Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

• the present invention relates to methods for enriching test samples for target nucleic acid molecules for further genetic screening.
• Another genetic variation is fetal gender, which can often be determined based on sex chromosomes X and Y.
• Some genetic variations may predispose an individual to any number of diseases such as, for example, diabetes, arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g., colorectal, breast, ovarian, lung). Identifying genetic variances can lead to diagnosis of, or determining predisposition to, a medical condition and inform medical decisions. Research and development efforts to discover genetic abnormalities that produce adverse health consequences have identified specific genes and/ or critical diagnostic markers for genetic based diseases.
• cfDNA cell-free DNA
• NIPS non-invasive prenatal diagnostic
• detection of fetal aneuploidy and autosomal recessive disorders with cfDNA has its challenges because only a small portion of the cfDNA in maternal plasma is derived from the fetus.
• a primary driver of NIPS sensitivity for aneuploidy in a given maternal plasma sample is the fetal fraction (FF). Flui, L. et al, Prenat Diagn. 2020; 40:155-163.
• FF values are between 4% and 30%. Wang, E. et al., Prenat Diagn. 2013; 33:662-666. Many laboratories fail samples with FF ⁇ 4% to diminish the risk of issuing false negative reports. Because the molecular and bioinformatic implementations of NIPS have evolved, diversified, and generally improved over time, sensitivity at progressively lower FF levels is platform- and laboratory dependent. Fiui, L. et al, Prenat Diagn. 2020; 40:155-163; Artieri, C.G. et al, Prenat Diagn. 2017; 37:482-490.
• cfDNA is a mixture of DNA which varies in properties (e.g. size, sequence, abundance) as well as tissue of origin ⁇ e.g., maternal vs. fetal).
• tissue of origin e.g., maternal vs. fetal
• cfDNA obtained from pregnant women contains DNA of both maternal and fetal origin
• cfDNA obtained from cancer patients contains DNA of both tumor and normal cellular origin
• cfDNA obtained from transplant patients contains DNA of both host and graft origin.
• properties of cfDNA e size
• fetal DNA has a smaller fragment size distribution of than maternal DNA. Fan, H. C., et al, (2010). Analysis of the Size Distributions of Fetal and Maternal Cell-Free DNA by Paired- End Sequencing. Clinical Chemistry. 56(8): 1279-1286.
• cfDNA has potential use as a diagnostic biomarker for other illnesses, such as schizophrenia and cancer.
• tumor DNA has a smaller size distribution of fragment sizes than DNA from normal tissues.
• the cfDNA in schizophrenia patients was composed of shorter DNA molecules and showed an apoptosis-like distribution pattern. Jang et al, Translational Psychiatry 2018; 8:104.
• fragment size eg., cfDNA
• a major factor influencing test performance is the relative proportion of the target DNA fraction (eg., fetal, tumor, graft) in a sample.
• the fetal fraction is the ratio of fetal DNA (i.e., the proportion of cfDNA fragments that originate from the placenta) to all DNA in a cfDNA sample.
• Nucleic acid methylation signature is another distinguishing characteristic that may be used. For example, methylation signature differences between mothers and fetuses have been observed. Hong-Dan, W., et al, Mol. Med. Rep. 2017;15(6): 3989-3998.
• an association between promoter hypermethylation and deactivation of genes involved in DNA repair resulting in certain cancer types has been shown. Jin, B. et al, Adv. Exp. Med. Biol. 2013; 754: 3-29.
• target DNA fractions with smaller fragment size distributions may be increased by enriching the mixture for smaller fragments using electrophoresis.
• larger fragments are discarded.
• the result is often a change in the proportion of target DNA in the mixture, but a loss of total mass.
• Target DNA fraction enrichments of 2x or greater are possible. For example, a cfDNA mixture that was previously 10% fetal DNA becomes 20% fetal DNA after enrichment for smaller DNA fragments by electrophoresis.
• Such enrichment processes, in this case via electrophoresis, is often referred to as “size selection.”
• the FX protocol represents an advance in NIPS because samples that would have had low FF on standard NIPS are molecularly transformed into samples that have high FF.
• assay improvement will increase the confidence that providers and patients have in their results with NIPS.
• An object of the present invention is to provide a method of enriching for and screening target nucleic acid fractions (e.g., DNA or RNA) in test samples.
• target nucleic acid fractions e.g., DNA or RNA
• the test samples are pooled from a plurality of test subjects.
• the distribution of nucleic acid fragments bearing characteristics of interest is determined using known methodologies followed by a sample specific calculation of the relative quantity of nucleic acid present, e.g., the relative abundance of nucleic acid present bearing the characteristic of interest.
• a numerical offset is calculated using the ratio of actual nucleic acid concentration in the library/predicted nucleic acid concentration in the library.
• the numerical offset value is used to calculate the weighted volume or weighted nucleic acid concentration from the sample specific libraries to be added to a second test sample for further selection or enrichment of fragments bearing the characteristic of interest.
• weighted volumes of sample specific libraries are mixed to create a second test sample where there are equal amounts of nucleic acid bearing the characteristic of interest for every sample specific library.
• selection or enrichment is performed on the second test sample using conventional techniques to produce a third test sample containing nucleic acid fragments bearing the characteristic of interest. Fragments that do not have the characteristic of interest may be discarded.
• the third test sample is sequenced using known sequencing techniques and using the sample specific labels (e.g., barcodes), sample specific nucleic acid is isolated and screened for anomalies.
• nucleic acid libraries are prepared/amplified for each sample of origin collected.
• unique markers e.g., labels, tags
• sequence-based barcodes can be added to the nucleic acid fragments in each sample on one or both ends that uniquely identify the sample of origin.
• the labeled ⁇ eg. barcoded) nucleic acid mixtures contained in the sample specific libraries are mixed 1:1 by volume (m ⁇ ) or mass (ng) ratio to produce a first test sample.
• selection is performed on the first test sample using conventional techniques to produce a second test sample containing nucleic acid fragments bearing the characteristic of interest (e.g., “target nucleic acid population”). Fragments that do not have the characteristic of interest can be discarded.
• the relative quantities of the target nucleic acid population for each sample of origin are determined using techniques including but not limited to sequencing. In other embodiments, relative quantities can be determined using other techniques, such as for example, quantitative PCR (qPCR), droplet digital PCR (ddPCR), or the like. In another embodiment, using the sample specific relative quantities, a numerical offset is calculated using the ratio of actual nucleic acid concentration in the library/ predicted nucleic acid concentration in the library.
• the numerical offset value is used to calculate the weighted volume or weighted nucleic acid concentration from the sample specific libraries to be added to a third test sample for further selection or enrichment of fragments bearing the characteristic of interest.
• weighted volumes of sample specific libraries are mixed to create a third test sample where there are equal amounts of nucleic acid bearing the characteristic of interest for every sample specific library.
• the third test sample is sequenced and screened the target nucleic acid population for genetic anomalies.
• a second selection (enrichment) is performed on the third test sample and isolating the target nucleic acid population in suspension to form a fourth test sample enriched for said target nucleic acid population and comprising substantially equal proportions from each sample of origin.
• the fourth test sample is sequenced and screened the target nucleic acid population for genetic anomalies.
• a method of enhancing the sensitivity and resolution of genetic diagnostic assays of pooled nucleic acid samples comprising the steps of: a. isolating and purifying nucleic acid from a plurality of test subjects to generate corresponding samples of origin to generate at least one sample of origin; b. preparing a library for each test subject wherein the nucleic acid fragments are barcoded and wherein each library corresponds to a specific sample of origin; c. adding a first number of nucleic acid units from each sample of origin to form a first pooled test sample; d. determining the fragment size distribution within each sample of origin; e. determining the abundance of a target nucleic acid population in each sample of origin; f.
• calculating a unique numerical offset value for each sample of origin g. adding a second number of nucleic acid units from each sample of origin based on the unique numerical offset value to form a second pooled test sample; and h. performing fragment size selection on the second pooled test sample and isolating the target nucleic acid population in suspension to form a third pooled test sample enriched for said target nucleic acid population, wherein said third pooled test sample is ready for diagnostic assay.
• nucleic acid is genomic DNA.
• nucleic acid is FFPE DNA.
• nucleic acid is RNA.
• nucleic acid is cell-free DNA.
• nucleic acid is isolated from whole blood.
• said unique numerical offset value is calculated by dividing the abundance of the target nucleic acid population determined in step e by the first number of nucleic acid units.
• step f is performed by quantitative PCR.
• FIG. 2 is a graph illustrating how the methods described herein (FX protocol) increase fetal fraction (FF) across all BMI levels.
• FX protocol fetal fraction
• FF fetal fraction
• FIG. 4 is plot illustrating the fold change difference in FF as a result of applying FX protocol as a function of the original FF without FX protocol. Dashed line indicates no change in FF and samples in the shaded region had increased FF with FX protocol.
• FIG. 5A is a schematic of the change in median depth per autosome as a result of FX protocol. The extent of the deviation from background is itself a measure of FF and is indicated as fhpositive ⁇
• FIG. 5D charts z-scores without FX protocol (circles) and with FX protocol (triangles) for the same samples as in FIG. 5B stratified by their screening results and summarized as individual samples.
• FIG. 6 is a plot illustrating how FX protocol improves the coefficient of variation (CV) for mapped reads versus procedures without FX protocol.
• FIG. 7 is a plot comparing assay sensitivity for a short microdeletion ( ⁇ 3MB; shaded in blue at left of each plot) under FX protocol (labeled “FFA” in this plot) and standard NIPS conditions.
• FFA fetal fraction of FFA
• standard NIPS standard NIPS
• Scatter points are the bin-level normalized depth; black scatter points show the rolling median of blue points (median over 25 bin window). This particular deletion is shorter than a typical 5p deletion, for which the median 3’ breakpoint is indicated (http://dbsearch.clinicalgenome.org/search/).
• the FF was 11% with FFA and 7% with standard NIPS.
• FIG. 8 are ROC curve graphs for different classes of chromosomal abnormalities showing that FX protocol (labeled “FFA” in this plot) enables near-perfect analytical sensitivity with near-perfect analytical specificity.
• FX protocol labeled “FFA” in this plot
• the sensitivity of common aneuploidies is higher with FX protocol, as is the aggregate sensitivity of RAAs.
• FIG. 9 is a chart showing the distribution of FFchrY values for samples called as female or male.
• solid lines indicate raw data
• dashed lines show best-fit traces for the female (Gaussian) and male (beta) populations. Only euploid samples are included.
• the arrow depicts one sample tested on both platforms, called female in standard NIPS and male with FX protocol (the fetus was confirmed to be male). After minimizing the number of estimated miscalls on each platform analytical miscalls are predicted to drop 318-fold with FX protocol.
• the term “subject”, generally refers to an animal, such as a mammal
• the subject can be a vertebrate, a mammal, a rodent ⁇ e.g., a mouse), a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets.
• a subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease ⁇ eg, cancer) or a pre - disposition to the disease, and/ or an individual that is in need of therapy or suspected of needing therapy.
• a subject can be a patient.
• a subject can be a microorganism or microbe ⁇ eg., bacteria, fungi, archaea, viruses).
• genomic information generally refers to genomic information from a subject, which may be, for example, at least a portion or an entirety of a subject’s hereditary information.
• a genome can be encoded either in DNA or in RNA.
• a genome can comprise coding regions ⁇ eg., that code for proteins) as well as non-coding regions.
• a genome can include the sequence of all chromosomes together in an organism.
• the human genome ordinarily has a total of 46 chromosomes. The sequence of all of these together may constitute a human genome.
• polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,
• nucleic acid and “oligonucleotide” are used interchangeably and generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component, tag, reactive moiety, or binding partner. Polynucleotide sequences, when provided, are listed in the 5' to 3' direction, unless stated otherwise.
• the term “gene” generally refers to a DNA segment that is involved in producing a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).
• base pair generally refers to a partnership ⁇ i.e., hydrogen bonded pairing) of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
• a base pair may include A paired with Uracil (U), for example, in a DNA/RNA duplex.
• barcode generally refers to a known nucleic acid sequence that allows some feature of a polynucleotide with which the barcode is associated to be identified.
• the feature of the polynucleotide to be identified is the sample from which the polynucleotide is derived.
• barcodes are about or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides in length. In some embodiments, barcodes are shorter than 10, 9, 8, 7, 6, 5, or 4 nucleotides in length. In some embodiments, barcodes associated with some polynucleotides are of different lengths than barcodes associated with other polynucleotides. In general, barcodes are of sufficient length and comprise sequences that are sufficiently different to allow the identification of samples based on barcodes with which they are associated.
• a plurality of barcodes may be represented in a pool of samples, each sample comprising polynucleotides comprising one or more barcodes that differ from the barcodes contained in the polynucleotides derived from the other samples in the pool.
• Samples of polynucleotides comprising one or more barcodes can be pooled based on the barcode sequences to which they are joined, such that all four of the nucleotide bases A, G, C, and T are approximately evenly represented at one or more positions along each barcode in the pool (such as at 1, 2, 3, 4, 5, 6, 7, 8, or more positions, or all positions of the barcode).
• the term “whole genome sequencing” refers to determining the complete DNA sequence of the genome at one time.
• a “whole genome sequence”, or WGS (also referred to in the art as a “full”, “complete”, or entire” genome sequence), generally refers to encompassing a substantial, but not necessarily complete, genome of a subject.
• WGS is used to refer to a nearly complete genome of the subject, such as at least 95% complete in some usages.
• fragment size distribution refers to any one value or a set of values that represents a length, mass, weight, or other measure of the size of molecules corresponding to a particular group (e.g. nucleic acid fragments from a particular chromosomal region).
• a size distribution relates to the rankings of the sizes (e.g., an average, median, or mean) of fragments of one chromosome relative to fragments of other chromosomes.
• a size distribution can relate to a statistical value of the actual sizes of the fragments of a chromosome.
• the term “library” or “sequencing library” generally refers to a nucleic acid ⁇ e.g., DNA or RNA) that is processed for sequencing, e.g., using massively parallel methods, e ., NGS.
• the nucleic acid may optionally be amplified to obtain a population of multiple copies of processed nucleic acid, which can be sequenced by NGS or other suitable technique.
• fraction multiplier technology or “FX technology” or “FX protocol” generally refers to the methods described herein to increase the yield of the target nucleic acid fraction ⁇ eg., cffDNA) thereby increasing sensitivity for detection of anomalies, such as, for example fetal anomalies arising from copy-number changes of any size across the genome.
• FX protocol leverages the reduced size of target nucleic acid molecules to increase the relative abundance of the target nucleic acid fraction.
• the methods may be referred to as “fetal fraction amplification” or “FFA”.
• a biological specimen is collected from test subjects ⁇ eg., blood plasma from pregnant females), the nucleic acid is isolated, purified, and libraries prepared and amplified using primers combined with sequence-based barcodes.
• nucleic acid is extracted from formalin fixed paraffin embedded tissue (FFPE DNA).
• FFPE DNA formalin fixed paraffin embedded tissue
• Formalin is commonly used as a fixative for long term tissue sample preservation and storage. While the fixation process adequately preserves the ultrastructure of the tissues, it results in various types of damage to DNA within the tissues.
• the DNA damage signature of FFPE DNA includes, hydrolysis of N-glycosyl bonds, deamination, oxidation, thymine dimers, nicks, and double stranded breaks.
• PCR primers can be designed to barcodes and a PCR reaction can be run to amplify sequences comprising the barcodes.
• the original samples are combined to a desired mixture ratio to generate a first pooled test sample.
• predetermined quantities of original samples are added to produce the first pooled test sample such that the units of each sample are substantially equivalent (eg., 1 : 1 : 1 :1, etc.) — an equal mixture ratio.
• “Units” can be defined as any appropriate unit of measurement, such as, for example nanograms (ng), microliters (m ⁇ ), or moles (mol).
• the distribution of nucleic acid fragments bearing a specific characteristic (eg. , fragment size, molecular weight, methylation state) for the first pooled test sample is assayed.
• a specific characteristic eg. , fragment size, molecular weight, methylation state
• paired-end sequencing can be used to deduce the fragment size distribution of each original sample within the first pooled test sample. Paired-end sequencing is known in the art and was originally described in Smith, M. W. et al. (1994). Genomic sequence sampling: a strategy for high resolution sequence-based physical mapping of complex genomes. Nature Genetics. 7: 40-47. Paired-end sequencing obtains information for both ends of each DNA molecule.
• sample specific relative quantities i.e., in units of choice
• DNA fragments within a target fragment size ranges eg., 100 to 165bp
• DNA fragments of specific target sizes e.g., 165bp
• Sample specific relative quantities can be calculated using amplification procedures, such as polymerase chain reaction (PCR), quantitative PCR (qPCR), droplet digital PCR (ddPCR), and isothermal amplification.
• PCR polymerase chain reaction
• qPCR quantitative PCR
• ddPCR droplet digital PCR
• a numerical offset value can be calculated by determining the ratio of sample specific relative quantity (units) / total units in first pooled test sample.
• the numerical offset value is used to calculate the weighted number of units (e.g., m ⁇ , ng, etc.) from the sample specific libraries to be added to a second pooled test sample for fragment size selection.
• the weighted number of units (e.g., based on mass, volume, etc.) of each sample of origin are mixed together to generate the second pooled test sample.
• the weighted number of units are mixed such that the units are in substantially equal proportions.
• unequal amounts (predetermined sample specific units) of one or more samples of origin are mixed together depending on the assay being performed.
• gel electrophoresis can be used to isolate, excise, and purify the desired nucleic acid size fraction and generate a third pooled test sample containing nucleic acid fragments within the target size range or of specific target size.
• nucleic acid electrophoretic separation followed by the recovery of the desired fragment lengths is used.
• Various known electrophoretic processes may be used for this purpose, but in one embodiment, the NIMBUS SelectTM workstation with Ranger TechnologyTM for high throughput nucleic acid size selection may be used.
• the numerical offset value is determined by performing fragment size selection on the first pooled sample omitting the fragment size distribution step described above via paired end sequencing, for example.
• a predetermined amount of the first pooled test sample is used to isolate, extract, and generate a second pooled test sample containing nucleic acid fragments within the target size range or of a specific target size.
• electrophoretic separation of nucleic acid followed by recovery of the desired lengths of nucleic acid fragments is used, as described above.
• the second pooled test sample is sequenced and the relative abundance of target fragments within each sample of origin may be inferred based on the number of reads assigned to a specific sample within sample specific bins then the sample specific relative quantities (i.e., in units of choice) of DNA fragments within a target fragment size ranges (eg., 100 to 165bp) within length bins or, alternatively, DNA fragments of specific target sizes (eg., 165bp) may be calculated for each original sample.
• Sample specific relative quantities can be calculated using amplification procedures, such as polymerase chain reaction (PCR), quantitative PCR (qPCR), droplet digital PCR (ddPCR), and isothermal amplification.
• a numerical offset value can be calculated by determining the ratio of sample specific relative quantity (units) / total units in first pooled test sample.
• the numerical offset value is used to calculate the weighted number of units (e.g., m ⁇ , ng, etc.) from the sample specific libraries to be added to a second pooled test sample for fragment size selection.
• a numerical offset value is determined based on the relative abundances observed from the sample specific sequencing reads. Aliquots from each sample of origin, adjusted based on the numerical offset value, are pooled to generate a third pooled test sample, and the fragment size selection/ sequencing steps repeated.
• the third pooled test sample which is also enriched for the target fragments.
• a second fragment size selection on the third pooled test sample can be performed using conventional techniques and the target nucleic acid population is isolated in suspension to form a fourth pooled test sample enriched for said target nucleic acid population and comprising substantially equal proportions from each said sample of origin.
• the third pooled test sample and/or the fourth pooled test sample is sequenced to screen the target nucleic acid population for genetic abnormalities.
• FX protocol can be combined with whole-genome sequencing
• WGS-based NIPS has been configured to identify novel microdeletions anywhere in the genome, but its sensitivity and resolution is limited to microdeletions exceeding 7Mb in length. Since many microdeletions span ⁇ 7Mb, increasing sensitivity for small regions across the genome could have great clinical value.
• the resolution limit of genome-wide copy number variant detection is driven by the relative amount of signal in a sample (dictated by the relative amount of FF in a sample and the size of the CNV) and the amount of noise present in a sample (dictated by depth to which a sample is sequenced) (e.g., it is more challenging to detect small deletions in samples with low FF). Attempts to increase resolution by deeper sequencing provides diminished returns and quickly yields and economically in viable screening test. Therefore, methods to increase FF (fetal fraction) are preferable under these circumstances; hence, the impact and applicability of FX protocol.
• FF fetal fraction
• Plasma was separated from a 10 ml whole-blood sample via centrifugation at 1600g for 10 min. using a two-step centrifugation process and plasma was transferred to microcentrifuge tubes and centrifuged at 16,000 x g for 10 min. The plasma was stored at -80 degree C before DNA extraction. DNA fragments were extracted from 0.6 ml cell-free plasma using the Circulating Nucleic Acid Kit (Qiagen, GE). An Ion Plus Fragment Library Kit (Life Technologies, USA) for the Ion Proton Platform was used to construct sequencing libraries for each plasma sample and the libraries quantified on a Qubit Fluorometer — each sample specific library containing substantially the same concentration of total DNA. Sample specific libraries are barcoded for sample of origin identification.
• Each library contains different amounts of DNA within a specific size range (100 to 165 bp) or a specific target size (165 bp). As shown in Table 1, samples 1-5 (remaining samples not shown) were mixed at a 1:1 ratio (10 ng each) to generate a first pooled test sample (FPTS).
• FPTS first pooled test sample
• an alternative extraction and library preparation protocol involves extraction of the target nucleic acid fraction (e.g., cffDNA) from plasma using silanol-coated magnetic beads (Dynabeads, ThermoFisher) to yield samples at a relatively uniform concentration and fragment size or length (e.g. 165 bp).
• the target nucleic acid fraction was quantified (PicoGreen, ThermoFisher) and converted into a barcoded next-generation (NGS) -competent sequencing library suitable for Illumina platform using manufacturer’s instructions. Libraries were amplified via 12 rounds of polymerase chain reaction (PCR) (KAPA HiFi HotStart PCR Kit, Roche) before magnetic bead— based PCR cleanup followed by another round of quantification.
• PCR polymerase chain reaction
• Fragment size distribution of each sample within the FPTS was determined and the relative amount of DNA (ng) within the target size range (100-165bp) was obtained.
• a 2 m ⁇ sample from each sample represented in the FPTS was analyzed for fragment size distribution using a Fragment Analyzer (Advanced Analytical Technologies, Ames Iowa).
• the proportion of DNA for each sample 1-5 within the target size range relative to the total units added (10ng) to the FPTS is calculated.
• the values obtained were used to calculate the total amounts of DNA needed from each of the five samples to have equal and predetermined DNA units (1 ng) within the target size range.
• Barcodes were deconvoluted via available software programs on the market to pair reads to sample of origin based on barcode sequences and then reads were screened for relevant medical condition or chromosomal abnormality, e.g., fetal aneuploidy.
• relevant medical condition or chromosomal abnormality e.g., fetal aneuploidy.
• plasma was extracted from 10 ml of whole blood samples (1,264 NIPS patient samples and 66 controls tested on 11 batches) using a two-step centrifugation process and plasma was transferred to microcentrifuge tubes and centrifuged at 16,000 x g for 10 min to remove residual cells and obtain cell free plasma which was stored at -80 degree C before DNA extraction.
• DNA fragments were extracted from 0.6 ml cell-free plasma using the Circulating Nucleic Acid Kit (Qiagen, GE).
• FX protocol leverages the reduced size of fetal-derived cfDNA molecules to increase the relative abundance of fetal cfDNA. The workflows were executed completely independently, each beginning with the extraction of cfDNA from replicate plasma aliquots.
• Fragment size distribution of each sample within the FPTS was determined and the relative amount of DNA (ng) within the target size range (100-165bp) was obtained.
• a 2 m ⁇ sample from each sample represented in the FPTS was analyzed for fragmentation size distribution using a Fragment Analyzer (Advanced Analytical Technologies, Ames Iowa).
• the proportion of DNA for each sample within the target size range relative to the total units added (10ng) to the FPTS was calculated.
• the values obtained were used to calculate the total amounts of DNA needed from each of the five samples to have equal and predetermined DNA units (1 ng) within the target size range.
• SPTS second pooled test sample
• SPTS were subjected to fragment size selection procedures using in this case E-Gel CloneWell Agarose Gels (Invitrogen, Carlsbad, CA, USA).
• a piece of E-Gels contains six effective wells and each well can run a mixed sample which contains five samples of the DNA sequencing library.
• DNA within target size range was retrieved from the bottom wells on the gel and the selected library (i.e., third pooled test sample (TPTS) was sequenced using an Ion Proton system (Life Technologies).
• FX protocol increases fetal fraction (FF)
• FIG. 4 shows the relative gain in FF conferred by FX protocol. Notably, 2395 of the 2401 samples tested (99.8%) had an increase in FF with FX protocol with an average FF increase of 2.3-fold.
• the relative sample-level gain in FF varied as a function of FF (FIG. 3): samples that were at low FF ( ⁇ 4%) with standard NIPS had the largest FF gain with an average of 3.9-fold higher FF after undergoing FX protocol.
• FF fetal fraction
• FF posi ti ve is directly proportional to the z-score of an aneuploid region, and a higher z-score means that aneuploidy is more likely to be detected. Therefore, if FX protocol increases FF posi ti ve of aneuploidy regions, then FX protocol also increases NIPS sensitivity.
• FX protocol yielded an increase in FF (FIG. 5B).
• An increase in FF posi ti ve without FX protocol is denoted by gray circles and an increase in FF posi ti ve with FX protocol is denoted by purple triangles.
• This upward shift in the distribution of FF was unchanged by FX.
• FX also increased z-scores for every tested aneuploid sample, whereas the z-score distribution for euploid samples was unchanged (FIG. 5C-D).
• Table 4 Intra-run repeatability. Six screen-positive samples (two each for T13, T18, and T21) and 50 screen negative samples were tested in duplicate on a single flow cell. The observed percent agreement was 100%.
• Table 5 Inter-run reproducibility. Nine samples that were screen-positive for common aneuploidies (three each for T13, T18, and T21) and 66 screen-negative samples were tested in duplicate on separate flowcells. The observed percent agreement was 100%.
• FNR false negative rate
• sensitivity is the analytical sensitivity estimated from the ROC analysis.
• sensitivity is the analytical sensitivity estimated from the ROC analysis.
• the number of FN per sample screened is the product of the FNR and the prevalence. Prevalence numbers can range based on age and other factors, thus prevalence values are approximate, expressed as 1 in x, where x is rounded to nearest hundred (for common aneuploidies and RAAs) or the nearest 1000 (for common microdeletions and 22qll.2). The five common microdeletions are lp, 4p, 5p, 15ql 1 , and 22qll.2.
• the rates of FNR and FN / sample screened are much lower when FX protocol is used to enhance the fetal fraction. See Table 6 below.
• Sex miscalls in NIPS arise from limitations that are either biological (e.g., true fetal mosaicism, vanishing twin) or technical (e.g., low FF). While the former poses inherent challenges (many sex miscalls occur at FF far greater than 4%), the latter can be mitigated by FX protocol due to its ability to increase the FF of all samples and thereby remove borderline calls.
• FIG. 9 shows distributions of FFchrY (i.e., the FF as measured from the NGS read density on chromosome Y) for male fetus and female fetus pregnancies as observed for standard NIPS and FX protocol.
• a goal of many next generation sequencing (NGS) based tests is to consolidate samples prior to sequencing in equal amounts. Ideally all samples would receive the exact number of reads they require to maintain test performance. In many cases, for consistency, this number of reads would be equal across all samples and the coefficient of variation (CV) for mapped reads would be 0. However, due to errors in the process (i.e. liquid handling, quantification, etc.) this is often not the case. This is even further exacerbated when pooled samples are size selected to isolate only a particular size range of nucleic acid.
• NGS libraries we consolidated (or pooled) in equimolar concentrations and a gel-based size selection to isolate fragments between 200-250 base pairs was performed by gel electrophoresis.
• the pooled NGS libraries were then sequenced on an Illumina sequencer and downstream analyses to determine the number of reads that mapped to the genome for every sample was performed. Referring to FIG.
• the distribution of the mean centered mapped reads is indicated in the left-hand boxplot below the label “without in-silico factors.” While every library was initially pooled at equimolar concentrations, since each sample had a different number of fragments within the 200-250 base pair size bin, the distribution of those reads is quite broad with the lowest read count samples receiving one third the number of reads as the highest read count samples. The CV for these samples was 0.16. [00142] The same equimolar pool was then sequenced on a small-scale Illumina sequencing platform and paired end data was used to determine the fragment length distribution for every sample in the pool.
• the relative amount of DNA that was present within the 200-250bp size range was determined which was used to create “factors” that can be applied to the original quantification value, named here “in silico factors.”
• the NGS libraries were re consolidated such that each library contained an equimolar amount of DNA within the 200-250 bp size range.
• This re-consolidated pool was then subjected to the same 200-250 bp gel-based size selection, Illumina sequencing, and analyses as outlined above.
• the distribution of mean centered mapped reads is indicated in the right-hand boxplot. Since samples were now pooled based on the number of molecules present in the 200-250bp size bin, the CV of mapped reads for this consolidated pool is much lower at 0.03.
• FX protocol has a dramatic impact on the performance of microdeletion screening in
• the resolution limit for novel gwCNV detection may need to be above 3 MB, but dbVar contains more than a thousand unique pathogenic microdeletions between 3 MB and 7 MB in size, a number of which are associated with clinically serious phenotypes, so any gains in resolution should increase the utility of NIPS for patients and providers.
• the FX protocol strategy described herein increases the FF of a sample at the molecular level via size selection upstream of sequencing, yet it is also possible to increase FF via algorithmic size selection downstream of sequencing.
• the bioinformatics pipeline could calculate each fragment’s length based on the respective mapping positions of its paired-end reads and upweight shorter fragments in the analysis.
• the disadvantage of this bioinformatic approach is that substantial resources would still be consumed by sequencing longer fragments — likely to be maternal-derived — that contribute little to fetal aneuploidy detection.
• all of the sequenced fragments have elevated likelihood of being fetal-derived.

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