A connector, a kit and a library construction method for multi-sample pooling library construction

Abstract

The present invention discloses a connector, a kit and a method for building a library for multi-sample pooling, wherein the connector comprises a connector positive chain and a connector negative chain, the sequence of the connector positive chain is shown in SEQ ID NO.1, and the sequence of the connector negative chain is shown in SEQ ID NO.2; the connector positive chain and the connector negative chain form a double-stranded connector after annealing. The scheme of the present invention designs a two-level label for the connector, which greatly increases the number of available connectors on the basis of effectively saving the cost of connector synthesis, provides enough connectors for large-scale sample pooling sequencing, and effectively solves the problem of insufficient number of connectors often occurring in multi-sample pooling sequencing. The unequal length connector of the inner label sequence of 4-10bp can be used for but not limited to the construction of second-generation sequencing libraries such as ordinary PCR product sequencing, multiple PCR product sequencing, large-scale sequencing identification of gene editing body offspring, low-depth resequencing, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a connector, a kit and a library construction method for multi-sample pooling library construction. Background Art

[0002] High-throughput sequencing technology has become one of the most important molecular biology analysis methods in the field of life sciences. It not only provides important data for basic life science research such as genome genetic information decoding, gene expression regulation, and species genetic evolution, but also plays an increasingly important role in many fields or scenarios such as animal and plant germplasm resource identification and molecular breeding, genetic diagnosis and gene therapy of human diseases, identification and detection of infectious bacteria or viruses, and environmental monitoring and governance.

[0003] Related technologies include high-throughput sequencing based on the MGI sequencing platform, using fragmented DNA fragments and bubble-shaped adapters to connect library construction adapters at both ends of the fragmented DNA fragments; after mixing equal volumes of every 8 connection products, magnetic bead double selection is performed to select the target fragment interval for the next step of library construction; low-cycle PCR amplification of the magnetic bead double selection products to form a sequencing library structure with sequencing primer binding sequences and circularization primer binding sequences on both sides of the target fragment interval; after equal amounts of PCR products are mixed, a circularization reaction is performed, and then the circularization products are digested with exonucleases ExoⅠ and ExoⅢ to remove uncircularized single-stranded and double-stranded DNA, thereby completing the preparation of the circularized library. In this technology, the library construction connector carries a 10-base-long Barcode tag sequence, and the MGI platform supplier provides a total of 192 Barcode tags for use; after connection through the connector, each sample thus carries a Barcode tag with a unique sequence; after library sequencing, in each sequencing lane, the sequencing data of the corresponding sample can be extracted by identifying the Barcode tag sequence, and one sequencing lane can identify and distinguish the sequencing data of 1-96 samples. However, the relevant technology only designs Barcode tags on the library construction connector, with a total of 96, 128, 192, etc.; one sequencing lane can only distinguish the sequencing data of 192 samples at most, which cannot meet the demand of placing more samples in one lane or one chip for sequencing at the same time; the library construction connector based on the relevant platform technology can only construct libraries for DNA fragments with different bases at the same position after fragmentation. If the gene sequences of a certain region of the DNA fragments to be used for library construction are identical, it will require additional dark reactions during the sequencing process, increasing sequencing costs, or directly causing sequencer errors and sequencing failures. This type of DNA fragment includes, but is not limited to: products amplified by the same PCR primers, DNA fragments produced by restriction endonucleases, etc. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a connector for multi-sample pooling library construction.

[0005] The present invention also provides a kit for building a library on the MGI platform.

[0006] The present invention also provides a method for constructing a second-generation sequencing library.

[0007] According to one aspect of the present invention, a connector for multi-sample pooling library construction is proposed, wherein the connector comprises a connector positive chain and a connector negative chain, the sequence of the connector positive chain is shown in SEQ ID NO.1, and the sequence of the connector negative chain is shown in SEQ ID NO.2; the connector positive chain and the connector negative chain form a double-stranded connector after annealing.

[0008] In some embodiments of the present invention, N at positions 39 to 48 in the sequence of the forward strand of the adapter is a forward sequence of an Index tag, with a length of 4-10 bases.

[0009] In some embodiments of the present invention, N at position 1 to position 10 in the sequence of the negative strand of the adapter is a reverse complementary sequence of the Index tag, with a length of 4-10 bases.

[0010] In some embodiments of the present invention, the sequence of the Index tag includes at least one of SEQ ID NO.6-SEQ ID NO.53.

[0011] According to a second aspect of the present invention, a kit for building a library on the MGI platform is provided, the kit comprising the above-mentioned linker.

[0012] In some embodiments of the present invention, the kit further comprises a universal primer, and the sequence of the universal primer is shown in SEQ ID NO.3.

[0013] In some embodiments of the present invention, the kit further comprises a Barcode Primer, and the sequence of the Barcode Primer is shown in SEQ ID NO. 4. The N at positions 18 to 27 of the Barcode Primer sequence shown in SEQ ID NO. 4 represents the sequence of the Barcode tag. Specifically, the Barcode tag includes but is not limited to 192 Barcode tags provided by the MGI platform supplier.

[0014] In some embodiments of the present invention, the kit further comprises a Splint Oligo sequence, and the SplintOligo sequence is shown as SEQ ID NO.5.

[0015] According to the third aspect of the present invention, a method for constructing a second-generation sequencing library is proposed, the method comprising the following steps: connecting the above-mentioned linker to the fragmented DNA to obtain linker-carrying DNA fragments; and performing PCR amplification on the linker-carrying DNA fragments to obtain a sequencing library.

[0016] In some embodiments of the present invention, the method for fragmenting DNA is ultrasonic shearing, fragmentation enzyme digestion or restriction endonuclease digestion.

[0017] In some embodiments of the present invention, before connecting the adapter to the fragmented DNA, a step of annealing two sequences of the adapter is also included.

[0018] In some embodiments of the present invention, during annealing, the added mass ratio of the two sequences is 1:1.

[0019] In some embodiments of the present invention, the annealing procedure is incubating at 92-98°C for 2-5 minutes, decreasing the temperature by 0.8-1.2°C every 85-95 seconds until the temperature reaches 20-28°C.

[0020] In some embodiments of the present invention, before the adapter is connected to the fragmented DNA, a step of performing end repair and A addition on the fragmented DNA is also included.

[0021] In some embodiments of the present invention, the primers used in the PCR amplification include universal primers and Barcode Primer.

[0022] In some embodiments of the present invention, the sequence of the universal primer is shown as SEQ ID NO.3.

[0023] In some embodiments of the present invention, the sequence of the Barcode Primer is shown as SEQ ID NO.4.

[0024] In some embodiments of the present invention, the method further comprises the step of purifying the sequencing library, wherein the purification is performed using magnetic beads.

[0025] In some embodiments of the present invention, the sequencing library further comprises a circularization step to obtain a circularized library.

[0026] In some embodiments of the present invention, the reaction system used for the cyclization is as follows:

[0027]

[0028] In some embodiments of the present invention, the reaction procedure used for the cyclization is to incubate the system used for the cyclization at 35-40° C. for 28-35 min.

[0029] In some embodiments of the present invention, the Splint Oligo sequence is shown as SEQ ID NO.5.

[0030] In some embodiments of the present invention, the method further comprises the step of purifying the circularized library, wherein the purification is performed using magnetic beads.

[0031] According to some embodiments of the present invention, at least the following beneficial effects are achieved: a primary label (48 Index label sequences) is set on the connector used for multi-sample pooling library construction in the scheme of the present invention, and a secondary label is set on the PCR primer (MGI platform supplier provides 192 Barcode label sequences. At the same time, as the number of Barcode labels released by the MGI platform supplier increases, the Barcode label sequences can also be increased accordingly, thereby further increasing the number of samples that can be pooled). The two-level labels are used to mark the samples in the library, so that each sequencing lane can distinguish up to 9216 (48×192 combinations) samples through labels; at the same time, in the design of the Index label sequence, base sequences of different lengths (4-10) are used, and the base balance between base sequences of unequal lengths is optimized, so that the connector system used can be used for library construction of various types of DNA fragments. The scheme of the present invention designs two-level tags in the sequencing library, which greatly increases the number of available adapters on the basis of effectively saving the cost of adapter synthesis, provides enough adapters for large-scale sample pooling sequencing, and effectively solves the problem of insufficient number of adapters that often occurs in multi-sample pooling sequencing. The unequal length adapters of the inner tag sequence of 4-10bp can be suitable for the construction of libraries with the same DNA fragments in a certain region of gene sequences, and can be used for but not limited to the construction of second-generation sequencing libraries such as ordinary PCR product sequencing, multiple PCR product sequencing, large-scale sequencing and identification of gene-edited progeny, whole genome resequencing, low-depth resequencing, and simplified sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0033] Figure 1 This is a schematic diagram of the joint structure in Example 1 of the present invention;

[0034] Figure 2 Schematic diagram of the library structure in Example 1 of the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0036] Example 1

[0037] This example prepares a method for constructing a two-stage tagged single-stranded circular library based on the MGI sequencing platform. The specific process is as follows:

[0038] 1. Experimental subjects: Materials for rice population purity identification and materials for selecting individual plants from rice blast-resistant populations, a total of 288 DNA samples.

[0039] 2. Joint design and synthesis preparation

[0040] (1) Synthesize the following sequence:

[0041] Sequence 1: 5'-TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTTNNNNNNNNNNT-3' (SEQ ID NO. 1), N from position 39 to position 48 is the forward sequence of the Index tag, with a length of 4-10 bases;

[0042] Sequence 2: 5'Phos-NNNNNNNNNNAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAACTCACGAGTAGGCTAGA-3' (SEQ ID NO. 2), wherein N from position 1 to position 10 is the reverse complementary sequence of the Index tag, with a length of 4-10 bases.

[0043] The Index tag sequence corresponding to each connector is shown in Table 1.

[0044] Table 1. 48 Index tag sequences of different lengths

[0045] Adapter number Index tag sequence Adapter number Index tag sequence 1 TACA (SEQ ID NO.6) 25 CTAG (SEQ ID NO.30) 2 ATGG (SEQ ID NO.7) 26 GAGAC (SEQ ID NO.31) 3 GGTA (SEQ ID NO.8) 27 ACTCG (SEQ ID NO.32) 4 CGAT (SEQ ID NO.9) 28 TGCTCG (SEQ ID NO.33) 5 ACTCA (SEQ ID NO.10) 29 GACGACGC (SEQ ID NO.34) 6 GACCA (SEQ ID NO.11) 30 ACTTGGC (SEQ ID NO.35) 7 CTGTA (SEQ ID NO.12) 31 TGAA (SEQ ID NO.36) 8 AGTGCC (SEQ ID NO.13) 32 CTGC (SEQ ID NO.37) 9 TCAGGA (SEQ ID NO.14) 33 AGATG (SEQ ID NO.38) 10 CTCACGC (SEQ ID NO.15) 34 TACCGA (SEQ ID NO.39) 11 GAACGCA (SEQ ID NO.16) 35 CTGGA (SEQ ID NO.40) 12 TCGTCAGG (SEQ ID NO.17) 36 GCTACCA (SEQ ID NO.41) 13 ACGTACCA (SEQ ID NO.18) 37 AGTCAAGCG (SEQ ID NO.42) 14 TGAACGC (SEQ ID NO.19) 38 CTGTCGCAC (SEQ ID NO.43) 15 CTGT (SEQ ID NO.20) 39 GACG (SEQ ID NO.44) 16 GACC (SEQ ID NO.21) 40 TCAAGACCG (SEQ ID NO.45) 17 ACTGG (SEQ ID NO.22) 41 AGCT (SEQ ID NO.46) 18 CTAGAG (SEQ ID NO.23) 42 CTACACGGAA (SEQ ID NO.47) 19 TGCA (SEQ ID NO.24) 43 TCTGGCAACG (SEQ ID NO.48) 20 CATCG (SEQ ID NO.25) 44 GAGACGACCA (SEQ ID NO.49) 21 ACTACA (SEQ ID NO.26) 45 GCTA (SEQ ID NO.50) 22 GACTGC (SEQ ID NO.27) 46 TGAGG (SEQ ID NO.51) 23 TGAGCAG (SEQ ID NO.28) 47 ATCCAC (SEQ ID NO.52) 24 GTGCAGAG (SEQ ID NO.29) 48 CAGT (SEQ ID NO.53)

[0046] (2) Preparation of joint

[0047] Add DNA annealing buffer to the oligo dry powder of sequence 1 and sequence 2 to dilute them, and obtain a 20μM solution of sequence 1 and sequence 2. Mix the oligo solutions of sequence 1 and sequence 2 in equal volumes, and place them on a PCR instrument for annealing to form a linker. The annealing program is: incubate at 95℃ for 3min, decrease 1℃ every 90sec until the temperature reaches 25℃, and store temporarily at 4℃ or store long-term at -20℃. The obtained linker concentration is 10μM. The linker structure is as follows Figure 1 shown.

[0048] 3. Construction of circularized library

[0049] (1) Genome enzyme cutting and interruption:

[0050] The genome was double-digested using restriction endonucleases TaqⅠ and MspⅠ. The double-digestion system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0051] Table 2

[0052] Component Volume <![CDATA[ddH2O]]> 5 μL 10×T4 DNA Ligase Buffer 3μL TaqⅠ(20U / μL) 1μL MspⅠ(20U / μL) 1μL Genomic DNA (50ng / μL) 20μL Total 30μL

[0053] Table 3

[0054] temperature time 37℃ 10min 65℃ 10min 80℃ 20min 4℃ Hold

[0055] After the enzyme digestion reaction, the genome is broken into DNA fragments of different lengths, with both ends of the fragments having 5' protruding sticky ends CG.

[0056] (2) End repair, adding A

[0057] The configuration of the end repair plus A reaction system is shown in Table 4.

[0058] Table 4

[0059]

[0060]

[0061] Use a pipette to gently pipette or shake to mix, and briefly centrifuge the reaction solution to the bottom of the tube.

[0062] Place the above PCR tube in a PCR instrument, set the reaction program shown in Table 5, and perform end repair / dA tail addition reaction.

[0063] Table 5

[0064] temperature time Heating cover 105℃ On 30℃ 20min 72℃ 20min 4℃ Hold

[0065] (3) Connector connection:

[0066] Connectors were connected to both ends of the double-enzyme digestion products to label each sample with a unique Index. The connection system is shown in Table 6, and the connection reaction procedure is shown in Table 7.

[0067] Table 6

[0068] Components volume <![CDATA[ddH2O]]> 3μL 10×T4 DNA Ligase Buffer 1μL Linker (10μM) 5μL T4 DNA Ligase (600U / μL) 1μL Double enzyme digestion product 30μL Total 40μL

[0069] Table 7

[0070] temperature time 23℃ 60min 4℃ Hold

[0071] (4) The ligation products are mixed into a sample library:

[0072] The ligation products with different Index tag sequences were grouped into 24, and 10 μL of each volume was taken to mix into a sample library. The ligation product library was purified using QIAquick PCR Purification Kit and eluted and recovered using 20 μL 1×TEBuffer.

[0073] (5) Select the target DNA fragment:

[0074] The Pippin HT high-throughput automated DNA fragment recovery system was used to select and recover DNA fragments in the range of 300-500 bp from the ligation product library, and 30 μL TE Buffer was used for elution and recovery; the Qubit TM The concentration of the product recovered by PippinHT was detected by dsDNA HS AssayKit and diluted to a concentration of 1 ng / μL with TE Buffer.

[0075] (6) PCR amplification:

[0076] Low-cycle PCR amplification of the target DNA fragments was performed using PCR common primer and 12 PCR Barcode primers (sequences are shown in Tables 10 and 11, wherein PCR common primer and PCR Barcode primer were diluted to a concentration of 10 μM by adding DNA annealing buffer, and each library was labeled with a unique Barcode. The PCR reaction system is shown in Table 8, and the reaction procedure is shown in Table 9.

[0077] Table 8

[0078] Element volume <![CDATA[ddH2O]]> 13μL KAPA HiFiHotStartReadyMix 25μL PCR common primer (10 μM) 1μL PCR Barcode primer (10 μM) 1μL Target DNA fragment 10μL Total 50μL

[0079] Table 9

[0080]

[0081] Table 10

[0082]

[0083]

[0084] In the Barcode Primer sequence shown in SEQ ID NO.4, N from the 18th to the 27th position is the sequence of the Barcode tag, and the Barcode tag can be any Barcode tag released by the MGI platform supplier. Currently, the MGI platform provides 192 Barcode tag sequences (the sequences used in this embodiment are shown in SEQ ID NO.54-SEQ ID NO.65). As the number of Barcode tags released by the MGI platform increases, the newly added Barcode tag sequences can also be used, thereby further increasing the number of samples that can be pooled.

[0085] The sequences of the barcode tags in the 12 PCR barcode primers used in this example are shown in Table 11.

[0086] Table 11

[0087] Barcode label MGI number Barcode tag sequence 01 GTGAGTGATG (SEQ ID NO.54) 02 GAGTCAGCTG (SEQ ID NO.55) 03 TGTCTGCGAA (SEQ ID NO.56) 04 ATTGGTACAA (SEQ ID NO.57) 13 AACCTAGATA (SEQ ID NO. 58) 14 TTGCCATCTC (SEQ ID NO. 59) 15 AGATCTTGCG (SEQ ID NO. 60) 16 CGCTATCGGC (SEQ ID NO. 61) 41 ATTCAACGGA (SEQ ID NO.62) 42 AACTGTACTG (SEQ ID NO.63) 43 GTACCTCAAT (SEQ ID NO. 64) 44 GACTTCTAAT (SEQ ID NO.65)

[0088] After the reaction was completed, 50 μL Ampure XP magnetic beads were added to purify and recover the PCR product, and eluted in 30 μL TE Buffer; TM The concentration of the PCR purified product was detected by dsDNA HS Assay Kit and diluted to a concentration of 5 ng / μL with TE Buffer; 1 μL of the PCR purified product was taken to the Qsep100 fully automatic nucleic acid protein analysis system for fragment interval detection.

[0089] (7) Preparation of circularized library:

[0090] 12 PCR libraries with different barcode labels, 3 μL of each library was mixed into 1 tube, the total DNA amount was 180 ng, and ddH2O was added to make the volume 40 μL; incubated at 95°C for 3 min in a PCR instrument, and immediately taken out and treated in an ice bath to form a single-stranded separation product; the cyclization system was prepared as shown in Table 12, and the cyclization reaction procedure was shown in Table 13.

[0091] Table 12

[0092] Components volume <![CDATA[ddH2O]]> 8μL 10×T4 DNA Ligase Buffer 6μL SplintOligo (10 μM) (sequence as shown in SEQ ID NO.5) 5μL T4 DNA Ligase (600U / μL) 1μL Single-stranded separation product 40μL Total 60μL

[0093] Table 13

[0094] temperature time 37℃ 30min 4℃ Hold

[0095] After the cyclization reaction is completed, DNA exonuclease is used to digest the uncyclized single-stranded DNA and double-stranded DNA. The exonuclease reaction system is shown in Table 14, and the reaction procedure is shown in Table 15.

[0096] Table 14

[0097] Components volume <![CDATA[ddH2O]]> 7.5μL 10×T4 DNA Ligase Buffer 1μL ExoⅠ(20U / μL) 1μL ExoⅢ(100U / μL) 0.5μL Cyclization product 60μL Total 70μL

[0098] Table 15

[0099]

[0100]

[0101] Add 10 μL 0.1M EDTA (pH 8.0) to terminate the exonuclease activity; add 160 μL Ampure XP magnetic beads to purify and recover the circularized library, and elute in 30 μL TE Buffer; use Qubit TM The concentration of the circularized library was detected by ssDNA Assay Kit and stored at -20°C for sequencing.

[0102] (8) Preparation of DNB and sequencing:

[0103] The prepared circularized library was subjected to DNB preparation according to the official kit of the MGI platform.

[0104] After DNB preparation, sequencing was performed on the MGISEQ-2000 or DNBSEQ-T7 platform. The sequencing results showed that the total data volume of 288 samples was 152G, and the average data quality value Q30% was 91.39. The sequencing data volume and quality were both good. The Q30% quality values ​​of the 1st to 3rd bases at the start position of the second-chain sequencing were 90.99, 96.5, and 89, respectively. After filtering and splitting the offline data, an average of 528M data was obtained for each sample, of which the lowest data volume was 474M and the highest data volume was 586M. The library diagram is shown below. Figure 2 shown.

[0105] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A connector for multi-sample pooling library construction, characterized in that: The linker comprises a linker positive chain and a linker negative chain, the sequence of the linker positive chain is shown in SEQ ID NO.1, and the sequence of the linker negative chain is shown in SEQ ID NO.2; the linker positive chain and the linker negative chain form a double-stranded linker after annealing; The N from the 39th to the 48th position of the sequence of the positive chain of the adapter is the forward sequence of the Index tag, which is 4-10 bases in length, and the N from the 1st to the 10th position of the sequence of the negative chain of the adapter is the reverse complementary sequence of the Index tag, which is 4-10 bases in length; The sequence of the Index tag is shown in SEQ ID NO.6-SEQ ID NO.53; The sequences of the Index tags are sequences of unequal length; The platform used for multi-sample pooling library construction is MGI; The adapter containing the Index tag can be used for library construction of DNA fragments with identical gene sequences in a certain region.

2. A kit for building a library on the MGI platform, characterized in that: The kit comprises the linker of claim 1.

3. The kit according to claim 2, characterized in that The kit also includes a universal primer, the sequence of which is shown in SEQ ID NO.

3.

4. The kit according to claim 2, characterized in that The kit further comprises a Barcode Primer, the sequence of which is shown in SEQ ID NO.

4.

5. The kit according to claim 2, characterized in that The kit further comprises Splint Oligo, the sequence of which is shown as SEQ ID NO.

5.

6. A method for constructing a second-generation sequencing library, characterized in that: The method comprises the following steps: connecting the linker described in claim 1 to fragmented DNA to obtain linker-carrying DNA fragments; and performing PCR amplification on the linker-carrying DNA fragments to obtain a sequencing library.

7. The construction method according to claim 6, characterized in that: Before connecting the adapter to the fragmented DNA, the method further includes a step of annealing the two sequences of the adapter.

8. The construction method according to claim 7, characterized in that: The annealing procedure is to incubate at 92-98°C for 2-5 minutes, and decrease the temperature by 0.8-1.2°C every 85-95 seconds until the temperature reaches 20-28°C.

9. The construction method according to claim 6, characterized in that: The construction method further comprises the step of circularizing the sequencing library.

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