 | rs12910273 | rs8032156 | rs4539564 |
rs4344704 | rs4778879 | rs28412916 | rs4778882 | rs4778651 | rs4779093 | rs12914832 | rs3848180 |
rs7166570 | rs7174089 | rs8035516 | rs8036531 | rs17768867 | rs4842890 | rs4843075 | rs49363 |
rs4843118 | rs4887197 | rs4887198 | rs16948824 | rs4243100 | rs2594328 | rs1433453 | rs11639000 |
rs2701365 | rs2701367 | rs4522384 | rs4932444 | rs2072267 | rs4702 | rs2386590 | rs12905835 |
rs12912474 | rs11073974 | rs4429270 | rs4525467 | rs12595110 | rs11639135 | rs8035412 | rs4777900 |
rs7171883 | rs59048995 | rs4777763 | rs8036747 | rs12591541 | rs4426305 | rs11074249 | rs12594904 |
rs290640 | rs290641 | rs12901554 | rs1036531 | rs4519318 | rs4965499 | rs4534823 | rs4273018 |
rs2727108 | rs8030813 | rs4246331 | rs62031069 | rs11248851 | rs761068 | rs1657143 | rs4786427 |
rs11076804 | rs2238457 | rs4786015 | rs4786026 | rs12922652 | rs10492834 | rs12447523 | rs4786777 |
rs6500721 | rs8059231 | rs1529921 | rs1865815 | rs4786069 | rs4786794 | rs34241869 | rs17140100 |
rs17140285 | rs4337300 | rs4520856 | rs4627375 | rs8045516 | rs17144146 | rs4787102 | rs4346186 |
rs11867018 | rs4780910 | rs4780486 | rs8061640 | rs4781019 | rs4781025 | rs4781070 | rs4781079 |
rs4781083 | rs4781096 | rs4628985 | rs4781110 | rs4781128 | rs4781223 | rs4510028 | rs2269446 |
rs4780469 | rs4353467 | rs3097191 | rs4513082 | rs4781357 | rs4780514 | rs1859057 | rs6497354 |
rs6497435 | rs1925503 | rs13335898 | rs4783503 | rs2239340 | rs4787924 | rs4787646 | rs9936902 |
rs891122 | rs4787297 | rs4787697 | rs4787701 | rs12931685 | rs205119 | rs4270178 | rs4390598 |
rs4404074 | rs4488450 | rs12448645 | rs4787393 | rs4494544 | rs4787915 | rs2289292 | rs11150628 |
rs4785369 | rs4416010 | rs4643309 | rs4457988 | rs4785098 | rs4783780 | rs6499091 | rs4783799 |
rs8063722 | rs4784376 | rs4622506 | rs4784465 | rs42313 | rs12922657 | rs4566154 | rs601194 |
rs1975629 | rs3743560 | rs11640080 | rs4335761 | rs7193838 | rs6498790 | rs11860558 | rs8047731 |
rs2407903 | rs8060455 | rs9302537 | rs12934454 | rs4429312 | rs4949128 | rs4353494 | rs4444361 |
rs233537 | rs4783718 | rs4788489 | rs4889197 | rs12447957 | rs7204888 | rs13337159 | rs11644674 |
rs4319778 | rs4887974 | rs3743683 | rs1110553 | rs7193219 | rs10514450 | rs4545829 | rs4427815 |
rs4888116 | rs4503787 | rs4888158 | rs12447549 | rs4889356 | rs4329917 | rs4328435 | rs4782642 |
rs4783222 | rs28378230 | rs4782742 | rs4783317 | rs7199681 | rs7192553 | rs4782749 | rs4782525 |
rs4782791 | rs34232206 | rs35215168 | rs4541064 | rs4782705 | rs9923691 | rs1800647 | rs4843926 |
rs299971 | rs182461 | rs4843477 | rs11646219 | rs4843529 | rs4843628 | rs7191387 | rs7191581 |
rs7500034 | rs4782511 | rs4782338 | rs4782395 | rs460879 | rs9747475 | rs8075977 | rs2733364 |
rs10852883 | rs2585405 | rs268449 | rs12937386 | rs10852912 | rs4275899 | rs9912329 | rs8079524 |
rs4792249 | rs4791495 | rs12937774 | rs4273078 | rs8080686 | rs10445273 | rs4614770 | rs2215083 |
rs12949495 | rs4315376 | rs6565410 | rs34434101 | rs7225645 | rs1975980 | rs8076645 | rs1829662 |
rs10459955 | rs1402724 | rs67290727 | rs7217537 | rs4471732 | rs8067440 | rs2854334 | rs1124838 |
rs114179497 | rs2470229 | rs396996 | rs373614 | rs8081280 | rs12603249 | rs4795924 | rs6503738 |
rs7213323 | rs2941503 | rs55999837 | rs11079199 | rs271661 | rs11079962 | rs8070947 | rs7503772 |
rs1800764 | rs17688563 | rs8068873 | rs9905519 | rs4254382 | rs2715828 | rs56023694 | rs9895895 |
rs4788853 | rs2620065 | rs8080915 | rs8076766 | rs11870955 | rs2598450 | rs4647887 | rs11868709 |
rs9900690 | rs11077415 | rs12600665 | rs1115834 | rs9319620 | rs12966458 | rs1032978 | rs11080902 |
rs1510026 | rs12454079 | rs12607181 | rs6506166 | rs595601 | rs7241235 | rs513953 | rs7239119 |
rs7228637 | rs16976241 | rs9965555 | rs3760550 | rs7227361 | rs1284408 | rs2155975 | rs10853460 |
rs11661488 | rs299255 | rs12605341 | rs12956367 | rs4800438 | rs7243495 | rs9946004 | rs7359704 |
rs525218 | rs9945987 | rs17720996 | rs11876474 | rs4482379 | rs9958174 | rs1602746 | rs12454084 |
rs12456568 | rs12969183 | rs12969524 | rs4293450 | rs4477797 | rs71372008 | rs11661698 | rs11661674 |
rs34436728 | rs35324628 | rs9630798 | rs12456276 | rs12606747 | rs1546103 | rs2055058 | rs12457108 |
rs67870886 | rs12458036 | rs11083351 | rs11083352 | rs1492806 | rs4436850 | rs4799615 | rs539773 |
rs12970933 | rs9963299 | rs12969289 | rs6507615 | rs4602126 | rs34093107 | rs2665561 | rs9958074 |
rs9965805 | rs3730668 | rs2276182 | rs4801031 | rs652402 | rs17833707 | rs11152081 | rs7236170 |
rs1012790 | rs9989529 | rs8084931 | rs1016366 | rs6566286 | rs12605850 | rs9945048 | rs7350994 |
rs8088842 | rs7236326 | rs4145852 | rs4530251 | rs10514047 | rs2702602 | rs2404869 | rs4891250 |
rs7240363 | rs4891252 | rs6566067 | rs6566069 | rs7229426 | rs11660762 | rs3898620 | rs7243155 |
rs624864 | rs17060086 | rs4890807 | rs13381082 | rs13381933 | rs1944001 | rs12458930 | rs4919838 |
rs2285853 | rs1056766 | rs4807395 | rs4807249 | rs4807291 | rs7246554 | rs4807567 | rs7256790 |
rs2287916 | rs12986314 | rs4804252 | rs4804386 | rs11669128 | rs1117268 | rs9305089 | rs10404348 |
rs8113029 | rs4804523 | rs4926199 | rs4926207 | rs4926214 | rs4808272 | rs7258240 | rs6512050 |
rs4808552 | rs4808069 | rs398668 | rs4808839 | rs1725932 | rs7256690 | rs12461406 | rs4805012 |
rs4806397 | rs4805218 | rs59345679 | rs4805303 | rs11083866 | rs741448 | rs8113496 | rs4805420 |
rs7246164 | rs8110447 | rs4805496 | rs4805590 | rs4804938 | rs2867078 | rs4805612 | rs4805677 |
rs407470 | rs10425595 | rs10424494 | rs4805827 | rs2216595 | rs7259371 | rs4528686 | rs8106453 |
rs7256182 | rs7256328 | rs12461297 | rs28840147 | rs4806155 | rs2733738 | rs4806187 | rs2242523 |
rs2967436 | rs12609806 | rs31728 | rs79666308 | rs7257602 | rs454904 | rs11083735 | rs1996409 |
rs8110348 | rs8109615 | rs4508518 | rs7253485 | rs4802208 | rs10853771 | rs4803792 | rs6509177 |
rs171140 | rs238405 | rs11673207 | rs4802388 | rs11083897 | rs11670990 | rs156640 | rs12984666 |
rs10420303 | rs2569478 | rs7259815 | rs9676916 | rs11671293 | rs7246847 | rs11881170 | rs2277779 |
rs6132436 | rs7273128 | rs6052751 | rs3729558 | rs4621230 | rs12625819 | rs4300912 | rs4816043 |
rs727684 | rs6056748 | rs2423468 | rs684169 | rs6078344 | rs13037956 | rs1223271 | rs16994839 |
rs7272683 | rs911135 | rs3790230 | rs6046143 | rs4814865 | rs3748486 | rs6035682 | rs6047212 |
rs6035877 | rs2024885 | rs2180356 | rs6013469 | rs6128822 | rs6029251 | rs1008069 | rs6029904 |
rs4239693 | rs6130136 | rs2903624 | rs2206465 | rs2206466 | rs6073063 | rs2235765 | rs4812744 |
rs4810415 | rs2239533 | rs7263437 | rs2426046 | rs226799 | rs421630 | rs4810824 | rs3091976 |
rs6012846 | rs232735 | rs2063539 | rs6097713 | rs958026 | rs6025094 | rs6014831 | rs8115191 |
rs1883799 | rs6026220 | rs6089455 | rs7271235 | rs2296086 | rs856952 | rs12479846 | rs7271530 |
rs117510755 | rs2155965 | rs4588273 | rs2822651 | rs2253231 | rs13050296 | rs2825496 | rs9980963 |
rs8132277 | rs7279915 | rs1786432 | rs2212626 | rs2826734 | rs2826737 | rs1980969 | rs2826949 |
rs2826963 | rs1735945 | rs244230 | rs219614 | rs462290 | rs13050132 | rs845017 | rs844974 |
rs844975 | rs915534 | rs2252828 | rs6517222 | rs947919 | rs12233305 | rs724545 | rs1209932 |
rs2070529 | rs2070530 | rs9975866 | rs7282360 | rs2837747 | rs1573415 | rs9976560 | rs1006757 |
rs2838441 | rs17004706 | rs2518751 | rs1867353 | rs369250 | rs5752500 | rs400946 | rs439792 |
rs5761533 | rs5752370 | rs5752495 | rs5762861 | rs8137254 | rs5998042 | rs2051564 | rs242901 |
rs242999 | rs4821237 | rs443705 | rs5755308 | rs2223432 | rs9610629 | rs8141354 | rs2267377 |
rs138457 | rs1980455 | rs735306 | rs760726 | rs9607599 | rs6001359 | rs2142833 | rs6001363 |
rs9611070 | rs7286979 | rs6002260 | rs2281330 | rs2294979 | rs5751085 | rs2267435 | rs133330 |
rs5758589 | rs695810 | rs695553 | rs16992816 | rs5012765 | rs3788629 | rs41487149 | rs5768019 |
rs7286517 | rs5767076 | rs34189568 | rs9616585 |
(二)实验思路及一般操作步骤
本实验方法思路如图1所示,可分为两块:
1、对器官移植患者的受体样本进行目标区域捕获测序,用于基因分型,基因分型实验步骤如图2所示;移植后受体血液样本分离的血细胞充当移植前受体样本,即受体基因组样本。
2、对移植后受体各个采血点的血浆cfDNA样本进行目标区域捕获和高深度测序,用
于分析评估各采血点血浆中供体cfDNA占总cfDNA的百分比,如图3所示。
以上各块的具体实验操作步骤如下:
1、基因组目标区域捕获进行SNP分型
基因组SNP分型实验流程如图2所示。先取1μg基因组DNA打断成主带为小片段DNA,打断后DNA片段进行末端补平,加接头,构建完成的文库,通过上述自主设计合成的液态芯片将目标区域进行富集,然后通过PCR扩增后纯化产物即可用于测序分析,其具体步骤如下:
1.1外周血样本基因组DNA的提取;
1.2取1μg基因组DNA,超声波打断为小片段DNA(随测序仪建库的不同而变化);
1.3把打断成小片段的DNA修复成平末端;
1.4加上接头后,用琼脂糖电泳切胶法选择DNA片段大小;
1.5PCR扩增目地片段,用芯片进行SNP位点捕获;
1.6PCR扩增,Agilent 2100检测文库质量合格后,进行高通量测序。
2、各采血点的血浆cfDNA检测
血浆cfDNA检测实验流程如图3所示。各采血点血浆分离后,提取cfDNA、末端修复、加接头、PCR扩增,自主合成芯片杂交,杂交产物通过PCR扩增,纯化后的产物即可用于测序分析,具体步骤如下:
2.1两步离心法分离各采血点血浆,避免基因组污染,提取血浆cfDNA;
2.2将cfDNA修复成平末端;
2.3加上接头并PCR扩增目地片段,用芯片进行SNP位点捕获;
2.4PCR扩增捕获的片段,Agilent 2100检测文库质量合格后,进行高通量测序。
二、获得测序数据后,将数据分析方法编写成一软件包,软件包实现的内容一般包括以下:
1.与参考基因组比对。以BGISEQ-100测序平台为例,对BGISEQ-100有效测序数据使用tmap工具比对到参考基因组上,得到精确的比对结果。其中tmap工具源自:https://github.com/iontorrent/TS/tree/master/Analysis/TMAP。其他测序平台候选比对工具有Burrows-Wheeler Aligner(BWA,参考文献:Li H.and Durbin R.(2009)Fast and accurate short read alignment with Burrows-Wheeler Transform.Bioinformatics,25:1754-60.)、Bowtie(参考文献:Langmead B,et al.Ultrafast and memory efficient alignment of short DNA sequences to the human genome.Genome Biol 10:R25.)等。
2.比对结果去除PCR重复片段。以BGISEQ-100测序平台为例,对tmap工具比对后的结果(bam格式)使用BamDuplicates工具去除PCR重复片段。其中,BamDuplicates工具源自Ion Torrent Systems,Inc.,其他测序平台候选去重工具有samtools rmdup及Picard MarkDuplicates(工具官网地址:http://broadinstitute.github.io/picard/index.html)等。
3.统计及质量控制。统计目标区域数据量占总数据量的比例、目标区域的平均测序深度、目标区域的覆盖率等,生成一系列质控指标用于判断测序数据的质量情况。
4.受体血细胞及血浆DNA按目标SNP位点进行展开
去重后的受体血浆DNA数据仅保留唯一比对的测序数据,分别对受体血细胞及血浆去重后的数据进行samtools pileup(参考文献:Li H.,Handsaker B.,Wysoker A.,Fennell T.,Ruan J.,Homer N.,Marth G.,Abecasis G.,Durbin R.and 1000Genome Project Data Processing Subgroup(2009)The Sequence alignment/map(SAM)format and SAMtools.Bioinformatics,25,2078-9.[PMID:19505943].软件官网地址:http://samtools.sourceforge.net/index.shtml),对pileup结果进行每个位点支持不同碱基的reads数进行统计。
前4步对进行目标区域捕获测序的受体血细胞及血浆样本均适用。对于血浆样本,在第3步去重后,还需去掉多比对的reads,只获取唯一比对的reads。
5.受体血细胞DNA样本基因分型
以BGISEQ-100测序平台为例,使用TVC工具(默认参数targetseq_germline_lowstringency_p1_parameters.json文件)(参考:http://ioncommunity.lifetechnologies.com/community/products/torrent-variant-caller)分别检测受体血细胞(或组织)的遗传性SNP(Germline SNP),得到部分基因分型位点。对TVC工具无法分型的位点,通过频率即支持reads的比例来分型,最大化基因分型位点,同时进行降噪处理,仅保留高质量的基因分型结果。其他测序平台候选基因分型工具有GATK(软件获取地址:https://www.broadinstitute.org/gatk/index.php)等,基因分型的具体操作步骤为:
(1)使用TVC工具对受体血细胞DNA数据进行分型,测序深度阈值为6。
(2)统计每个位点的基因型。对于TVC工具无法分型的位点,根据统计等位基因频率来分型。将频率>95%的位点定义为纯合子,将频率在25%(包含25%)至95%(包含95%)间的位点定义为杂合子。对于有多种基因型的杂合子,取最大频率的两种碱基作为其基因型。
(3)仅保留等位基因频率为100%、测序深度大于等于平均测序深度的纯合子位点。
6.统计受体血浆特定位点频率统计
在保留的受体血细胞纯合子位点中,统计受体血浆DNA中不同等位基因的频率,获取血浆频率列表。要求血浆中最多允许2种等位基因,并且不同等位基因的reads支持数至少为2条。
7.受体血浆中供体比例统计
与受体血细胞不同的等位基因碱基大部分可认为来自供体,极小一部分是由于测序或比对错误导致,而供体在该位点可能为杂合子或者纯合子,具体表型未知,可通过频率来判断。以10%供体比例、1000X平均测序深度的血浆样本为例,若在某位点中参考碱基为A,受体血细胞全部为A碱基的reads,受体血浆存在T碱基reads,T碱基reads可能来自供体。若供体为杂合子,则T支持reads条数理应为5,检测T的频率比例为5%,A支持reads条数理应为995,检测A的频率比例为95%;若供体为纯合子,则T支持reads条数理应为10,检测T的频率比例为10%,A支持reads条数理应为990,检测A的频率比例为90%。基于每个可区分供受体位点中供体的频率可代表供体含量的假设,在获取的不同等位基因频率列表中,理论上可明显聚类两类,这两类值存在两倍关系。使用K-means(k=2)聚类,将频率按均值迭代聚成两类,使用均值进行卡方检验,判断两倍关系的显著性,输出两类均值。
8.生成报告。对质控(QC),供体比例结果进行汇总生成一张Excel表,数据解读人员在此Excel表格基础上对数据进行解读。
图4显示以上数据分析流程。
目前已有的高通量测序检测混合cfDNA样本中的比例相对少的供体的cfDNA含量的方法或成本过高,或依赖于供体遗传样本。本发明提出一种技术路线为:1)对移植后受体血液样本进行血浆分离,受体血细胞(或组织)DNA通过目标区域捕获测序进行基因分型,保留纯合子位点;2)对移植后受体血浆DNA进行目标区域捕获测序,统计受体血细胞为纯合子的位点中不同等位基因的频率,通过K-means聚类及卡方检验等模型,计算其中的供体cfDNA的比例。本发明将该技术路线编码成一体化操作、可独立部署、高效运行的软件包,目的之一在于提供一种基于高通量测序法的无供体依赖性的器官移植免疫排斥监测的分析方法及软件包。
综合以上,该示例设计了一个新的芯片和相应的实验方法以及数据分析检测方法,至少有以下四个方面的有益效果:
(1)无需供体样本即可进行检测,应用广泛;运用高通量测序技术的检测方法中,首次通过统计受体血细胞为纯合子的位点中不同等位基因的频率进行免疫排斥检测,摆脱了
对供体样本的依赖,特别适用于追溯供体样本有一定难度的器官移植患者。因而本实验方法应用更加广泛,能够辅助检测多种器官移植免疫排斥反应如肺移植、心脏移植、肝移植、肾移植等。
(2)检测结果更加准确。本实验方法要求高深度的捕获测序,检测值能准确的反映血浆cfDNA中真实供体含量,使结果更加准确;而且,首次提出可区分供受体位点中供体的频率可反映供体含量的假设,创新性地运用等位基因频率检测的方法,可以取代传统加权公式,来计算供体cfDNA比例,检测值准确反映真实供体cfDNA含量。对移植后血浆进行高深度的捕获测序,而非传统超低深度的全基因组测序,以及严格的降噪处理,检测供体比例更准确。
(3)数据分析方法可利用灵活、一体化的软件包实现,可独立部署、高效运行。
(4)低创或无创的检测、可接受的成本,可作为一种便捷、早期、无创、准确的移植排斥监测技术,可作为临床免疫排斥检测的辅助或补充手段。
实施例二
实施例设计思路如下:取2个正常人血样(取自志愿者),一个为供体,另一个为受体,混合出待测样本,进行模拟实验。采取的血样分离血细胞与血浆,受体血细胞(无需供体血细胞)提取基因组DNA后,打断DNA并进行目标区域捕获测序,用于基因分型;供体和受体血浆提取cfDNA后,Agelint 2100测定其浓度,供受体的cfDNA按3.5%、5.5%、8%、10%比例人为混合,然后将混合的cfDNA建库捕获测序(本实施例所用测序仪为BGISEQ-100测序平台),用以检测本实验方法的可靠性。按照实施例一中的实验步骤,本实施例步骤也分为两步,1、受体基因组目标区域捕获测序;2、各混合cfDNA目标区域捕获测序。具体如下:
实施例中接头、PCR扩增引物由Invitrogen公司合成,所使用的C0T1DNA购买于Invitrogen公司。所用试剂信息如下表所示:
1、基因组目标区域捕获测序
1)分离血浆和血细胞
①抗凝管取血(5ml)后,颠倒混匀5-6次充分混匀;
②水平离心机,1600g,4℃离心10min;
③将上清(约1.5ml)分装到2ml管中,下层即为血细胞;
④16000g,4℃离心10min去除残余细胞,将上清转放新的1.5ml管中,标记后-80℃保存。
2)基因组DNA的提取
取200μl分离的血细胞进行基因组DNA提取,具体步骤参见试剂盒说明书。
3)样品打断(Fragmentation)
①在Bioruptor仪制冷水槽中加Milli-Q的水,水面介于MAX线与MIX线之间;
②开启制冷开关并浆制冷仪的温度设置为4℃;
③当制冷水槽的水温达到4℃时开启Bioruptor仪器,制冷水槽中的水会被运送到打断运行槽中,并开始循环流动;
④用Nuclease-free water或(1×TE)将1μg的g DNA稀释到100μL,混匀后用移液器小心的转入打断小管中;点击“set”,按下表设置参数:ON 30s,OFF 30s,5个Cycle;将打断管放入打断转盘装置,并放进打断槽中。点击“run”按钮,盖上仪器盖子,样本开始打断;仪器停止后将样品取出,涡旋振荡10s混匀瞬离后冰浴3min,重复步骤④共6次;
⑤取2μL的样品用于电泳检测打断效果,主带位于150-250bp左右视为合格。
4)打断后DNA的纯化(Agencourt AMPure beads)
①使用前将磁珠置于室温下平衡30min;
②将100μL打断后的DNA转入1.5mL的EP管中,加入1.8倍体积的磁珠(180μL),用移液器吹打10次混匀;
③室温下静置10min使磁珠与DNA充分结合,然后瞬时离心3秒;
④将EP管放到磁力架上至液体澄清,用移液器小心的去除上清;
⑤保持EP管在磁力架上,加入500μL 70%的乙醇洗涤磁珠表面,以除去盐离子以及未吸附的DNA等,除去乙醇,重复一次;
⑥瞬时离心,尽量完全去除乙醇,并将磁珠置于开盖置于磁力架上,至磁珠表面没有光泽(约10分钟);
⑦加入25μL Elution Buffer,轻轻将磁珠从管壁冲洗下来并吹打10次混匀;
室温静置10min以使DNA完全从磁珠上洗脱下来;
⑧将EP管放到磁力架上至液体澄清。将25μL洗脱下来的DNA转入一个新的EP管中。
5)末端修复
在1.5ml的离心管中配制末端修复反应体系:
上述100μL反应混合物轻微振荡混合均匀,瞬时离心,在Thermomixe或水浴锅中20℃温浴30min。
6)末端修复产物的纯化(Agencourt AMPure beads)
加入1.8倍体积的磁珠(180μL)纯化,用22μL Elution Buffer洗脱
7)Adapter的连接(Adapter Ligation)
在1.5ml的离心管中配制Adapter连接反应体系,体系如下表。
上述100μL反应混合物轻微振荡混合均匀,瞬时离心后置于Thermomixer中20℃温浴15min。
8)Agencourt AMPure beads纯化连接产物
加入1.5倍体积的磁珠(150μL)纯化,用32μL Elution Buffer洗脱
9)片段选择
①每个样品称取1.3g的琼脂糖于65ml的1×TAE中;
②点样之前加入1μl上样缓冲液再次检查胶孔是否漏液;
③使用NEB 50bp DNA Ladder,须取出1μl与2μl上样缓冲液充分混匀后点样;
④将来自步骤5.8的样品分别至少与10μl上样缓冲液充分混合;
⑤先将步骤1的第3)步所得到的样品跑入胶中,再100V电压下电泳120min;
⑥100ml的电泳缓冲液1×TAE,加入10μl核酸染料EB充分混匀待用;
⑦电泳结束后取出凝胶,放到染胶盘中染色10min;
⑧在凝胶系统中拍照存档;
⑨以Marker为参照,切230bp-250bp回收,再分别切210bp-230bp和250bp-270bp作为备份;
⑩完成切胶后将剩下的胶块放在保鲜膜或PE手套上,用凝胶成像系统中拍照并存档。确认一切没有问题后可将所剩凝胶丢弃至垃圾桶中;
10)片段凝胶回收(QIAquick Gel Extraction Kit)
①往需回收的凝胶中加入6倍体积(600μl)缓冲液QG。
②50℃孵育10min,期间颠倒混匀3~5次,以帮助凝胶溶解。
③往步骤5.10.2的溶液中加入1倍体积(100μl)预冷的异丙醇,充分混匀。
④将步骤5.10.3的溶液加入到核酸吸附柱(MinElute Spin Column)中,室温静置2min,17900g离心1min。
⑤将步骤5.10.4的滤液重新加入到吸附柱中,室温静置2min,17900g离心1min,弃滤液。
⑥往吸附柱中加入500μl缓冲液QG,17900g离心1min,弃滤液。
⑦往吸附柱中加入750μl缓冲液PE,室温静置2~5min,17900g离心1min,弃滤液,重新17900g离心1min。
⑧将吸附柱转移到新的1.5ml离心管中,环吸后室温静置数分钟以晾干吸附柱中残留的液体。
⑨往核酸吸附柱的膜中间悬空加入35μl缓冲液EB,室温静置4min,17900g离心1.5min。
11)片段浓度测定(Qubit)
12)Non-Captured样品LM-PCR
在0.2mL管中配制PCR反应体系:
置于PCR仪中按照下列程序反应:
72℃ 20min,95℃ 5min,8个循环的95℃ 30s/58℃ 30s/70℃ 1min、72℃ 5min、4℃ Hold
13)PCR产物的纯化
加入1.5倍体积的Agencourt AMPure beads(150μL)纯化,用32μL Elution Buffer洗脱。
14)混合(Pooling)
将各文库等比例Pooling成750ng。
15)杂交
①将heat block调到95℃
②将分装好的4.5μLExome Library从-20℃冰箱中拿出,放在冰上化冻。
①在一个1.5mL的PE管中加入:
②盖好管盖,用干净的50ml注射器针在分装的EP管盖上戳一个孔,将上述样品文库和block的混合物置于浓缩仪中蒸干,温度设置为60℃;
③使用新的离心管管盖替换戳孔的管盖,标记,并分别加入以下两种试剂:
④将样品震荡混匀后置于离心机上全速离心10秒。将离心后样品转移至95℃heatblock中10分钟使DNA变性;
⑤将样品取出,震荡混匀后室温条件下全速离心10秒;
⑥将上述杂交混合物转入分装好的4.5μL Exome Library中;
⑦震荡混匀后置于离心机上全速离心10秒;
⑧放在PCR仪上57℃杂交24h,PCR仪热盖应设置保持在105℃;
16)捕获序列的洗涤和洗脱
①提前从冰箱中拿出链霉素磁珠,vortex磁珠1min,使其充分混匀;
②在1.5mL的EP管中加入100μL磁珠(1个样品);
③将EP管置于磁力架上至液体澄清,用移液器小心的去除上清;
④保持EP管在磁力架上,加入200μL(2倍体积)的Streptavidin Dynabead Binding and Wash Buffer;
⑤从磁力架上取下EP管,vortex 10s混匀,将EP管重新放回磁力架至液体澄清,用移液器小心的去除上清,用移液器小心的去除上清,重复洗两次;
⑥用100μL的Streptavidin Dynabead Binding and Wash Buffer悬浮磁珠,并将其转入0.2mL的小管中;
⑦用磁力架结合磁珠,直到液体澄清,用移液器小心的去除上清,现在磁珠可以用来结合捕获的DNA了。
①将杂交混合物吸出来(记录杂交后剩余体积)加到5.2准备好的磁珠中
②用移液器吹打10次混匀。
③将小管放在PCR仪上57℃孵育45min(PCR仪热盖应设置保持在105℃,每隔10min拿出来vortex 3s以防止磁珠沉淀。
①孵育45min后,将混合物从0.2mL的小管中转入1.5mL的EP管中,将EP管置于磁力架上至液体澄清,用移液器小心的去除上清。
②加100μL预热到57℃的1X Wash Buffer I,vortex 10s混匀,将EP管置于磁力架上至液体澄清,用移液器小心的去除上清
③从磁力架上取下EP管,加入200μL预热到47℃的1X Stringent Wash Buffer,用移液器吹打10次混匀。57℃孵育5min,将EP管置于磁力架上至液体澄清,用移液器小心的去除上清。重复本次操作两次,即总共用1X Stringent Wash Buffer洗三次;
④加200μL室温下放置的1X Wash Buffer I(不用47℃预热的),vortex 2min混匀,如果液体溅到管盖上,用手指轻弹EP管使其集中到管低。将EP管置于磁力架上至液体澄清,用移液器小心的去除上清;
⑤加200μL室温下放置的1X Wash Buffer II,vortex 1min混匀。将EP管置于磁力架上至液体澄清,用移液器小心的去除上清。
⑥加200μL室温下放置的1X Wash Buffer III,vortex 30s混匀。将EP管置于磁力架上至液体澄清,用移液器小心的去除上清。
⑦从磁力架上取下EP管,加入30μL ΜltraPureWater。
17)Captured样品LM-PCR
在1.5mL管中为每个样品按下表配制PCR反应体系:
置于PCR仪中按照下列程序反应。
95℃ 5min、12个循环的95℃ 15s/58℃ 15s/70℃ 1min、72℃ 2min、
4℃ Hold
①将PCR混合物(100μL)转入1个1.5mL的EP管中,将EP管放到磁力架上至液体澄清,将上清转移到一个新的EP管中,弃链霉素磁珠。
②上清中加入1.5倍体积的磁珠(150μL)进行纯化,用52μL Elution Buffer洗脱;
加入1.5倍体积的磁珠(75μL)进行纯化,用32μL Elution Buffer洗脱;
18)文库检测
使用Agilent 2100Bioanalyzer检测文库产量
2、各混合cfDNA目标区域捕获测序
1)cfDNA提取
①取200μl血浆到2ml的离心管中,加缓冲液GA到100μl终体积。
②加入20μl Proteinase K溶液,涡旋混匀。
③加入200μl的缓冲液GB,轻轻颠倒混匀,56℃孵育10min,并不时摇动样品。简短离心以去除管盖内壁的液滴。
④加入200μl的无水乙醇。如果室温超过25℃,将乙醇置冰上预冷。轻轻颠倒混匀样品,室温放置5min,简短离心以去除管盖内壁的液滴。
⑤将上一步所得溶液添加到一个吸附柱CR2中(吸附柱放入收集管中),12,000rpm离心30sec,弃废液,将吸附柱CR2放回收集管中。
⑥向吸附柱CR2中加入500μl缓冲液GD,12,000rpm离心30sec,弃废液,将吸附柱CR2放回收集管中。
⑦向吸附柱CR2中加入600μl漂洗液PW,12,000rpm离心30sec,弃废液,将吸附柱CR2放回收集管中。
⑧重复操作步骤⑦。
⑨12,000rpm离心2min,倒掉废液。将吸附柱CR2置于室温放置2-5min,以彻底晾干吸附材料中残余的漂洗液。
⑩将吸附柱CR2转入一个干净的离心管中,向吸附膜中间位置悬空滴加20-50μl洗脱缓冲液TB,室温放置2-5min,12,000rpm(~13,400×g)离心2min,将溶液收集到离心管中。
2)Qubit HS测定核酸浓度(2100检测)
3)末端修复与纯化
①按照下列的配比准备反应混合物:
在Thermomixer中20℃,反应30min。
②磁珠纯化
加入1.8倍体积的磁珠(90μL)进行纯化,用24μL Elution Buffer洗脱;
4)DNA Adaptor连接与连接产物纯化
①按照下列的配比准备反应混合物
在Thermomixer中,20℃反应20min。
②磁珠纯化
加入1.2倍体积的磁珠(84μL)进行纯化,用32μL Elution Buffer洗脱;
③Qubit测定核酸浓度
5)PCR反应与纯化
①PCR体系和反应条件,扩增体系:
反应程序:72℃20min、95℃5min、15个循环的95℃30s/60℃30s/70℃30s/70℃5min、12℃∞。
②PCR产物的磁珠纯化
加入1倍体积的磁珠(100μL)进行纯化,用32μL Elution Buffer洗脱;
③Qubit测定核酸浓度
6)后续杂交洗脱等实验步骤同基因组捕获测序
3、结果评价和分析
我们抽取两个自愿者的血液样本,一个做为供体(15ml血液),另一个做为受体(25ml血液),分离血浆和血细胞后,供体得到6.6ml血浆,7.5ml血细胞,受体得到11.4ml血浆,12ml血细胞。得到的样本用于下述实验。
3.1基因组DNA的SNP位点分型实验结果及分析
1)基因组DNA提取
取200μl分离的血细胞用于提取DNA,用Qubit进行核酸浓度检测,提取的结果如表2所示,结果显示提取正常中,可用于下步实验。
表2两志愿者的基因组DNA提取结果
2)打断、加接头及胶回收
取1μg供体基因组DNA超声波打断后,加接头并用琼脂糖电泳进行DNA片段大小选择,我们切取230-250bp和250-270bp大小的片段,其中一份作为备份,胶回收核酸浓度(Qubit检测)如表3所示,胶回收的核酸总量达到杂交捕获的要求,可进行下步实验。
表3打断后片段选择结果
3)PCR后进行液态芯片杂交捕获
进行一个PCR扩增后,取750ng进行杂交捕获:
4)出库浓度
目的序列杂交捕获下来,洗脱,进行PCR扩增后即可进行下一步的上机测序,出库浓度如下表4所示,出库浓度符合0.3K大小芯片杂交正常水平,2100结果正常,可用于测
序分析。
表4基因组DNA杂交出库结果
3.2血浆cfDNA捕获测序检测结果
我们人工模拟了3.5%、5.5%、8%、10%供体比例的实验,即把两个正常人的血浆cfDNA样本按上述比例混合在一起,然后用高通量测序进行检测。
1)血浆提取cfDNA的提取
供体用6.6ml血浆进行提取,受体用11.4ml血浆进行提取,得到的结果为表5所示,正常人的血浆cfDNA浓度较低,结果显示提取正常。
表5血浆cfDNA提取结果
2)按模拟浓度比例混合cfDNA
按我们设计的模拟浓度3.5%、5.5%、8%、10%进行混合,具体操作如下:
3)末修,加接头及PCR
混合好的血浆cfDNA,末端修复后,加上不同的接头,进行一个PCR扩增后,纯化浓度如表6所示,结果正常,可用于下步测序分析。
表6血浆建库PCR纯化结果
4)PCR后进行液态芯片杂交捕获
目地序列杂交捕获下来,洗脱,进行PCR扩增后即可进行下一步的上机测序,出库浓度如下表7所示,出库浓度符合0.3K大小芯片杂交正常水平,2100结果正常,可用于测序分析。
表7血浆cfDNA杂交出库结果
3.3结果分析与画图
上述的文库用BGISEQ-100测序平台进行测序,得到的数据通过生物信息学分析,得了各个点的供受体cfDNA比值,画成线性图后,结果如图5所示,我们可以看出,其符合线性规律(R2=0.9999),证明我们发明的不依赖供体样本的实验方法应用于监测器官移植免疫排斥的灵敏度优于普通方法。
实施例三
为验证运用等位基因频率检测方法的技术可行性,进行已知供体比例的模拟验证试验。以BGISEQ-100测序平台为例,在该实施例中选择正常受体(样品名R)血细胞样品进行目标区域捕获测序,对混合了供体血浆DNA的受体血浆同样进行目标区域捕获测序,混合比例分别为3.5%、5.5%、8%、10%,样品名分别于混合比例命名,对测序有效数据通过tmap比对、BamDuplicates去重、质量控制(QC)、受体血细胞基因分型、受体血浆频率统计、供体比例计算,最终获得4个采血点的供体含量检测报告,以评估器官移植排斥程度。
本检测系统各部流程方法都已整合到软件Donor_cfDNA中,本软件的运行环境为Unix/Linux操作系统,通过Unix/Linux命令行运行。
具体操作步骤如下:
在LINUX操作系统计算机终端中输入以下命令:
perl Donor_cfDNA_main.pl-l list-o result
Donor_cfDNA_main.pl命令行参数见表8的参数说明。
表8参数说明
一个完整的list表举例如下:
>RD
receptor 1.bam
3.5 6.bam
5.5 7.bam
8 8.bam
10 10.bam
该list表示名为RD的模拟实验,需检测混合了供体DNA比例分别为3.5%、5.5%、8%、
10%采样点文库的供体比例。
部分分析结果如表9和表10所示。
表9统计及质量控制分析
注:上表为结果节选
表10供体cfDNA比例结果
表10中每列标签解释及意义如下:
从上述结果来看,检测供体比例与实际供体含量虽然不是完全相等的关系(呈线性关系),但数值较接近,理论上加大血浆的目标区域平均测序深度至500X,甚至1000X,检测比例值更精确。说明使用该方法技术上可行,可用于或辅助用于检测移植物供体cfDNA含量,进而移植后受体急性排斥的动态监测。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例,本领域的普通技术人员可以理解:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。
Claims (18)
- 一种确定受体cfDNA样本中供体来源的cfDNA的比例的方法,其特征在于,包括:获取第一测序数据和第二测序数据,所述第一测序数据为受体基因组DNA的至少一部分的测序结果,包括多个第一读段,所述第二测序数据为受体cfDNA的至少一部分的测序结果,包括多个第二读段;将所述第一测序数据和所述第二测序数据分别与参考序列进行比对,对应获得第一比对结果和第二比对结果;基于所述第一比对结果进行SNP检测,获得第一分型结果,所述第一分型结果包括多个一级纯合基因型SNP,表示所述一级纯合基因型SNP在所述第一测序数据中的基因型为AA;基于所述第二比对结果中比对上二级纯合基因型SNP的第二读段的量,确定所述供体来源的cfDNA的比例,所述二级纯合基因型SNP为满足以下条件的一级纯合基因型SNP的至少一部分:第二比对结果中比对上该位点的第二读段中包含不支持等位基因A的第二读段。
- 一种确定受体cfDNA样本中供体来源的cfDNA的比例的方法,其特征在于,包括:获取第一测序数据,所述第一测序数据为受体基因组DNA的至少一部分序列的测序结果,包括多个第一读段;将所述第一测序数据与参考序列进行比对,获得第一比对结果;基于所述第一比对结果进行SNP检测,获得第一分型结果,所述第一分型结果包括多个一级纯合基因型SNP,表示所述一级纯合基因型SNP为AA;获取第二测序数据,所述第二测序数据为受体cfDNA的至少一部分的测序结果,包括多个第二读段;将所述第二测序数据与所述参考序列进行比对,获得第二比对结果;基于所述第二比对结果中比对上二级纯合基因型SNP的第二读段的量,确定所述供体来源cfDNA的比例,所述二级纯合基因型SNP为满足以下条件的一级纯合基因型SNP的至少一部分:第二比对结果中比对上该位点的第二读段中包含不支持等位基因A的第二读段。
- 权利要求1或2的方法,其特征在于,所述获取第一测序数据和/或第二测序数据, 包括:获取所述受体基因组DNA样本和/或所述受体cfDNA样本,所述基因组DNA样本包含基因组DNA,所述cfDNA样本包含cfDNA;对所述基因组DNA和/或cfDNA进行捕获,获得第一目的片段和/或第二目的片段;对所述第一目的片段和/或第二目的片段进行测序,获得所述第一测序数据和/或所述第二测序数据。
- 权利要求3的方法,其特征在于,对所述基因组DNA进行捕获,包括:对所述基因组DNA进行片段化,获得第一DNA片段;对所述第一DNA片段进行末端修复,获得第一修复片段;对所述第一修复片段进行测序接头连接,获得第一连接产物;对所述第一连接产物进行大小选择,获得预定大小的第一连接产物;对所述预定大小的第一连接产物进行扩增,获得第一扩增产物;以及对所述第一扩增产物进行所述捕获,以获得所述第一目的片段。
- 权利要求4的方法,其特征在于,所述第一DNA片段的大小为150-250bp。
- 权利要求4的方法,其特征在于,所述预定大小的第一连接产物的大小为210-270bp。
- 权利要求3的方法,其特征在于,对所述cfDNA进行捕获,包括:对所述cfDNA进行末端修复,获得第二修复片段;对所述第二修复片段进行测序接头连接,获得第二连接产物;对所述第二连接产物进行扩增,获得第二扩增产物;以及对所述第二扩增产物进行所述捕获,以获得所述第二目的片段。
- 权利要求3-7任一方法,其特征在于,利用液相芯片进行所述捕获,所述捕获的区域包括以下合并(i)-(iii)中的至少之二后的非冗余位点:(i)人类群体等位基因频率数据库中的杂合度为0.48-0.5的SNP位点,(ii)在千人基因组数据库的东亚人群中的次等位基因频率为0.5,或者在该数据库中的所有群体中的平均次等位基因频率为0.4-0.5的SNP位点,(iii)在HapMap数据库的CHB子库中的次等位基因频率大于0.4,或者次等位基因频率为0.5,或者杂合度为0.48-0.5的SNP位点。
- 权利要求8的方法,其特征在于,所述捕获的区域包括表1所示的位点。
- 权利要求8的方法,其特征在于,所述测序的平均深度为不小于200×。
- 权利要求1或2的方法,其特征在于,在所述获得第一比对结果和/或第二比对结 果之后,对所述第一比对结果和/或所述第二比对结果进行去重,分别以去重后的第一比对结果和/或去重后的第二比对结果替代所述第一比对结果和/或所述第二比对结果。
- 权利要求1或2的方法,其特征在于,所述基于第一比对结果进行SNP检测,获得第一分型结果,包括进行以下a或者进行以下a和b:a.依据所占的比例大于95%的那一类第一读段,确定该位点的基因型,b.依据所占的比例大于等于25%且小于等于95%的多类第一段读段中的所占比例最大的前两类第一读段,确定该位点的基因型,所述多类第一读段之间的区别在于其共同比对上的位点的相应位置上的碱基不同。
- 权利要求1或2的方法,其特征在于,所述一级纯合基因型SNP为等位基因频率为100%、和/或测序深度大于等于平均测序深度的位点。
- 权利要求1或2的方法,其特征在于,在进行所述基于第二比对结果中比对上二级纯合基因型SNP的第二读段的量,确定所述供体来源cfDNA的比例之前,去除所述第二比对结果中的非唯一比对的第二读段。
- 权利要求1或2的方法,其特征在于,所述基于第二比对结果中比对上二级纯合基因型SNP的第二读段的量,确定所述供体来源cfDNA的比例,包括:对所述量进行聚类,获得聚类结果,依据所述聚类结果中呈两倍关系的两类量中的至少一类确定所述供体来源cfDNA的比例。
- 一种确定受体cfDNA样本中共体来源的cfDNA的比例的装置,其特征在于,包括:数据输入单元,用于输入数据;数据输出单元,用于输出数据;处理器,用于执行可执行程序,所述可执行程序包括完成权利要求1-15任一方法;存储单元,与所述数据输入装置、所述数据输出装置和所述存储器相连,用于存储数据,其中包括所述可执行程序。
- 一种监测器官移植排斥的方法,其特征在于,包括:分别于不同时间点对受体进行采血,获得多个血液样本;利用权利要求1-15任一方法确定每个所述血液样本中供体来源cfDNA的比例;基于确定的多个所述供体来源cfDNA的比例,进行所述监测。
- 一种监测器官移植排斥的装置,其特征在于,包括:样本获取单元,用以分别于不同时间点对受体进行采血,获得多个血液样本;供体cfDNA比例确定单元,与所述样本获取单元相连,用以利用权利要求1-15任一方法确定每个所述血液样本中供体来源cfDNA的比例;监测单元,与所述供体cfDNA比例确定单元相连,用以基于确定的多个所述供体来源cfDNA的比例,进行所述监测。
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