WO2016195382A1 - Séquençage nucléotidique de prochaine génération utilisant un adaptateur comprenant séquence de code à barres - Google Patents

Séquençage nucléotidique de prochaine génération utilisant un adaptateur comprenant séquence de code à barres Download PDF

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WO2016195382A1
WO2016195382A1 PCT/KR2016/005817 KR2016005817W WO2016195382A1 WO 2016195382 A1 WO2016195382 A1 WO 2016195382A1 KR 2016005817 W KR2016005817 W KR 2016005817W WO 2016195382 A1 WO2016195382 A1 WO 2016195382A1
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sequence
adapter
dna
sequencing
barcode
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방두희
이지현
김하영
안진우
황병진
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연세대학교 산학협력단
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • 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
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/301—Endonuclease
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/50—Other enzymatic activities
    • C12Q2521/531—Glycosylase
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/191—Modifications characterised by incorporating an adaptor
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/185—Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals

Definitions

  • the present invention relates to a method for preparing a DNA library using an adapter comprising a barcode sequence and a method for analyzing the next generation sequencing.
  • DNA sequence information is very important to understand life phenomenon and obtain information related to disease.
  • the key to the translation of DNA sequence information, or genome sequencing, is to identify individual differences and ethnic characteristics or to identify congenital causes, including chromosomal abnormalities, in diseases associated with genetic abnormalities, and to identify genetic defects in complex diseases such as diabetes and hypertension. Is to find.
  • sequencing data is very important because information such as gene expression, gene diversity and its interactions can be widely used in the field of molecular diagnosis and treatment.
  • Next generation sequencing has been used as a method for genome sequencing since 2007, and the development of NGS has made it easier and less expensive to analyze than the conventional method.
  • Next generation genome sequencers that implement next-generation sequencing include Roche / 454, Illumina / Solexa, and Life Technologies (ABI) SOLiD. These next-generation sequencing instruments can read more than 80 million sequences in seven hours. These advances have made it possible to utilize next-generation sequencing methods that were previously used only for research due to the enormous cost of testing in medical clinical tests.
  • the present invention is to solve the above technical problem, to significantly improve the reliability of sequencing (ultra high depth), to provide an improved next-generation sequencing method that can detect even small frequency variation.
  • the present inventors have completed the present invention by confirming that by using an adapter including a barcode sequence, it is possible to reduce the error rate and improve the reliability of the next generation sequencing method.
  • Next-generation sequencing can be divided into two steps.
  • the first step is to prepare a DNA library that makes the DNA to be analyzed by a next-generation sequencing device, and the second step is to analyze the prepared DNA library by a next-generation sequencing device.
  • the step of preparing the DNA library includes the following.
  • Illumin's next-generation sequencing device is described as an example. In the present specification, for convenience of explanation, only the analyzer of Illumina Inc. is used as an example, and other conventional next-generation sequencing devices may be used without limitation.
  • genomic DNA to be analyzed is fragmented to a certain length through sonication. However, if DNA is fragmented, such as ctDNA, for example, proceed to the next step without this decomposition process.
  • a base A is conjugated to the 3 'end of fragmented double stranded DNA.
  • Adapter conjugation Conjugate adapters to double stranded DNA fragments for sequencing by next-generation sequencing instruments.
  • adapters eg, adapters from NEB
  • base sequences typically required for Illumina platform NGS are conjugated.
  • the uracil region present in the adapter is cleaved using the USER (Uracil-Specific Excision Reagent) enzyme, so that the double stranded DNAs connected to each other can be separated by the adapter. .
  • Polymerase Chain Reaction Amplify the DNA to be analyzed by PCR.
  • index PCR is performed using an index primer inserted with an index base sequence of 8bp to distinguish between different samples that are sequenced together in one lane of one sequencing substrate.
  • index nucleotide sequence introduction and amplification can be performed in the DNA to be analyzed.
  • the DNA molecule pool generated through the above process is called 'DNA library'.
  • next generation sequencing DNA library conjugation of an adapter for next generation sequencing to a double stranded DNA fragment of the genomic DNA to be analyzed is an essential step for sequencing.
  • the method of preparing a DNA library using an index primer has an advantage in that it enables to distinguish between different samples while using a conventional adapter.
  • current next-generation sequencing methods fragment the genomic DNA under analysis into fragments that the sequencer can read, such as between 100 and 500 mer, and these DNAs after the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the present invention provides a new design of adapters and primers to replace the adapters and index primers used in the preparation of the next generation sequencing DNA library for solving the problem, the method of producing a DNA library and the next generation sequencing using the same An analytical method and kit for preparing a DNA library are provided.
  • the present invention comprises the steps of: conjugating adapters for next generation sequencing at both ends of a double stranded DNA fragment of the genomic DNA to be analyzed; Separating the adapter-conjugated double stranded DNA fragments into single strands; And amplifying the DNA fragments separated into single strands through polymerase chain reaction (PCR) using two PCR primers.
  • PCR polymerase chain reaction
  • the adapter comprises an adapter flanking sequence, a barcode sequence consisting of 4 to 30 arbitrary nucleic acids, a complementary sequence to the PCR primer,
  • One of said PCR primers comprises an index sequence, another one does not comprise an index sequence,
  • the DNA nucleic acid molecule generated by the PCR includes an index sequence and a barcode sequence, wherein the index sequence serves to distinguish a sample to which the genomic DNA to be analyzed is analyzed during next generation sequencing, and the barcode sequence is Provided is a method for producing a next-generation sequencing DNA library, which serves to provide a barcode to a single-stranded DNA fragment of the genomic DNA to be analyzed.
  • the present invention in the next generation sequencing method for the DNA library,
  • next generation sequencing method comprising recovering one or more reads having different barcode sequences from reads treated as duplicate reads among reads aligned at the same position in the reference genome and returning to sequencing.
  • the method for preparing the next-generation sequencing DNA library and the next-generation sequencing method according to the present invention include adapter flanking sequences, barcode sequences consisting of 4 to 30 random nucleic acids, and complementary to PCR primers. It is characterized by using an adapter comprising a host sequence.
  • the conventional adapter is a loop is conjugated to the adapter at both ends of the double-stranded DNA fragment of the genomic DNA to be analyzed.
  • Adapters consist of sequences optimized for use with sequencers and primers.
  • the adapter refers to a sequence that allows the sequencing device to recognize the DNA molecule to be analyzed, and allows the analysis of the DNA molecule to be bonded onto the substrate of the sequencing device and at the same time enables the amplification of the DNA molecule on the substrate. .
  • An adapter flanking sequence refers to one of the adapter sequences that is in direct contact with both ends of the DNA fragment of interest, in particular.
  • Conventional adapters consist solely of adapter flanking sequences.
  • the adapter is single stranded, and the 5 'and 3' ends of the adapter are joined to each of both strands of the double stranded DNA fragment of the genomic DNA to be analyzed.
  • double-stranded DNA fragments of the genomic DNA to be analyzed must be separated into single strands. Therefore, an uracil position in the flanking sequence is first cleaved by an enzyme that cuts the uracil position, thereby forming a Y-shape at both ends of the double-stranded DNA fragment.
  • the adapter is in a state of being joined.
  • Amplification of DNA fragments by PCR is carried out through a primer comprising an index, which includes a sequence complementary to the consensus sequence, the index sequence and the flanking sequence of the adapter for next-generation sequencing.
  • the consensus sequence for the next generation sequencing means that the consensus sequence should be included in all DNA libraries for NGS.
  • Common sequencing for next-generation sequencing serves as a sequence for immobilizing DNA nucleic acid molecules on the sequencer. Therefore, the specific sequence structure or length may vary depending on the type of sequencer, but the presence of the sequencer is essential. . In this specification, in order to distinguish common sequences for the next generation sequencing located at the front (5 'direction) and the rear (3' direction) of the DNA fragment, these are respectively the first common sequence for the next generation sequencing and the next generation.
  • the second consensus sequence for sequencing was named.
  • common sequences for next-generation sequencing using Illumina's sequencers are usually represented by arbitrary abbreviations such as P5 and P7, and accordingly used in the following drawings. Therefore, in the present specification, it can be understood that the first common sequence for the next generation sequencing and the second common sequence for the next generation sequencing are interchangeable with P5 and P7, respectively.
  • DNA nucleic acid molecules prepared using conventional adapters and index primers include consensus sequences, index sequences, flanking sequences, and DNA fragments for next generation sequencing.
  • the index number of samples can be two (2) (a kind of index to be used), the number of samples that can be may be exist in the both ends of DNA fragment DNA nucleic acid molecules that have such dual-index separated This can be advantageous when you put them in one sequencer and analyze them.
  • the index since the index merely serves to enable discrimination between samples, it is inherently different from the role of the barcode of the present invention, and thus does not play a role in reducing sequencing errors at all.
  • an adapter according to the present invention comprises an adapter comprising adapter flanking sequences, a barcode sequence consisting of 4 to 30 arbitrary nucleic acids, and a complementary sequence to a PCR primer. It is characterized by using.
  • the adapter flanking sequence located in front (5 'direction) of a barcode sequence can use the same flanking sequence used by a conventional adapter.
  • the adapter flanking sequence of the NEB company was used, but since the adapter flanking sequence has been developed optimized adapter flanking sequence according to the base sequence analyzer, those skilled in the art It is apparent that the adapter flanking sequence can be changed accordingly.
  • the adapters of the present invention comprise a barcode sequence that includes 4 to 30 arbitrary sequences per strand of the DNA fragment, such that the combination of any of these sequences serves as a barcode assigned to the DNA fragment strand. Barcode sequences are 4-30, 6-20, 6-16, 6-10, 6-8, specifically 6, 8, 10 or 16 nucleic acids. It may be made. For example, with eight barcode sequences, one of 4 8 barcodes can be assigned to each of the DNA fragment strands.
  • the adapters used for the preparation of the DNA library do not have the same sequence, but rather form a set of adapters having substantially different sequences, i.e., adapter pools. do.
  • conventional adapters differ in that they use an adapter pool consisting of adapters of the same sequence.
  • the common sequence, index sequence, adapter for the next generation sequence analysis In addition to flanking sequences, DNA fragments will contain barcode sequences.
  • the barcode sequence according to the embodiment of the present invention is identical to the nucleic acid molecule obtained using a conventional dual index primer, but has a structure in which the position of one of the indexes is replaced by the barcode sequence.
  • the barcode sequence since the barcode sequence is introduced in place of the existing index, it does not interfere with the adapter flanking sequence and thus does not cause a reduction in the length of the DNA sequence sequenced. As a result, there is no change in the length of DNA fragments that can be interpreted through NGS, even when using conventional adapters.
  • the "second common sequencing for the next generation sequencing" is located behind the barcode sequence (3 'direction) (see P7 in FIG. 2).
  • “complementary sequence for the common sequence for next-generation sequencing” ie, complementary sequence for P7; FIG. 2. Only P7-comp) is used as a primer.
  • the conventional index primer ie, the common sequence for the next generation sequencing (see P5 in FIG. 2), the index sequence and the adapter A sequence complementary to the flanking sequence of.
  • the adapter when conjugating an adapter according to the invention to a DNA fragment, the adapter is single stranded, such that the 5 'and 3' ends of the adapter are joined to each of both strands of the double stranded DNA fragment of the genomic DNA to be analyzed. do.
  • the adapter-conjugated double-stranded DNA fragment is separated into single strands, a portion of the adapter is conjugated to each single-stranded DNA fragment by an enzyme that cuts uracil positions present in the adapter flanking sequence. .
  • separation of double stranded DNA fragments into single strands can be performed by treating uracil DNA glycosylase and DNA glycosylase-lyase endonucleases.
  • the adapter when the adapter is linear rather than looped, one end of the single-stranded DNA fragment is joined to a part of the adapter including the adapter flanking sequence, and the other end is an adapter flanking sequence, 4 to 4 A portion of the adapter comprising a barcode sequence consisting of 30 arbitrary nucleic acids, the complementary sequence to a PCR primer, is conjugated.
  • the adapter fragment conjugated DNA fragments according to the present invention have an asymmetric adapter conjugation state at the 5 'and 3' ends.
  • the adapter forms a dimer through binding between the adapters and not the DNA fragments. You can prevent it.
  • the adapter may have a length of 60 to 90 mer, which is a length including a barcode sequence consisting of 4 to 30 arbitrary nucleic acids.
  • the adapter according to the present invention may further be inserted two to three arbitrary nucleic acids.
  • Adapter flanking sequences are already optimized according to the type of sequencer. However, if the barcode is inserted directly in the middle of the flanking sequence, the sequencing quality may be degraded due to random sequence insertion. Therefore, in order to prevent this problem, any sequence 2-3mer may be put together before and after the barcode sequence.
  • Such any nucleic acid may also vary depending on the type of sequencer.
  • the position at which two to three arbitrary nucleic acids are inserted can be inserted without limitation as long as it is not a position adjacent to the barcode sequence, eg, one to two nucleic acid sites before and after the barcode sequence. At this time, the sequence consisting of two to three nucleic acids to be inserted is to improve the quality of the analysis by reducing the errors that can occur when the sequencing instrument immediately read the random barcode sequence during sequencing, affecting the function of the sequence It does not affect.
  • the following example used an adapter consisting of a nucleic acid sequence represented by SEQ ID NO: 1, but this is only an example and the present invention is not limited thereto.
  • U means deoxy uracil
  • the second position C located at the front of the 3 'end is cytosine (C) including phosphorothioate, in which the oxygen atom of the phosphate group is substituted with a sulfur atom. to be.
  • C cytosine
  • the adapter is split into two parts so that double stranded DNA fragments can be separated into single strands.
  • phosphorothioate cytosine plays a role in preventing DNA degradation by DNA exonuclease.
  • NNNNNNNN in the adapter sequence of SEQ ID NO: 1 represents a barcode sequence consisting of eight arbitrary nucleic acids (any base of A, G, C, T).
  • the PCR primers used in the method for preparing the next generation sequencing DNA library are as follows.
  • Primers used in the conventional dual index primer system as can be seen in Figure 1, the primer comprises a sequence complementary to the consensus sequence, index sequence, and adapter flanking sequence for next-generation sequencing.
  • Two primers are used per sample for the genomic DNA to be analyzed, namely two primers each comprising index 1 and index 2.
  • the index sequence serves as a label for distinguishing between different samples.
  • the method for producing a next-generation sequencing DNA library in the case of using two primers per sample for the genomic DNA to be analyzed, it is the same as a conventional dual index primer system, 1 of PCR primers
  • the species differs in that it comprises an index sequence and another species does not comprise an index sequence.
  • the adapter used in the present invention includes a barcode sequence because the adapter itself includes a barcode sequence so that the barcode can be inserted in place of one of two indexes in the DNA nucleic acid molecule produced by the method for preparing a DNA library.
  • a primer that does not contain an index is used for the end of the DNA fragment to which the adapter is conjugated.
  • the PCR primers used in the method for preparing the next-generation sequencing DNA library according to the present invention include one index sequence, a sequence complementary to the first common sequencing sequence and the adapter flanking sequence for next-generation sequencing. And the other one does not include an index sequence and includes a second common sequence for next generation sequencing.
  • the adapter fragment containing the barcode sequence of the DNA fragment is not included in the index. Only the “common sequence for next-generation sequencing" shows an example in which it was used as a primer.
  • One end of the DNA fragment containing no barcode sequence but only the adapter flanking sequence is conjugated to a conventional index primer, that is, complementary to the common sequence, index sequence and flanking sequence of the adapter for next-generation sequencing. Primers comprising the sequence are used.
  • the index sequence may be appropriately selected from the range of 4 to 30 depending on the sequencing apparatus used, for example, 4 to 30, 6 to 20, 6 to 16, 6 to 10 Dog, 6 to 8, specifically 6, 8, 10 or 16 may be composed of a nucleic acid.
  • primers comprising an index sequence consisting of eight nucleic acids were used.
  • the DNA nucleic acid molecule generated by PCR using the adapter and primer according to the present invention comprises an index sequence and a barcode sequence, wherein the index sequence is a genomic DNA to be analyzed in the next generation sequencing It serves to distinguish the sample belonging, the barcode sequence is to serve to assign a barcode to the single-stranded DNA fragment of the genomic DNA to be analyzed.
  • the sample and the primer according to the present invention are simply treated as a replica lead in addition to the function of classifying the sample through the index, in addition to the function of distinguishing the sample through the index. The reads that have been discarded can be recovered and returned to sequencing to increase the depth of sequencing.
  • the DNA library manufacturing method according to the present invention may be used for next generation sequencing, but this is only one example, and it is obvious that the method may be applied to any analysis requiring DNA library preparation.
  • the double stranded DNA fragment may be between 100 and 500 mer in length.
  • the length of the double stranded DNA fragment used for the preparation of the DNA library can be appropriately adjusted depending on the type of sequencing instrument used. For example, when using Illumina's sequencing device, the double stranded DNA fragment can be adjusted to have a length of 200 to 400 mer.
  • the double stranded DNA fragment may be one in which adenosine is conjugated to three ends after end repair of the DNA fragment before adapter conjugation.
  • the method for preparing a DNA library according to the present invention may further comprise the step of capturing and separating analytical target region from DNA nucleic acid molecules generated by PCR.
  • Target capture is a representative method of analyzing various variations by capturing only specific regions of the genome, not the entire genome.
  • Target capture methods include various methods such as in-solution capture, hybridization capture, molecular inversion probe (MIP) capture, and multiplexing PCR.
  • MIP molecular inversion probe
  • hybridization-based target region capture may be performed through a panel capable of capturing only the sequence of the target region in order to sequence only the gene region to be used for analysis.
  • the target capture is performed after generating the DNA library using the adapter according to the present invention, since the analysis can be performed at a very high depth for a specific area to be analyzed, detection of a mutation that was not detected using a conventional adapter Becomes possible.
  • the genomic DNA of interest may be ctDNA. According to the present invention it is possible to detect even a small amount of variation in the ctDNA. ctDNA is only described as an advantageous example according to the present invention, the genomic DNA to be analyzed in the present invention is not limited.
  • an adapter that provides a barcode to a double-stranded DNA fragment of the genomic DNA to be analyzed, it minimizes the loss of read length that can be analyzed at one time, and significantly increases the number of reads, and at a low frequency.
  • the advantage is that it can detect even rare variations that exist.
  • the adapter according to the present invention it is possible to detect mutations present in ctDNAs (Circulating Tumor DNAs) that are present in very small amounts in the blood, which were difficult to detect using conventional diagnostic techniques.
  • ctDNAs Circulating Tumor DNAs
  • variations in ctDNAs can be detected only by using the adapter according to the present invention. Therefore, the cancer can be diagnosed by a simple blood collection without damaging the living body, and at the same time, the detection of ctDNA remaining in the blood during the treatment period or after surgery can be more easily diagnosed.
  • the genomic DNA library generation process proceeds in the same manner as before by changing the adapter to the adapter according to the present invention after genomic DNA fragmentation.
  • a probe or primer that captures only the KRAS gene is used to enrich and sequence only the KRAS gene region in the entire genome.
  • the adapter according to the present invention than when using the conventional adapter A significant improvement in depth can be expected by the number of leads discarded when used. This increase in depth allows for the detection of variations that could not be detected due to the lower upper limit of depth when using conventional adapters.
  • DNA library prepared according to the method of the present invention can be used for next-generation sequencing. Specifically, by comparing the analysis and the reference sequence with the next-generation sequencing device, sequence information of which sequence the DNA to be analyzed can be confirmed.
  • A, T, C, G bases and primers labeled with fluorescent substances, and DNA polymerase are added.
  • the DNA strand constituting the cluster is used as a template, and the DNA base sequences complementary to each other are synthesized using fluorescent labels A, T, C, and G bases. It can be confirmed which base sequence was made.
  • an adapter comprising a barcode sequence consisting of any of 4 to 30 nucleic acids is used to assign different barcode sequences to reads derived from different libraries. Accordingly, even in the case of reads having the same size and sequence, which are to be removed, if the derived libraries are different, the reads are recovered to the reads for analysis, thereby significantly increasing the number of reads and averaging the reads capable of reading a region of the genome to be analyzed. You can improve the depth, which is the number.
  • next-generation sequencing method for a DNA library prepared according to the present invention comprising recovering reads having different barcode sequences among reads treated as duplicate reads and returning them to the analysis.
  • the present invention also provides a method for distinguishing rare mutations by using a process of selecting mutation positions that are frequently detected in a gene of interest based on a sequencing database of cancer patients.
  • the mutation statistics of TCGA The Cancer Genome Atlas
  • TCGA The Cancer Genome Atlas
  • Experimental error by calculating the error probability matrix for 10 types of genotypes (AA, AT, AC, AG, CT, CG, CC, TT, TG, GG) Used to distinguish between
  • P (site type) is a prior probability (prior probability) is information that tells how much the variation occurs for each specific type of cancer in a large database such as TCGA empirically by position in the genome
  • site type) / P (Obs) is estimated using the maximum likelihood method.
  • Obs), which represents the posterior probability, is obtained by multiplying P (Obs
  • the specific method is as follows.
  • the reference is diploid in humans, so if a particular position has a genotype of A / C, then the observed sequencing read is a base of A at that particular position, It is calculated as (0.5 * 0.9 + 0.5 * 0.025), which corresponds to the P (Obs
  • the present invention provides an adapter pool for preparing a DNA library comprising a plurality of adapters including an adapter flanking sequence, a barcode sequence consisting of 4 to 30 arbitrary nucleic acids, and a complementary sequence to a primer for PCR.
  • the adapters are each characterized by comprising different barcode sequences.
  • the present invention also provides an adapter pool for preparing a DNA library comprising a plurality of adapters including adapter flanking sequences, barcode sequences consisting of 4 to 30 arbitrary nucleic acids, and complementary sequences for primers for PCR;
  • a primer for PCR comprising a sequence complementary to an index sequence, a consensus base sequence for next-generation sequencing, and an adapter flanking sequence
  • It does not include an index sequence, and comprises a primer for PCR containing a common nucleotide sequence for next-generation sequencing,
  • the DNA nucleic acid molecule in the DNA library generated by PCR includes an index sequence and a barcode sequence at the same time, wherein the index sequence serves to distinguish the sample to which the genomic DNA to be analyzed belongs in the next generation sequencing, and the barcode The sequence serves to assign a barcode to a single stranded DNA fragment of the genomic DNA to be analyzed.
  • kits for preparing DNA libraries are provided.
  • kit for preparing the DNA library may further include an enzyme that cuts the uracil position present in the adapter flanking sequence.
  • the present invention in the next-generation sequencing analysis, by using an adapter having a barcode sequence introduced therein, the number of reads can be significantly increased, so that detection errors due to sequencing errors can be relatively reduced.
  • the advantage is that you can detect even rare variations that exist at very low frequencies.
  • the introduced barcode sequence replaces the existing index sequence, it does not cause a reduction in the length of the nucleotide sequence that can be read by the sequencing device, thereby preventing the loss of data.
  • it can be used for various next generation sequencing by applying to adapters suitable for various next generation sequencing instruments currently used.
  • Figure 1 shows a schematic diagram of a method for preparing a DNA library using a conventional adapter (NEB existing adapter).
  • Figure 2 shows a schematic diagram of a method for preparing a DNA library using a barcode-introduced adapter according to the present invention.
  • Figure 3 shows the differences in the structure of the resulting DNA nucleic acid molecules when the DNA library is prepared using a conventional adapter (NEB's existing adapter) and a barcode-introduced adapter according to the present invention.
  • Figure 4 shows the results of electrophoresis of the fragmented Cas9-GFP plasmid on 1.5% agarose gel.
  • Figure 5 shows the results of electrophoresis on 1.5% agarose gel to confirm the successful creation of the library.
  • Lanes 1 to 6 in Fig. 5 represent the following.
  • FIG. 6 shows the result of checking the ratio of the replication lead according to the range of the selected fragment area when the library is generated using the adapter and the existing adapter according to an embodiment of the present invention.
  • Figure 7 shows the result of confirming the depth increase according to before and after regeneration from the replication lead list when the barcode is different when the library is created using the adapter according to an embodiment of the present invention.
  • FIG 8 shows a process for making a virtual rare variant sample to see the efficiency of the adapter according to the invention in the sample with the rare variant.
  • FIG. 9 shows a result of confirming a depth increase according to before and after regeneration of a lead from a duplicate read list due to a different barcode in a virtual rare mutation sample using an adapter according to an embodiment of the present invention.
  • FIG. 10 shows a result of confirming a ratio of replication leads between virtual rare mutation samples using an adapter according to an embodiment of the present invention.
  • FIG. 11 shows that a rare variation is detected in a virtual rare variation sample using an adapter according to an embodiment of the present invention.
  • Figure 12 shows the frequency of genetic variation and sequencing error peaks when the sequencing read according to the barcode sequence and position information of the present invention in a plasma sample as a clinical sample was analyzed without recovery.
  • FIG. 13 shows that the similarity of barcode sequences of sequencing reads having the same position information at a specific position of the KRAS gene is significantly different, so that among the sequencing reads that are actually removed by PCR duplication, the reads that are lost due to PCR duplication are It is worthwhile.
  • FIG. 15 shows the depth comparison before and after sequencing read recovery using barcodes and location information from KRAS capture clinical samples.
  • FIG. 16 shows changes in mutant peaks when sequencing reads were retrieved by combining barcode sequences and location information of the present invention in a KRAS capture clinical sample. This shows that after recovering the sequencing reads, the peaks of the genetic variation are maintained and the peaks due to the sequencing errors are lowered due to the increase in the sequencing depth, so that the variation peaks with the frequency similar to the sequencing error peaks can be observed.
  • Figure 17 shows the observation of the peaks after the application of the sequencing error removal method with statistical probability.
  • Figure 18 shows the results of the verification by Sanger sequencing in the tissue samples of the patient to verify for the amino acid 12, 13th variation of the KRAS gene found in the clinical sample.
  • the uracil reference first half nucleotide sequence is composed of 65 bp of base which is 32 bp longer than the conventional NEB company adapter nucleotide sequence (33 bp). At this time, the 65 bp also includes a random nucleotide sequence 8 bp.
  • the uracil reference late base sequence was constructed identically to the base sequence of the existing NEB's adapter. However, the sequence of the adapter may vary depending on the sequencing instrument used.
  • the adapter of the present invention was synthesized with 78 nmole, and 100 ⁇ M of adapter was prepared by adding 780 ⁇ L of Nuclease-Free water to the buffer.
  • the first synthesized adapter has a random three-dimensional structure, so for proper folding, 5 ⁇ L and 100 ⁇ M adapter 5 ⁇ L, 10 X T4 DNA Ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT, pH 7.5@25°C) 40 ⁇ L of a mixed solution of Nuclease-Free water was added to the Thermal Cycler. At this time, the thermal cycler program executed is incubated at 95 ° C. for 3 minutes in order to solve the random three-dimensional structure of the adapter. Then, the temperature is lowered by 1 ° C. per second for proper folding of the linear adapter. It was allowed to reach °C. This procedure completed a correctly folded 10 ⁇ M adapter solution.
  • 10 X T4 DNA Ligase buffer 500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT, pH 7.5@25°C
  • the sequence of the prepared adapter was as shown in SEQ ID NO: 1.
  • U means deoxy uracil
  • the second position C located at the front of the 3 'end is cytosine (C) including phosphorothioate, in which the oxygen atom of the phosphate group is substituted with a sulfur atom. to be.
  • FIG. 1 A method of producing a library generated using an NEB adapter is shown in FIG. 1.
  • a total of 9271 bp of Cas9-GFP plasmid 28 ⁇ g was fragmented so as to be 300 bp fragments by 75 seconds ultrasonic sonication (sonication).
  • the size (bp) distribution of fragmented Cas9-GFP was confirmed by electrophoresis of fragmented Cas9-GFP around 300bp. 1.5% agarose gel was used for electrophoresis. At this time, electrophoresis was performed by loading fragmented Cas9-GFP into two compartments of 1.5% agarose gel. As a result of electrophoresis, it was found that DNA fragmentation proceeded according to the intended size distribution.
  • DNA loaded in one compartment on 1.5% agarose gel was cut at 50bp before and after 300bp and DNA loaded at the other compartment was cut at 150bp before and after 300bp.
  • QIAGEN gel purification kit Agarose gel purification was carried out using.
  • the adapter conjugation process was performed using a NEB adapter and two inventive adapters for each of the three samples.
  • Adapter bonding was performed using two types of adapters for a total of three samples, resulting in six samples.
  • Electrophoresis was performed on 1.5% agarose gel to confirm the successful generation of the library. As a result, as shown in Figure 5, it can be seen that the library was successfully generated through the DNA size.
  • the index PCR was performed using the forward index primer and the reverse index primer in six different combinations.
  • Index PCR outputs were purified using beads (Sera-mag SpeedBeads, PEG-8000, 0.5M EDTA pH 8.0, 1.0M Tris pH 8.0, Tween 20, 5M NaCl) to obtain an elution solution after purification. Index PCR output elution solution was quantified using Broad Range Qubit.
  • volume ( ⁇ L) was calculated to have six samples each had the same amount of library molecules, and then the corresponding volume ( ⁇ L) was taken from each sample and merged into one pool. .
  • Example 1 when the DNA library was generated using the adapter of Preparation Example 1 and the existing NEB company adapter, the proportion of the replication lead occupied according to the selected fragment region range was confirmed.
  • the ratio of the reproducible reads to the reproducible reads from the duplicate read list and the ratio of the duplicate reads to the same position in the reference genome but different barcodes ranged from the fragment region. Confirmed according to.
  • the lead corresponding to about 50% to 66% of the total lead is the replication lead, and the lead corresponding to the average of 99.7% is reproduced from the replication lead using the adapter of Preparation Example 1. I could see that.
  • the DNA of the NA12878 cell line sample was mixed with the DNA of the SW480 cell line sample having the G12V mutation of the KRAS gene in a specific ratio as a wild type of the KRAS gene.
  • DNA libraries of NA12878 and SW480 were prepared.
  • Each DNA library used the Hiseq 2500 Next Generation Sequencing Machine to obtain sequence information and then mixed NA12878 and SW480 NGS data in a specific ratio to mimic NGS data for rare variant samples. Data from SW480 with G12V mutations were mixed at 1%, 0.5%, 0.25% relative to the data of NA12878.
  • DNA was extracted from plasma samples of colorectal cancer patients.
  • the extracted DNA was subjected to a library generation process using the adapter of Preparation Example 1.
  • the process of creating an isolated pool was carried out through a target capture process that captures only the KRAS gene sequence from the library.
  • KRAS is the most common mutation in colorectal cancer.
  • KRAS capture pool was used to obtain sequence information using Hiseq 2500 next generation sequencing instrument. Sequence information was first used to analyze genetic variation without considering barcode sequences.
  • the barcode nucleotide sequence information of the nucleotide sequence is added to the position where the ID information of the nucleotide sequence is displayed and programmed for later use in the sequencing recovery process. As a result, as shown in Fig. 12, the variation of the 13th amino acid sequence of KRAS (1.4%) and other peaks identified by sequencing errors were found.
  • Peaks due to sequencing errors are derived from one or two nucleotide sequences per position, but appear to be low in frequency, but are difficult to distinguish from rare variations detected by plasma. This disadvantage is noticeable as more leads are recognized and removed as duplicate leads.
  • the sequencing read recovery process was performed to determine whether it is possible to reduce the peak due to the sequencing error.

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Abstract

La présente invention concerne un procédé de construction de bibliothèque d'ADN qui utilise un adaptateur comprenant une séquence de code à barres, et un procédé de séquençage de nucléotides de prochaine génération. Selon la présente invention, lors du séquençage de nucléotidiques de prochaine génération, l'adaptateur à code à barres est utilisé et, de ce fait, le nombre de liens est sensiblement augmenté par rapport à un procédé classique, ce qui permet de détecter même des mutations rares qui sont présentes à une fréquence extrêmement faible.
PCT/KR2016/005817 2015-06-01 2016-06-01 Séquençage nucléotidique de prochaine génération utilisant un adaptateur comprenant séquence de code à barres WO2016195382A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3431611A1 (fr) * 2017-07-21 2019-01-23 Menarini Silicon Biosystems S.p.A. Procédé amélioré et kit pour la génération de bibliothèques d'adn pour un séquençage massivement parallèle
CN109797197A (zh) * 2019-02-11 2019-05-24 杭州纽安津生物科技有限公司 一种单链分子标签接头及单链dna建库方法及其在检测循环肿瘤dna中应用
CN110719958A (zh) * 2017-04-06 2020-01-21 王磬 构建核酸文库的方法和试剂盒
CN111005075A (zh) * 2019-12-20 2020-04-14 北京科迅生物技术有限公司 用于双样本共建测序文库的y型接头和双样本共建测序文库的方法
WO2022114732A1 (fr) * 2020-11-27 2022-06-02 연세대학교 산학협력단 Procédé permettant de réaliser un groupe par connexion d'informations de brins générés pendant un processus de pcr et suivi de l'ordre de génération de brins générés
WO2022199242A1 (fr) * 2021-03-25 2022-09-29 南方医科大学 Ensemble de lieurs de code à barres et procédé de construction et de séquençage de bibliothèque de méthylation d'adn représentative à cellules uniques multiples à flux de milieu

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11505826B2 (en) * 2017-07-12 2022-11-22 Agilent Technologies, Inc. Sequencing method for genomic rearrangement detection
US11339424B2 (en) 2017-09-06 2022-05-24 Dxome Co., Ltd. Method for amplification and quantitation of small amount of mutation using molecular barcode and blocking oligonucleotide
KR101967879B1 (ko) * 2017-11-30 2019-04-10 사회복지법인 삼성생명공익재단 핵산 서열분석에서 uid 핵산 서열의 순결도를 측정하는 방법
WO2020014331A1 (fr) * 2018-07-13 2020-01-16 Coral Genomics, Inc. Procédés d'analyse de cellules
KR102187795B1 (ko) * 2019-04-30 2020-12-08 (주)디엑솜 디옥시우리딘을 이용한 차세대 염기서열 분석을 위한 라이브러리 제조 방법
KR20220122095A (ko) 2021-02-26 2022-09-02 지니너스 주식회사 분자 바코딩 효율을 향상시키기 위한 조성물 및 이의 용도
WO2022181858A1 (fr) * 2021-02-26 2022-09-01 지니너스 주식회사 Composition pour améliorer l'efficacité du codage à barres moléculaire et son utilisation
WO2022216133A1 (fr) * 2021-04-09 2022-10-13 주식회사 셀레믹스 Procédé simplifié de préparation de bibliothèque de séquençage de nouvelle génération

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120238738A1 (en) * 2010-07-19 2012-09-20 New England Biolabs, Inc. Oligonucleotide Adapters: Compositions and Methods of Use
KR20130018575A (ko) * 2011-08-01 2013-02-25 연세대학교 산학협력단 핵산분자의 제조방법
US20140031240A1 (en) * 2012-07-03 2014-01-30 Foundation Medicine, Inc. Tm-enhanced blocking oligonucleotides and baits for improved target enrichment and reduced off-target selection
WO2014071361A1 (fr) * 2012-11-05 2014-05-08 Rubicon Genomics Marquage par code-barre d'acides nucléiques
WO2014096394A1 (fr) * 2012-12-23 2014-06-26 Hs Diagnomics Gmbh Procédés et jeux d'amorces pour séquençage par pcr à haut rendement

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106103743A (zh) 2014-01-07 2016-11-09 Imppc私人基金会 用于产生双链dna文库的方法和用于鉴定甲基化胞嘧啶的测序方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120238738A1 (en) * 2010-07-19 2012-09-20 New England Biolabs, Inc. Oligonucleotide Adapters: Compositions and Methods of Use
KR20130018575A (ko) * 2011-08-01 2013-02-25 연세대학교 산학협력단 핵산분자의 제조방법
US20140031240A1 (en) * 2012-07-03 2014-01-30 Foundation Medicine, Inc. Tm-enhanced blocking oligonucleotides and baits for improved target enrichment and reduced off-target selection
WO2014071361A1 (fr) * 2012-11-05 2014-05-08 Rubicon Genomics Marquage par code-barre d'acides nucléiques
WO2014096394A1 (fr) * 2012-12-23 2014-06-26 Hs Diagnomics Gmbh Procédés et jeux d'amorces pour séquençage par pcr à haut rendement

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIM, HA YOUNG: "Method for Rare Mutations Detection based on High Depth Sequencing using Barcode Inserted Adapter", MASTER'S THESIS, February 2016 (2016-02-01), pages i - xii, 1-62 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3607065A4 (fr) * 2017-04-06 2020-04-15 Qing Wang Procédé et kit de construction d'une bibliothèque d'acides nucléiques
CN110719958A (zh) * 2017-04-06 2020-01-21 王磬 构建核酸文库的方法和试剂盒
CN110719958B (zh) * 2017-04-06 2023-07-18 康博国际有限公司 构建核酸文库的方法和试剂盒
WO2019016401A1 (fr) * 2017-07-21 2019-01-24 Menarini Silicon Biosystems S.P.A. Méthode améliorée et kit pour la génération de bibliothèque d'adn pour le séquençage massivement parallèle
CN110959045A (zh) * 2017-07-21 2020-04-03 美纳里尼硅生物系统股份公司 生成大规模平行测序的dna文库的改进的方法和试剂盒
EP3431611A1 (fr) * 2017-07-21 2019-01-23 Menarini Silicon Biosystems S.p.A. Procédé amélioré et kit pour la génération de bibliothèques d'adn pour un séquençage massivement parallèle
US11859249B2 (en) 2017-07-21 2024-01-02 Menarini Silicon Biosystems S.P.A. Method and kit for the generation of DNA libraries for massively parallel sequencing
CN110959045B (zh) * 2017-07-21 2024-04-16 美纳里尼硅生物系统股份公司 生成大规模平行测序的dna文库的改进的方法和试剂盒
CN109797197A (zh) * 2019-02-11 2019-05-24 杭州纽安津生物科技有限公司 一种单链分子标签接头及单链dna建库方法及其在检测循环肿瘤dna中应用
CN111005075A (zh) * 2019-12-20 2020-04-14 北京科迅生物技术有限公司 用于双样本共建测序文库的y型接头和双样本共建测序文库的方法
CN111005075B (zh) * 2019-12-20 2023-04-21 北京科迅生物技术有限公司 用于双样本共建测序文库的y型接头和双样本共建测序文库的方法
WO2022114732A1 (fr) * 2020-11-27 2022-06-02 연세대학교 산학협력단 Procédé permettant de réaliser un groupe par connexion d'informations de brins générés pendant un processus de pcr et suivi de l'ordre de génération de brins générés
WO2022199242A1 (fr) * 2021-03-25 2022-09-29 南方医科大学 Ensemble de lieurs de code à barres et procédé de construction et de séquençage de bibliothèque de méthylation d'adn représentative à cellules uniques multiples à flux de milieu

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