WO2018110940A1 - Procédé permettant de mesurer la complexité d'une banque en vue d'un séquençage de nouvelle génération - Google Patents

Procédé permettant de mesurer la complexité d'une banque en vue d'un séquençage de nouvelle génération Download PDF

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WO2018110940A1
WO2018110940A1 PCT/KR2017/014549 KR2017014549W WO2018110940A1 WO 2018110940 A1 WO2018110940 A1 WO 2018110940A1 KR 2017014549 W KR2017014549 W KR 2017014549W WO 2018110940 A1 WO2018110940 A1 WO 2018110940A1
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nucleic acid
library
polynucleotide
complexity
pcr
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Korean (ko)
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정종석
손대순
박웅양
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삼성전자 주식회사
사회복지법인 삼성생명공익재단
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Publication of WO2018110940A1 publication Critical patent/WO2018110940A1/fr

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/191—Modifications characterised by incorporating an adaptor

Definitions

  • the present invention relates to a method for measuring complexity of a library for next generation nucleic acid sequencing and a device using the same.
  • NGS Next generation sequencing
  • QC quality control
  • QC is performed prior to entering the nucleic acid sequencing, and it is determined whether to proceed with nucleic acid sequencing with the prepared library.
  • QC is performed by a method provided by the manufacturer of the library.
  • the generated nucleic acid sequence data ie, reads
  • the quality of data generation prior to analysis such as mutations, gene mutations, gene expression, and the like.
  • one of the factors determining the quality and determining the quality at each stage of the next-generation nucleic acid sequencing is the complexity of the library.
  • the complexity of the library can be measured during nucleic acid sequencing or after nucleic acid sequencing is completed to determine whether to perform, stop, and utilize the generated nucleic acid sequence data. have.
  • fragmenting nucleic acid extracted from a target sample comprising:
  • It provides a method for measuring the complexity of the library for nucleic acid sequencing, comprising the step of calculating the ratio of the second Ct value to the first Ct value to determine the complexity of the first library.
  • Nucleic acid sequencing of the “nucleic acid sequencing library” may be next generation sequencing (NGS).
  • NGS next generation sequencing
  • massive parallel sequencing or the term “second-generation sequencing”.
  • NGS refers to a technique for fragmenting a full-length genome in chip-based and PCR-based paired end forms, and performing the nucleic acid sequencing analysis at a very high speed based on hybridization.
  • NGS is a technique for simultaneous nucleic acid sequencing of a large amount of fragments of nucleic acid, and can perform NGS-based targeted nucleic acid sequencing or panel nucleic acid sequencing.
  • NGS is, for example, 454 platform (Roche), GS FLX titanium, Illumina MiSeq, Illumina HiSeq, Illumina Genome Analyzer, Solexa platform, SOLiD System (Applied Biosystems), Ion Proton (Life Technologies), Complete Genomics, Helicos Biosciences Heliscope, Pacific Biosciences' single molecule real time (SMRT TM) technology, or a combination thereof.
  • library refers to a collection of nucleic acid fragments.
  • the library is, for example, a genomic library, a complementary DNA library, or a randomized mutant library.
  • library complexity refers to the number of unique fragments that exist in that library. Complexity may be influenced by the amount of nucleic acid that is a starting material, the amount of nucleic acid lost during library preparation, the amount of nucleic acid amplified by PCR, and the like. The complexity of the library can be expressed at a relative level.
  • the method includes fragmenting the nucleic acid extracted from the target sample.
  • the target sample can be from an individual or a cell.
  • the subject may be a mammal, including humans, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents.
  • the cell may be a cell or cell line derived from an individual.
  • the target sample may be a biological sample.
  • the biological sample may be obtained from, for example, blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, tears, or a combination thereof.
  • the biological sample may be a sample of eukaryotic cells, prokaryotic cells, viruses, bacteriophage, or the like derived from various species.
  • the nucleic acid may be a genome or a fragment thereof.
  • the term “genome” is a term that collectively refers to the entirety of a chromosome, chromatin, or gene.
  • the genome or fragment thereof may be isolated DNA, eg, cell-free DNA (cfDNA). Methods for extracting or separating nucleic acids from a target sample can be performed by methods known to those skilled in the art.
  • Extracting nucleic acid from the target sample may be performed by a method known to those skilled in the art.
  • the fragmentation may be a physical, chemical or enzymatic cleavage of the genome.
  • fragmentation is cleavage of the genome with restriction enzymes.
  • the method may further comprise selecting the size of the fragmented nucleic acid.
  • the step of size selection can be performed by electrophoresis, centrifugation, chromatography, or a combination thereof.
  • the fragmented nucleic acid has a length of about 10 bp (base pair) to about 2000 bp, about 20 bp to about 1500 bp, about 50 bp to about 1000 bp, about 100 bp to about 800 bp, about 150 bp to about 600 bp, Or about 300 bp to about 600 bp.
  • the method includes ligating a first polynucleotide at one or more ends of the fragmented nucleic acid to produce a first library for nucleic acid sequencing.
  • the preparing of the first library may further include end-repair and 3′-adenosine tailing of the fragmented nucleic acid.
  • the first polynucleotide may be an adapter.
  • the adapter may be a polynucleotide comprising a primer sequence for enriching a target nucleic acid in NGS.
  • the adapter may be a polynucleotide known to those skilled in the art.
  • the adapter may comprise a universal sequence for nucleic acid sequencing. For example, it is an adapter included in a library preparation kit for nucleic acid sequencing.
  • Ligation refers to the binding of ends between nucleic acid fragments.
  • the ligation can be performed using DNA ligase.
  • the first library may be a library prepared for nucleic acid sequencing.
  • the method includes preparing a second library by spiking a second polynucleotide to the first library.
  • the spike may be to mix the first library with a small amount of the second polynucleotide.
  • the second polynucleotide comprises a first region wherein at least two consecutive nucleotides comprise a nucleic acid sequence identical to a target nucleic acid sequence, and at least one end of the first region, wherein the second polynucleotide comprises at least two consecutive nucleotides from at least one end of the target nucleic acid sequence; It may comprise a second region comprising different nucleic acid sequences.
  • the target nucleic acid sequence may comprise genetic variation used for companion diagnostics (CDx).
  • CDx companion diagnostics
  • the length of the second polynucleotide is about 20 nucleotides (hereinafter referred to as 'nt') to about 500 nt, about 30 nt to about 450 nt, about 40 nt to about 400 nt, about 50 nt to about 350 nt, about 60 nt to about 300 nt, about 70 nt to about 250 nt, about 80 nt to about 200 nt, about 90 nt to about 190 nt, about 100 nt to about 180 nt, about 110 nt to about 170 nt, about 120 nt To about 160 nt, about 130 nt to about 150 nt, or about 150 nt.
  • 'nt' nucleotides
  • the first region may comprise a nucleic acid sequence wherein two or more consecutive nucleotides are the same as the target nucleic acid sequence.
  • the first region is about 10 nucleotides (hereinafter referred to as 'nt') to about 490 nt, about 20 nt to about 440 nt, about 30 nt to about 390 nt, about 40 nt to about 340 nt, about 50 nt to About 290 nt, about 60 nt to about 240 nt, about 70 nt to about 150 nt, about 80 nt to about 180 nt, about 90 nt to about 170 nt, about 100 nt to about 160 nt, about 110 nt to about 150 nt, about 120 nt to about 150 nt, about 130 nt to about 150 nt, about 140 nt to about 150 nt, or about 142 nt.
  • the second region is located at both ends of the first region, each second region comprising a sequence different from at least 2 consecutive nucleotides from the 5 'end of the target nucleic acid sequence and a sequence different from at least 2 consecutive nucleotides from the 3' end.
  • the length of the second region is about 2 nt to about 15 nt, about 2 nt to about 13 nt, about 2 nt to about 10 nt, about 2 nt to about 8 nt, about 2 nt to about 6 nt, about 2 nt To about 4 nt, about 3 nt, or about 4 nt.
  • the second polynucleotide may comprise a second region, a first region, and a second region, for example in the 3 'direction from its 5'-end.
  • the second polynucleotide may further comprise two or more contiguous nucleotides identical to the first polynucleotide at one or more ends thereof.
  • the second polynucleotide is, for example, two or more contiguous nucleotides equal to the first polynucleotide in its 3 'direction from its 5'-end, two equal to the second region, first region, second region, and first polynucleotide It may comprise a continuous nucleotide.
  • the method includes performing a first polymerase chain reaction (PCR) using a first primer set complementary to a second library and a first polynucleotide to yield a first threshold cycle (Ct) value. do.
  • PCR polymerase chain reaction
  • the PCR is, for example, quantitative PCR (qPCR), digital PCR (digital PCR (dPCR), hot start PCR, touchdown PCR, nested PCR, booster (booster) ) PCR, multiplex PCR, real-time PCR, differential display PCR (D-PCR), rapid amplification of cDNA ends (RACE), inverse PCR (inverse) polymerase chain reaction (IPCR), vectorette PCR, and TAIL-PCR (thermal asymmetric interlaced PCR).
  • qPCR quantitative PCR
  • digital PCR digital PCR
  • dPCR digital PCR
  • hot start PCR hot start PCR
  • touchdown PCR nested PCR
  • booster (booster) booster
  • multiplex PCR multiplex PCR
  • real-time PCR real-time PCR
  • D-PCR differential display PCR
  • RACE rapid amplification of cDNA ends
  • IPCR inverse PCR
  • vectorette PCR vectorette PCR
  • TAIL-PCR thermoasymmetric interlaced PCR
  • the first primer set may be a polynucleotide complementary to the first polynucleotide.
  • the first primer set may be a universal primer set.
  • the second primer set may be a polynucleotide complementary to a second polynucleotide.
  • the second primer set may be a polynucleotide that is complementary to the second polynucleotide but not complementary to the first polynucleotide.
  • a probe complementary to the target nucleic acid can be further used for detection of the amplified nucleic acid.
  • the probe may be one or more of its ends is labeled with a fluorescent material, quantum dots, FRET and the like.
  • the threshold cycle (Ct) value refers to the number of cycles in the PCR that initially represent the amplified signal above the background signal. For quantitative PCR, this refers to the number of cycles representing the threshold of the fluorescence signal. Since the Ct value is inversely correlated with the copy number of the original nucleic acid as starting material in the amplification reaction, the Ct value can be used to calculate the copy number of the nucleic acid in the target sample.
  • the first Ct value may represent the total read of the second library.
  • read refers to nucleic acid sequence information of nucleic acid fragments obtained by nucleic acid sequence analysis.
  • the method includes performing a second PCR using a second primer set complementary to a second library and a second polynucleotide to yield a second Ct value.
  • the second Ct value may represent a read of a second polynucleotide in a second library.
  • the first PCR, the second PCR, or a combination thereof may be performed by quantitative PCR (qPCR) or digital PCR (dPCR).
  • qPCR quantitative PCR
  • dPCR digital PCR
  • the first PCR and the second PCR may be performed simultaneously or sequentially.
  • the method includes calculating the ratio of the second Ct value to the first Ct value to measure the complexity of the first library.
  • Another aspect includes fragmenting nucleic acid extracted from a target sample
  • the second polynucleotide comprises a first region wherein at least two consecutive nucleotides comprise a nucleic acid sequence identical to a target nucleic acid sequence, and at least one end of the first region, wherein the second polynucleotide comprises at least two consecutive nucleotides from at least one end of the target nucleic acid sequence; Comprising a second region comprising different nucleic acid sequences;
  • the complexity of the target sample, nucleic acid, nucleic acid sequencing, fragmentation, first polynucleotide, ligation, addition, second polynucleotide, PCR, Ct value, library, and library is as described above.
  • the method includes fragmenting the nucleic acid extracted from the target sample.
  • the method includes ligating a first polynucleotide at one or more ends of the fragmented nucleic acid to produce a first library for nucleic acid sequencing.
  • the method includes adding a second polynucleotide to the first library to prepare a second library.
  • the method includes performing nucleic acid sequencing with a first primer complementary to the second library and the first polynucleotide to obtain a total read of the second library.
  • the first primer may be one primer or a set of primers.
  • the method includes selecting a read of the second polynucleotide from the total reads obtained to obtain a read of the second polynucleotide.
  • the method includes calculating the ratio of the number of reads of the second polynucleotide to the total number of reads to determine the complexity of the first library.
  • the method can monitor the complexity of the first library in real time during or after nucleic acid sequencing.
  • the library is prepared in a simple and accurate manner in real time during the preparation of the nucleic acid sequencing library, during the nucleic acid sequencing process after the library preparation, or after completion of the nucleic acid sequencing.
  • the complexity of can be measured.
  • FIG. 1A is a schematic diagram illustrating the principle of a method of measuring the complexity of a library for NGS according to one aspect
  • FIG. 1B is a schematic diagram showing the ratio of artificial sequence reads among the entire reads when the complexity of the library is high or low.
  • ⁇ adapter
  • ⁇ artificial sequence
  • 2 is a graph showing Ct values in quantitative PCR according to library complexity.
  • 3 is a graph showing the ratio of artificial sequence reads among total reads according to library complexity.
  • Genes KRAS, IDH1, BRAC1, ALK, and ERBB2, including variants known to be utilized in companion diagnostics (CDx) as target sequences for next generation sequencing (NGS), and regions of these genes was selected. About 150 bp of reference sequence was selected based on the selected position.
  • the selected reference sequence and the nucleic acid sequence are identical, but replace the 4 bp from the 5 'end and the 4 bp from the 3' end with an artificial sequence, and the nucleic acid fragment including the adapter nucleic acid sequence of the library at both ends (hereinafter , Called “artificial sequence containing nucleic acid fragments” were prepared by gene synthesis methods.
  • nucleic acid sequences excluding the adapter nucleic acid sequence from the nucleic acid sequences of the selected genes, reference sequences, and artificial sequence containing nucleic acid fragments are shown in Table 1 below.
  • Chromosome No. 12 Exon No. 3: 3: Chromosome 12: 25380168-25380346 5'-AGGAATCCTGAGAAGGGAGAAACACAGTCTGGATTATTACAGTGCACCTTTTACTTCAAAAAAGGTGTTATATACAACTCAACAACAAAAAATTCAATTTAAAAATGGGCAAAGGACTTGAAAAGACATTGTTCCTGCTCCAAAGATGAC-3 '(SEQ ID NO: 1) -3 '(SEQ ID NO: 2) 2 IDH1: Chromosome No. 12 Exon No.
  • Chromosome 2 209113048-209113359 5'-AGATAATGGCTTCTCTGAAGACCGTGCCACCCAGAATATTTCGTATGGTGCCATTTGGTGATTTCCACATTTGTTTCAACTTGAACTCCTCAACCCTCTTCTCATCAGGAGTGATAGTGGCACATTTGACGCCAACATTATGCTTCTTTA-3 '(SEQ ID NO: 3) -3 '(SEQ ID NO: 4) 3
  • BRAC1 Chromosome 17 Exon number: 15: Chromosome 17: 41222945-41223255 5'-TCAATTCTGGCTTCTCCCTGCTCACACTTTCTTCCATTGCATTATACCCAGCAGTATCAGTAGTATGAGCAGCAGCTGGACTCTGGGCAGATTCTGCAACTTTCAACTTTCAATTGGGGAACTTTCAATGCAGAGGTTGAAGATGGTATG-3 '(SEQ ID NO: 5) -3 '(SEQ ID NO: 6) 4 ALK: Chromosome No.
  • Chromosome 2 29446208-29446394 5'-GGTCACTGATGGAGGAGGTCTTGCCAGCAAAGCAGTAGTTGGGGTTGTAGTCGGTCATGATGGTCGAGGTGCGGAGCTTGCTCAGCTTGTACTCAGGGCTCTGCAGCTCCATCTGCATGGCTTGCAGCTCCTGGTGCTTCCGGCGGTACA-3 '(SEQ ID NO: 7) -3 '(SEQ ID NO: 8) 5 ERBB2: Chromosome No. 17 Exon No.
  • Chromosome 17 37864574-37864787 5'-CAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACA-3 '(SEQ ID NO: 9) -3 '(SEQ ID NO: 10)
  • HapMap mixed samples in which a total of 10 samples of human genome samples HapMap NA07014, NA10840, NA18595, NA18957, NA18488, NA18511, NA18867, NA18924, NA19108, and NA19114 were mixed at the same molar concentration ratio to prepare a library for NGS. 50 ng or 200 ng was prepared. Prepared mixed samples were subjected to sequential fragmentation, end-repair, 3'-adenosine tailing, and adapter ligation of the human genome using Kapa hyper prep kits for illumine (Kapa Biosystems) according to the method provided by the manufacturer. It was.
  • Example 1.1 50 atmoles of each of the artificial sequence containing nucleic acid fragments prepared in Example 1.1 were spiked into the library to which the adapter was ligated.
  • the library to which the artificial sequence-containing nucleic acid fragment was added was subjected to a pre-capture polymerase chain reaction (PCR), followed by target enrichment.
  • PCR pre-capture polymerase chain reaction
  • the target concentrated library was then subjected to post-capture PCR.
  • Real-time PCR was performed using a quantitative PCR (qPCR) kit for measuring KAPA Illumina library concentrations, and Ct (cycle threshold) values were calculated from real-time qPCR results.
  • Ct cycle threshold
  • the calculated Ct value represents the number of reads derived from the artificial sequence containing nucleic acid fragments.
  • a library having changed the library complexity during the library preparation process was prepared.
  • the product ligated in the adapter ligation step was purified once, using an adapter of 30 ⁇ M and according to this method
  • the prepared library was used as a negative control.
  • complexity can be achieved by using two purifications of the ligated product in the ligation step, using 3 ⁇ M of adapter (ie 1/10 dilution), or a combination thereof. Reduced libraries were prepared.
  • the library of the negative control and the library of artificially reduced complexity were subjected to real-time qPCR in the same manner as above to calculate the Ct value.
  • the calculated Ct value according to the complexity of the library is shown in FIG. 2.
  • the complexity of the library decreased, the Ct value of the artificial sequence containing nucleic acid fragments decreased.
  • the Ct value of the total reads did not change significantly, despite the complexity of the library.
  • the nucleic acid sequences of the prepared libraries were analyzed and the raw read data analyzed was used to calculate the total number of reads and the ratio of artificial sequence reads among the total reads. According to the change in complexity, the calculated total number of reads and the ratio of artificial sequence reads among the total reads are shown in FIG. 3.
  • "50 ng” means that the amount of human genomic DNA HapMap mixed sample at the time of library preparation is 50 ng.
  • the number of total reads and the number of artificial sequence reads did not correlate with library complexity, but it was confirmed that the ratio of artificial sequence reads among all reads correlated inversely with library complexity.

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Abstract

L'invention concerne un procédé permettant de mesurer la complexité d'une banque. Selon la présente invention, la complexité d'une banque peut être mesurée de manière simple et précise pendant un processus de production de la banque en vue d'un séquençage en temps réel, pendant un processus de séquençage après la production de la banque, ou après l'achèvement du séquençage.
PCT/KR2017/014549 2016-12-13 2017-12-12 Procédé permettant de mesurer la complexité d'une banque en vue d'un séquençage de nouvelle génération WO2018110940A1 (fr)

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US20130065768A1 (en) * 2011-09-12 2013-03-14 Sequenta, Inc. Random array sequencing of low-complexity libraries
US20140324359A1 (en) * 2013-04-25 2014-10-30 University Of Southern California Predicting the molecular complexity of sequencing libraries
US20160122748A1 (en) * 2014-10-30 2016-05-05 The Board Of Trustees Of The Leland Stanford Junior University Scalable method for isolation and sequence-verification of oligonucleotides from complex libraries

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