Low-depth whole genome sequencing and targeted sequencing combined library building method

Abstract

A low-depth whole genome sequencing and targeted sequencing combined library building method, comprising the following steps: using a restriction endonuclease for sample compatibility to shear a nucleic acid sample to obtain a long DNA fragment; using a random primer with a universal sequence and a gene-specific primer pair having a universal sequence to amplify the DNA fragment to obtain an amplified product; and using an adapter primer pair to carry out second amplification on the amplified product to obtain a DNA library for sequencing.

Description

Library construction method combining low-depth whole-genome sequencing and targeted sequencing

[0001] The present invention claims priority to Chinese patent application No. 2023107758048, filed with the Patent Office of China on June 28, 2023, entitled “Library construction method combining low-depth whole-genome sequencing and targeted sequencing”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biotechnology and relates to gene sequencing, and specifically to a library construction method combining low-depth whole-genome sequencing and targeted sequencing. Background Art

[0003] With the continuous iteration and advancement of sequencing technologies, from Sanger sequencing to high-throughput sequencing, sequencing costs have been reduced, throughput has been increased, and speed has been increased. This allows for accurate detection of gene copy number variations, indels, point mutations, and other genomic features. However, different types of mutations require different sequencing requirements. Detection of copy number variations requires genome-wide sequencing, detecting insertions, deletions, inversions, or duplications of DNA fragments larger than 1 kb in length. Therefore, uniform genome coverage is required, while sequencing depth is not required; 0.6x is generally sufficient. For insertions, deletions, and single-nucleotide polymorphisms (SNPs), only the target region is of interest, requiring a sequencing depth of at least 20x, while non-target regions are not subject to this requirement. Therefore, in practice, to reduce costs, whole-genome sequencing or targeted sequencing is often chosen, depending on the specific situation (CN 111379032 A). Therefore, if information on copy number variations, indels, and SNPs is desired simultaneously, whole-genome sequencing and targeted sequencing must be performed separately. This approach is not only time-consuming, cumbersome, and costly. In response to this situation, two companies have made technical improvements for different application directions, which can perform whole genome and targeted sequencing simultaneously. Beijing Berry Genomics combines whole genome sequencing and targeted sequencing in single-cell applications. By using random primers and specific primers with universal sequences to simultaneously amplify the cell genomic DNA, and then using labeled adapter primers for a second round of amplification, a sequenceable library is obtained. This method can simultaneously detect copy number variations and gene mutations in single cells. Another company is Wuhan BGI, which combines whole genome sequencing and targeted sequencing in pathogen detection. Its main principle is to first construct a whole genome library using the nucleic acid of the sample. Subsequently, universal primers for adapters and specific primers with universal adapters are used to amplify and enrich the whole genome and target region at the same time, and then primers with complete adapters and universal primers for adapters are used to simultaneously amplify the target region and the whole genome region, so that the target region has a complete adapter. A sequenceable library is obtained, achieving the purpose of simultaneously detecting known pathogens and unknown pathogens (CN 111690639 B).

[0004] The simultaneous amplification method using random primers and specific primers with universal sequences has disadvantages. On the one hand, this technology is mainly targeted at single-cell applications and has a narrow scope of application. On the other hand, the library constructed by this method is difficult to process. One reason is that many adapter dimers and multimers are generated during the library construction process and are difficult to remove, resulting in a reduction in the effective sequencing data volume. Another reason is that because whole-genome libraries are constructed by random primer PCR, fragment length is uncontrollable and fragment screening is required, which may result in data loss. Without fragment screening, the library will be more diffuse, affecting the sequencing quality. The method of combining whole-genome sequencing with targeted sequencing first constructs a whole-genome library for the sample, and then performs specific amplification of the target region within the library. A disadvantage of this method is that when the target pathogen is present in low levels, constructing the whole-genome library first may result in a reduction in the target pathogen nucleic acid. At the same time, the nucleic acid fragmentation required for whole-genome library construction may cause the target region to be cut off, resulting in failure of specific primer amplification and undetectable results.

[0005] Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a library construction method that combines low-depth whole-genome sequencing and targeted sequencing with wide application, simple operation, short time and low cost. It can simultaneously detect copy number variation, point mutation and insertion and deletion mutation under low-depth sequencing data volume, and can be applied to genotyping in genetic breeding, infection analysis of pathogens and detection of drug resistance sites.

[0007] The technical solutions for achieving the above-mentioned objectives include the following.

[0008] In a first aspect, the present invention provides a library construction method combining low-depth whole-genome sequencing and targeted sequencing, comprising the following steps: Step 1: shearing a nucleic acid sample using a restriction endonuclease adapted to the sample to obtain longer DNA fragments;

[0009] Step 2: Use random primers and gene-specific primers to amplify the DNA fragments in step 1 in proportion to obtain amplified products;

[0010] Step 3: Use the adapter primer to perform a second amplification on the amplified product of step 2 to obtain a DNA library for sequencing.

[0011] The above-mentioned library construction method can solve the problem of scattered and unfocused library fragments and greatly reduce the risk of target fragments being interrupted.

[0012] Based on the genomic sequence and the restriction endonuclease recognition sites, restriction endonucleases capable of breaking the genome into long fragments are screened to obtain a long DNA fragment product 1. Using gene-specific primers with universal adapter sequences and random primers with universal adapter sequences, DNA fragment product 1 is amplified in the first round of PCR at an optimized ratio to obtain DNA fragment 2. A second round of PCR amplification of DNA fragment 2 is performed using primers with tag sequences to obtain a DNA library for sequencing.

[0013] In some embodiments, the restriction endonuclease can be one or a mixture of several. For example, a restriction endonuclease that can recognize methylation modifications, such as BamHI-HF, McrBC, AbaSI, FspEI, LpnPI, MspJI, etc., can be selected to break the double-stranded DNA near the base by recognizing the methylation-modified bases on the genome. A restriction endonuclease that can recognize the terminator sequence AATAAA in the chromosome coding region, such as SspI-HF and SspI, can also be used to break the double-stranded DNA.

[0014] In some embodiments, the two enzymes mentioned above can also be used in combination.

[0015] In some embodiments, after the genomic nucleic acid is fragmented, gene-specific primers with universal sequences and random primers with universal sequences are mixed in different proportions for the first round of PCR amplification. Preferably, the amount of gene-specific primers with universal sequences is lower than that of random primers with universal sequences. For example, the amount ratio of gene-specific primers to random primers with universal sequences is in the range of 1:1 to 1:20, which can obtain information on specific sites without affecting the sequencing effect of uniformity of whole-genome coverage. Preferably, it is 1:5 to 20, more preferably 1:10 to 15, or the mixing ratio can be adjusted according to the ratio of the size of the target region to be specifically amplified to the size of the genome.

[0016] Then, using adapters with indexed sequences, the first-round PCR products are amplified a second time to create a library ready for sequencing. Only a small amount of data is needed to obtain low-depth whole-genome information and information on targeted regions of interest, enabling simultaneous analysis of copy number, point mutations, indels, and other information, or comparative analysis of paternal and maternal genetic information.

[0017] In some embodiments, the universal sequence random primer comprises three parts, the first part is a universal adapter sequence, which can be conventionally designed according to different sequencing platforms, the second part is a random sequence of 6 Ns, and the third part is three G bases for increasing the Tm value.

[0018] In some embodiments, the gene-specific primer comprises two parts: a first part is a universal adapter sequence, which is routinely selected based on different sequencing platforms; and a second part is a target-specific primer, which is designed based on the target region of interest.

[0019] In a second aspect, the present invention provides the application of any of the above-mentioned library construction methods combining low-depth whole-genome sequencing and targeted sequencing in genotyping in genetic breeding, pathogen detection, and detection of drug-resistant sites.

[0020] The library construction method described in the present invention, which combines low-depth whole-genome sequencing with targeted sequencing, can simultaneously detect copy number variations, point mutations, and insertion-deletion mutations, etc., with low-depth sequencing data volume. It also has the advantages of simple operation and short library construction time. The constructed library is of high quality, free of residual adapter dimers, with concentrated library fragments and good peak shape.

[0021] The library construction method combining low-depth whole-genome sequencing and targeted sequencing described in the present invention has low sequencing costs, can perform genetic typing analysis, and can also simultaneously detect copy number variation, structural variation, insertion and deletion, and point mutations. It can be applied to genotyping in genetic breeding, pathogen infection analysis, and detection of drug-resistant sites.

[0022] In a third aspect, the invention further provides a library construction method combining low-depth whole-genome sequencing and targeted sequencing, comprising the following steps:

[0023] Step 1.1: First, a portion of genomic DNA is taken from the sample to be tested and amplified using one or more pairs of specific primers 1 to obtain one or more DNA fragments 1 of 600 bp or longer (specific amplification products);

[0024] Step 1.2: The DNA fragment 1 is mixed with the original genomic DNA in a certain ratio, and then the fragment is repaired and A is added, and an adapter is added, and the ligation product is eluted and purified to obtain an eluted product;

[0025] Step 1.3: Perform PCR using full-length adapter primers with indexed sequences to generate a sequenceable library.

[0026] Or include the following steps:

[0027] Step 2.1: First, a portion of genomic DNA is taken from the sample to be tested, and one or more pairs of specific primers 2 are used to amplify the target region to obtain one or more DNA amplified fragments 2 (specific amplification products);

[0028] Step 2.2: Simultaneously, another portion of genomic DNA is taken from the sample to be tested and subjected to the steps of fragmentation, end repair, A addition, and adapter addition to obtain DNA fragment product 3 (adapter product);

[0029] Step 2.3: Mix the DNA amplified fragment 2 and the DNA fragment product 3 in a certain ratio, add the full-length adapter primer with the tag sequence for amplification, and obtain a sequenceable library.

[0030] In some embodiments, the length of the amplified DNA fragment 2 is within the range of 100 bp-1000 bp, preferably within the range of 200 bp-500 bp.

[0031] In some embodiments, the specific amplification primers 1 include a forward specific primer 1 and a reverse specific primer 1, each of which is 17 to 25 bp in length, and is completely complementary to the genome at both ends, or is a degenerate primer.

[0032] In some embodiments, the adapter described in step 1.2 is a universal short adapter without a tag, comprising two oligonucleotide chains S1 and S2, wherein S1 and S2 are partially reverse-complementary to form a double strand, and the 3' end of S1 protrudes one more T base than the 5' end of S2, which can complementarily pair with the DNA fragment after the end is repaired and A is added to complete the adapter connection.

[0033] In some embodiments, the full-length adapter primer with a tag sequence described in step 1.3 comprises a forward full-length adapter primer F1 and a reverse full-length adapter primer F2, wherein the 3' end of F1 is the S1 sequence, the 5' end of F1 is the P5 sequence, the 5' end of F2 is the P7 sequence, and the 3' end of F2 is the reverse complementary sequence of S2.

[0034] In some embodiments, the specific primer 2 comprises a forward specific primer 2 and a reverse specific primer 2, wherein the 5' end of the forward specific primer 2 carries a universal adapter sequence 1, and the 5' end of the reverse specific primer 2 carries a universal adapter sequence 2.

[0035] In some embodiments, the adapter in step 2.2 is a universal short adapter without a tag, comprising two oligonucleotide chains S1 and S2, wherein S1 and S2 are partially reverse-complementary to form a double strand, and the 3' end of S1 protrudes one more T base than the 5' end of S2, which can complementarily pair with the DNA fragment after the end is repaired and A is added to complete the adapter connection.

[0036] In some embodiments, the full-length adapter primers with tags in step 2.3 include a forward full-length adapter primer F1, a reverse full-length adapter primer F2, and a reverse full-length adapter primer F3, wherein the 3' end of F1 is an S1 sequence, the 5' end of F1 is a P5 sequence, the 5' end of F2 is a P7 sequence, the 3' end of F2 is a reverse complementary sequence of S2, the 5' end of F3 has a P7 sequence, and the 3' end of F3 is the same as the universal adapter sequence 2 carried at the 5' end of the reverse specific primer 2.

[0037] In some embodiments, in step 1.2, the specific amplification product is mixed with the original genomic DNA at a mass ratio of 1:1-20, preferably 1:5-20, more preferably 1:10-15, or 1:10-20, or the mixing ratio can be adjusted according to the ratio of the size of the target region of specific amplification to the size of the genome. The ratio of the size of the target region of specific amplification: the size of the genome is used for testing, and then adjusted according to the deviation in the amount of sequencing data. If the amount of data in the target region of specific amplification is small, the mixing ratio of the specific amplification product is increased according to the gradient of the number 5. Otherwise, the ratio of the original genomic DNA is increased.

[0038] In some embodiments, in step 2.2, the genome fragmentation plus adapter product and the specific amplification product are mixed in a mass ratio of 1 to 20:1, preferably 5 to 20:1, more preferably 10 to 20:1, or 10 to 15:1, or the mixing ratio can be adjusted according to the ratio of the size of the target region of specific amplification to the size of the genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG1 is a flow chart of Scheme 1 of a library construction method combining genome sequencing and targeted sequencing according to the present invention.

[0040] FIG2 is a flow chart of Scheme 2 of a library construction method combining genome sequencing and targeted sequencing according to the present invention.

[0041] Figure 3 is a schematic diagram of the short linker in the library construction method.

[0042] FIG4 is a nucleic acid quality control graph in Example 1.

[0043] FIG5 is a schematic diagram of the Labchip quality control results of nucleic acid fragment length in Example 1.

[0044] FIG6 is a schematic diagram of the Labchip quality control results of nucleic acid fragment length in Example 2.

[0045] FIG7 is a schematic diagram of the Labchip quality control results of nucleic acid fragment length in Example 3. DETAILED DESCRIPTION

[0046] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0047] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] Definitions To facilitate understanding of this technology, certain terms and phrases are defined below.

[0050] Sequencing depth refers to the ratio of the total number of sequenced bases (bp) to the genome size and is one of the indicators for evaluating sequencing quality. Sequencing depth is positively correlated with genome coverage, and the error rate or false positive results associated with sequencing decreases with increasing sequencing depth.

[0051] Coverage: Sequencing coverage is the percentage of bases in a genome that are sequenced. Sequencing coverage is one of the indicators of sequencing randomness. The relationship between sequencing depth and coverage can be determined using the Lander-Waterman model (1988). When the sequencing depth reaches 5X, approximately 99.4% of the genome is covered.

[0052] Low-depth whole-genome sequencing: Detects and analyzes chromosome copy number variations based on a high-throughput sequencing platform, capable of detecting chromosomal aneuploidy and microdeletions / microduplications larger than 50kb / 100kb.

[0053] After obtaining the library construction method combining low-depth whole-genome sequencing and targeted sequencing of the present invention, we also tried other library construction methods combining whole-genome detection and targeted sequencing:

[0054] The first approach (see Figure 1) is to combine low-depth whole-genome sequencing with targeted sequencing for library construction, which includes the following steps:

[0055] Step 1: First, a portion of genomic DNA is taken and amplified using one or more pairs of specific primers 1 to obtain one or more DNA fragments 1 of 600 bp or longer;

[0056] Step 2: The DNA fragment 1 is mixed with the original genomic DNA in a certain ratio, and then the fragment is repaired at the end and A is added, an adapter is added, and the ligation product is eluted and purified to obtain an eluted product;

[0057] Step 3: Perform PCR using full-length adapter primers with tag sequences to obtain a sequence-ready library.

[0058] The above-mentioned specific amplification primer 1 includes a forward specific primer 1 and a reverse specific primer 1, both of which are 17-25bp in length, and can be completely complementary to the genome head to tail, or be a degenerate primer. The degenerate primer is a primer that may contain several bases that are not completely complementary to the genomic DNA. The above-mentioned adapter is a universal short adapter without a tag sequence, comprising two oligonucleotide chains S1 and S2, S1 and S2 partially reverse complementary to form a double strand, and the 3' end of S1 protrudes one more T base than the 5' end of S2, which can be complementary to the DNA fragment after the end repair and A addition to complete the adapter connection. The full-length adapter primer with the tag sequence includes a forward full-length adapter primer F1 and a reverse full-length adapter primer F2, the 3' end of F1 is the S1 sequence, the 5' end of F1 is the P5 sequence, the 5' end of F2 is the P7 sequence, and the 3' end of F2 is the reverse complementary sequence of S2.

[0059] The second approach (see Figure 2) is to combine low-depth whole-genome sequencing with targeted sequencing for library construction, which includes the following steps:

[0060] Step 1: First, a portion of genomic DNA is taken and amplified using one or more pairs of specific primers 2 to obtain one or more DNA amplified fragments 2. The length of the DNA amplified fragments 2 ranges from 100 bp to 1000 bp, preferably from 200 bp to 500 bp. The position of the primers can be designed according to the sequencing length of different applications to optimize the length of the DNA amplified fragments 2.

[0061] Step 2: At the same time, another portion of genomic DNA is taken for fragmentation, end repair, A addition, and adapter addition to obtain DNA fragment product 3;

[0062] Step 3: Mix the DNA amplified fragment 2 and the DNA fragment product 3 in a certain ratio, add the full-length adapter primer with the tag sequence for amplification, and a sequenceable library can be obtained.

[0063] The specific primers 2 include a forward specific primer 2 and a reverse specific primer 2. The 5' end of the forward specific primer 2 carries a universal adapter sequence 1, and the 5' end of the reverse specific primer 2 carries a universal adapter sequence 2. The universal adapter sequence 1 and sequence 2 are partially identical or completely different, preferably completely different, and cannot complementarily pair with genomic DNA.

[0064] The adapter in step 2 is a short, untagged universal adapter consisting of two oligonucleotide chains, S1 and S2. S1 and S2 are partially reverse-complementary, forming a double-stranded chain. Specifically, a portion of the 3' end of S1 and a portion of the 5' end of S2 are reverse-complementary, and the 3' end of S1 has an extra T base overhanging the 5' end of S2. This T base can complementarily pair with the DNA fragments after end-repair and A addition, completing the adapter ligation step. Oligonucleotide chain S1 and forward-specific primer 2 have the same universal adapter sequence 1 at their 5' ends. The aforementioned tagged full-length adapter primers include forward full-length adapter primer F1, reverse full-length adapter primer F2, and reverse full-length adapter primer F3. Forward full-length adapter primer F1 is identical to forward full-length adapter primer F1 in scheme 1. Reverse full-length adapter primer F2 is identical to reverse full-length adapter primer F2 in scheme 1. F3 has a P7 sequence at its 5' end, and its 3' end is identical to the universal adapter sequence 2 at the 5' end of reverse-specific primer 2.

[0065] The structure of the short connector is shown in Figure 3.

[0066] After research, it was found that the library construction method that combines low-depth whole-genome sequencing and targeted sequencing can successfully build a library and reduce the cost of library construction, obtaining more sequencing information with less sequencing time and sequencing cost.

[0067] Based on this, we conducted further research and obtained a library construction method that combines low-depth whole-genome sequencing and targeted sequencing, including the following steps:

[0068] Step 1: Use a restriction endonuclease adapted to the sample to shear the nucleic acid sample to obtain a longer DNA fragment: According to the need to build a library of nucleic acid samples, select a restriction endonuclease that can recognize methylation modifications, such as AbaSI, FspEI, LpnPI, MspJI, etc., where the restriction endonuclease can be one or a mixture of several. By recognizing the methylated bases on the genome, the DNA double strand is interrupted near the base. You can also use a restriction endonuclease that can recognize the terminator sequence AATAAA in the chromosome coding region, such as SspI-HF, SspI, to interrupt the DNA double strand. The restriction endonuclease can be one or a mixture of several, and the above two restriction endonucleases can also be used in combination.

[0069] Step 2: Amplify the DNA fragments from step S1) using random primers and gene-specific primers in proportion to obtain amplified products: After fragmenting the genomic nucleic acid using restriction endonucleases, longer DNA fragments are obtained. These fragments are used as templates. Gene-specific primers with universal sequences and random primers with universal sequences are mixed in varying ratios for the first round of PCR amplification. The gene-specific primers with universal sequences are used in a lower ratio than the random primers with universal sequences. The ratio of gene-specific primers to random primers with universal sequences is in the range of 1:1 to 1:20, preferably 1:5-20, and more preferably 1:10-15. Alternatively, the mixing ratio can be adjusted based on the ratio of the target region size to the genome size. Preferably, the ratio of the target region size to the genome size is used for testing, and then adjusted based on the amount of sequencing data. If the amount of data in the target region of specific amplification is low, the mixing ratio of the specific amplification products is increased by a gradient of 5. Otherwise, the ratio of the original genomic DNA is increased. Various configurations can be used within this range to achieve the best amplification effect. Through this amplification, it was found that the sequencing effect of obtaining information on specific sites would not affect the uniformity of whole-genome coverage.

[0070] The universal sequence random primer consists of three parts: the first part is a universal adapter sequence, which can be designed according to different sequencing platforms; the second part is a random sequence of six N bases; and the third part is three G bases, which is used to increase the Tm value. The gene-specific primer consists of two parts: the first part is a universal adapter sequence, which is selected according to different sequencing platforms; and the second part is a targeted specific primer, which is designed according to the target region of interest.

[0071] Step 3: A second amplification of the amplified product from Step 2 is performed using adapter primers to generate a DNA library for sequencing. Then, using adapters with indexed sequences, a second amplification of the first-round PCR product is performed to generate a library ready for sequencing. At this point, only a small amount of data is required to obtain low-depth whole-genome information and information on the targeted region of interest, enabling simultaneous analysis of copy number, point mutations, indels, and other information, or comparative analysis of paternal and maternal genetic information.

[0072] Through the above-mentioned library construction method, it is possible to simultaneously detect copy number variation, structural variation, point mutation and other mutation types with very little data (low-depth sequencing).

[0073] Those skilled in the art will appreciate that the steps included in the above-mentioned library construction method do not need to be limited to a specific order. Those skilled in the art can make appropriate adjustments to the order based on their common sense. For example, obtaining specific amplification products and genome fragmentation and adapter products can be performed simultaneously or sequentially, as long as the library construction method of the present invention can be achieved.

[0074] The present invention is further described and illustrated below through specific examples, but is not intended to limit the scope of protection of the present invention.

[0075] Seq in the following abbreviations corresponds to SEQ ID NO:, for example, Seq1 corresponds to SEQ ID NO:1.

[0076] Example 1. Three clinical sputum cultures with known reference results were used to conduct experiments to identify Mycobacterium tuberculosis strains and detect drug resistance sites in the Rpob gene.

[0077] Sample information:

[0078] Follow these steps:

[0079] Sputum samples were used to extract DNA nucleic acid using the bacterial genomic DNA extraction kit (YDP302) from Tiangen Biochemical Technology Co., Ltd.

[0080] Take 30ng of extracted nucleic acid and use NEB's restriction endonuclease BamHI-HF (Cat. No. R3136V) for enzyme digestion. The enzyme cutting site is: 5'..G^GATC C..3'

[0081] 3'..C CTAG^G..5'

[0082] Prepare the reaction system according to the following table:

[0083] 1. Run the program: 37℃ for 20 min, hold at 4℃.

[0084] 2. After the reaction, 1.2x DNA purification magnetic beads were used for purification and 15 μL was eluted to obtain DNA fragment products with a length range of 500 bp-10 kb, with a main peak size of approximately 2600 bp. The nucleic acid quality control spectrum is shown in Figure 4.

[0085] 3. Specific primers with fixed sequences (Seq3-Seq6) and random primers with fixed sequences (Seq1 and Seq2) were mixed at a molar ratio of 1:15 to obtain a primer mixture.

[0086] 4. Prepare the reaction system according to the following table:

[0087] Primer mix

[0088] Among them, the italic part in the above sequence is a fixed sequence, and NNNNNN is a random sequence, arbitrarily selected from ATCG. 1. Run the program:

[0089] 2. After the reaction is completed, purify using 0.8x DNA purification magnetic beads and elute with 20 μL of water.

[0090] 3. Prepare the reaction system according to the following table:

[0091] Adapter primer:

[0092] *Thiolation modification prevents exonuclease cleavage, retains more primers, and improves PCR efficiency. It is also possible not to add thiolation modification.

[0093] 4. Run the program:

[0094] 5. After the reaction is completed, purify using 0.8x DNA purification magnetic beads, elute with 30 μL of water, and take 1 μL for quantification using qubit to obtain a sequenceable library.

[0095] 6. Qubit quality control results:

[0096] The results of Labchip quality control of nucleic acid fragment length are shown in Figure 5.

[0097] 7. Analyze the results

[0098] 1) Sequencing data quality control:

[0099] 3M bases of raw data (number of bases) were randomly sampled from each sample for data quality control. The Q30 of all samples met the requirements, and the data volume of the target regions of the three samples accounted for 15%-21.5% of the total data volume.

[0100] 2) Whole-genome analysis results:

[0101] Whole-genome data analysis revealed that all three samples detected the target strain, Mycobacterium tuberculosis, and provided the likely locations of mutations. The top three most reliable results all pointed to the Rpob gene.

[0102] 3) Specific primer analysis results

[0103] According to the targeted region data analysis, samples T24 and T324 were both positive for S531L (TCG→TTG), with sequencing depths greater than 1000x. Sample T884 was negative, with sequencing depths greater than 1000x at each locus. The results are reliable and consistent with expectations. In the targeted data analysis, each sample was accurately analyzed to the target region, providing correct results.

[0104] Combining the results of 1), 2), and 3) above, low-depth whole-genome combined with targeted sequencing can effectively identify pathogens and correctly identify drug-resistant sites in the target area, obtaining more analytical results with a small amount of data.

[0105] The above-mentioned library construction method can solve the problem of scattered and unfocused library fragments and greatly reduce the risk of target fragments being interrupted.

[0106] Conclusion: The low-depth whole-genome combined with targeted sequencing of the library construction scheme of the present invention is feasible, and the detection results are consistent with the theoretical results.

[0107] Example 2: Three clinical sputum cultures with known reference results were used to conduct experiments to identify Mycobacterium tuberculosis strains and detect the Rpob gene.

[0108] Sample information:

[0109] Follow these steps:

[0110] Sputum samples were used to extract DNA nucleic acid using the bacterial genomic DNA extraction kit (YDP302) from Tiangen Biochemical Technology Co., Ltd.

[0111] 1. Take 10ng of extracted DNA nucleic acid and use the PCR amplification (amplified product length 601 bp) was performed using C2P4 Blood Direct MμLtiplex PCR Master Mix (13606) and specific primers as follows:

[0112] 1) Prepare the reaction according to the following system:

[0113] 2) Run the program:

[0114] 3) Equilibrate DNA purification magnetic beads (N411 from Novozymes) at room temperature in advance.

[0115] 4) After the reaction is complete, purify the reaction product using 1.8x DNA purification magnetic beads, elute 10 μL, and take 1 μL for qubit double-stranded DNA quantification to obtain the specific amplification product (DNA fragment 1);

[0116] 2. Mix the specific amplification product and the original extracted nucleic acid at a mass ratio of 1:15. The mixing volume is shown in the table of Qubit quality control results in step 4.

[0117] 3. Use the VAHTS Universal Plus DNA Library Prep Kit for Illumina V2 (ND627) from Nanjing Novozymes Biotechnology Co., Ltd. for enzyme digestion (end repair and A addition), addition of universal adapters, and PCR amplification. The steps are as follows:

[0118] 1) Prepare the reaction system according to the following table:

[0119] 2) Operating program: 37°C for 18 min, 65°C for 30 min, hold at 4°C.

[0120] 3) After the reaction is complete, prepare the reaction system according to the table below:

[0121] 4) Run the program: 20℃ for 15 min, hold at 4℃.

[0122] 5) Equilibrate DNA purification magnetic beads (N411 from Novozymes) at room temperature in advance.

[0123] 6) After the reaction is complete, purify the reaction product using 0.9x DNA purification magnetic beads and elute with 20 μL of water to obtain the eluted product. Prepare the PCR reaction system according to the following table:

[0124] PCR primers:

[0125] Run the program:

[0126] 7) Purify with 0.8x DNA purification magnetic beads, elute with 30 μL of water, and take 1 μL for qubit double-stranded DNA quantification to obtain a library product that can be sequenced.

[0127] 4. Qubit quality control results

[0128] The results of Labchip quality control of nucleic acid fragment length are shown in Figure 6.

[0129] 5. Analysis results:

[0130] 1) Sequencing data quality control:

[0131] 3M bases of raw data (number of bases) were randomly sampled from each sample for data quality control. The Q30 of all samples met the requirements. The data volume of the target areas of the three samples accounted for 0.4%-0.6% of the total data volume, which was less than the expected data volume, but still met the analysis requirements.

[0132] The smaller-than-expected data volume is due to the relatively small size of the specific amplified fragment, 600 bp. During the fragmentation step, some fragments were broken and unable to be constructed, resulting in a smaller percentage of valid target-specific amplification data. Subsequently, the specific amplification fragments may be increased, or the specific amplification primers may be replaced to increase the size of the specific amplified fragments.

[0133] 2) Whole-genome analysis results

[0134] In whole-genome data analysis, the target strain, Mycobacterium tuberculosis, was detected in all three samples, and the corresponding possible mutation locations were indicated. The background of ribosome 23s was high, and all the confidence scores of 100 were derived from ribosome 23s data. In addition to ribosome data, the top three confidence scores also pointed to the Rpob gene.

[0135] 3) Specific primer analysis results

[0136] According to the targeted region data analysis, samples T24 and T324 were both positive for S531L (TCG→TTG), with sequencing depths greater than 40x. Sample T884 was negative, with sequencing depths greater than 40x at each locus. The results are reliable and consistent with expectations. In the targeted data analysis, each sample was accurately analyzed to the target region, providing correct results.

[0137] Combining the results of 1), 2), and 3) above, low-depth whole-genome combined with targeted sequencing can effectively identify pathogens and correctly identify drug-resistant sites in the target area, obtaining more analytical results with a small amount of data.

[0138] Conclusion: This library construction scheme is feasible for low-depth whole-genome combined with targeted sequencing, and the detection results are consistent with the theoretical results.

[0139] Example 3: Three clinical sputum cultures with known reference results were used to conduct experiments to identify the tuberculosis strain and detect the Rpob gene.

[0140] Sample information:

[0141] Follow these steps:

[0142] Sputum samples were used to extract DNA nucleic acid using the bacterial genomic DNA extraction kit (YDP302) from Tiangen Biochemical Technology Co., Ltd.

[0143] 1. Take 20 ng of the extracted DNA nucleic acid and use the VAHTS Universal Plus DNA Library Prep Kit for Illumina V2 kit (ND627) from Nanjing Novozymes Biotechnology Co., Ltd. for enzyme digestion and add universal adapters as follows:

[0144] 1) Prepare the reaction system according to the following table:

[0145] 2) Operating program: 37°C for 18 min, 65°C for 30 min, hold at 4°C.

[0146] 3) After the reaction is complete, prepare the reaction system according to the table below:

[0147] 4) Run the program: 20℃ for 15 min, hold at 4℃.

[0148] 5) Equilibrate DNA purification magnetic beads (N411 from Novozymes) at room temperature in advance.

[0149] 6) After the reaction is complete, purify the reaction product using 1.8x DNA purification magnetic beads, elute 10 μL, and take 1 μL for qubit double-stranded DNA quantification to obtain the genome fragmentation and adapter product;

[0150] Take 10ng of the extracted DNA nucleic acid and use Yisheng Bio's PCR amplification was performed using C2P4 Blood Direct MμLtiplex PCR Master Mix (13606) and specific primers. The lengths of the amplified products were 235, 216, 180, and 199 (two pairs of four primers were combined in pairs to obtain four amplified products: seq3 and seq4 obtained a product of 180 bp in length, seq5 and seq6 obtained a product of 235 bp in length, seq3 and seq6 obtained a product of 199 bp in length, and seq4 and seq5 obtained a product of 216 bp in length).

[0151] Here are the steps:

[0152] Prepare the reaction according to the following system:

[0153] Run the program:

[0154] 2. Equilibrate DNA purification magnetic beads (N411 from Novozymes) at room temperature in advance.

[0155] 3. After the reaction is completed, the reaction product is purified using 1.8x DNA purification magnetic beads, eluted with 10 μL, and 1 μL is taken for qubit double-stranded DNA quantification to obtain the specific amplification product (DNA amplification fragment 2);

[0156] 4. Mix the fragmented and adapter-added products with the specific amplification products and amplify them to prepare the library:

[0157] 1) Mix the fragmented and adapter-added products with the specific amplification products at a mass ratio of 15:1;

[0158] 2) Amplify using KAPA hot start HiFi high-fidelity enzyme premix and full-length adapter primers with tag sequences.

[0159] 3) Prepare the system according to the following table: (The volume of x and y is in the table of the Qubit quantitative quality control results in point 4)

[0160] Adapter primer:

[0161] Run the program:

[0162] 4) After the reaction is complete, purify the reaction product using 0.8x DNA purification magnetic beads, elute 30 μL, and take 1 μL for qubit double-stranded DNA quantification to obtain a library product that can be sequenced.

[0163] 5. Qubit quantitative quality control results

[0164] The Labchip quality control results for nucleic acid fragment length are shown in Figure 7.

[0165] 6. Analysis results:

[0166] Sequencing data quality control:

[0167] 3M bases of raw data (number of bases) were randomly sampled from each sample for data quality control. The Q30 of all samples met the requirements. The data volume of the target areas of the three samples accounted for 13%-22% of the total data volume, which met the analysis requirements.

[0168] 2) Whole-genome analysis results

[0169] Whole-genome data analysis revealed that the target strain, Mycobacterium tuberculosis, was detected in all three samples, with the corresponding likely mutation locations identified. Given a background containing a low concentration of 23s ribosomes, the top three most reliable mutations were linked to the Rpob gene.

[0170] 3) Specific primer analysis results

[0171] According to the targeted region data analysis, samples T24 and T324 were both positive for S531L (TCG→TTG), with sequencing depths greater than 1000x. Sample T884 was negative, with sequencing depths greater than 1000x at each locus. The results are reliable and consistent with expectations. In the targeted data analysis, each sample was accurately analyzed to the target region, providing correct results.

[0172] Combining the results of 1), 2), and 3) above, low-depth whole-genome combined with targeted sequencing can effectively identify pathogens and correctly identify drug-resistant sites in the target area, obtaining more analytical results with a small amount of data.

[0173] Conclusion: This library construction scheme is feasible for low-depth whole-genome combined with targeted sequencing, and the detection results are consistent with the theoretical results.

[0174] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A library construction method combining low-depth whole genome sequencing and targeted sequencing, characterized in that: The following steps are involved: Step 1: Use a restriction endonuclease that is suitable for the sample to shear the nucleic acid sample to obtain a longer DNA fragment; Step 2: using random primers with universal sequences and gene-specific primers with universal sequences to amplify the DNA fragments in step 1 in proportion to obtain amplified products; Step 3: Use the adapter primer to perform a second amplification on the amplified product of step 2 to obtain a DNA library for sequencing.

2. The library construction method according to claim 1, characterized in that: The restriction endonuclease is selected from at least one of the following: BamHI-HF, McrBC, AbaSI, FspEI, LpnPI, MspJI, SspI-HF, SspI.

3. The library construction method according to claim 2, characterized in that: The restriction endonuclease is at least one selected from BamHI-HF, McrBC, AbaSI, FspEI, LpnPI, and MspJI, and / or at least one selected from SspI-HF, SspI.

4. The library construction method according to claim 1, characterized in that: The random primer with a universal sequence is composed of a universal linker sequence, a random sequence of 6 Ns, and three G bases in sequence.

5. The library construction method according to claim 1, characterized in that: The usage ratio of the gene-specific primers with universal sequences to the random primers with universal sequences is 1:1-20.

6. The library construction method according to claim 5, characterized in that: The usage ratio of the gene-specific primers with universal sequences to the random primers with universal sequences is 1:5-20.

7. The library construction method according to any one of claims 1 to 6, characterized in that: The gene-specific primer with a universal sequence consists of a universal linker sequence and a specific primer targeting a target region.

8. Application of the library construction method combining low-depth whole genome sequencing and targeted sequencing as described in any one of claims 1-7 in genotyping in genetic breeding, pathogen detection, and detection of drug resistance sites.

9. A library construction method combining whole genome sequencing and targeted sequencing, characterized in that: The following steps are involved: Step 1.1: First, take part of the genomic DNA from the sample to be tested, use one or more pairs of specific primers 1 to amplify the target region, and obtain one or more specific amplification products of 600 bp or more in length; Step 1.2: The specific amplification product is mixed with the original genomic DNA in a certain ratio, and then the end is repaired and A is added, a linker is added, and the connection product is eluted and purified to obtain an eluted product; Step 1.3: Use the eluted product as a template and perform PCR using a full-length adapter primer with a tag sequence to obtain a library that can be sequenced; Or including the following steps: Step 2.1: Take a portion of genomic DNA from the sample to be tested, and use one or more pairs of specific primers 2 to amplify the target region to obtain one or more specific amplification products; Step 2.2: Take another part of the genomic DNA from the sample to be tested, perform end repair, add A, and add adapters to obtain genomic fragmentation and adapter products; Step 2.3: The genome fragmentation plus adapter product and the specific amplification product are mixed in a certain ratio, and the full-length adapter primer with the tag sequence is added for amplification to obtain a sequenceable library.

10. The library construction method according to claim 9, characterized in that: The length of the DNA amplified fragment 2 is within the range of 100 bp to 1000 bp.

11. The library construction method according to claim 10, characterized in that: The length of the DNA amplified fragment 2 is within the range of 200 bp to 500 bp.

12. The library construction method according to claim 9, characterized in that: The specific amplification primers 1 include a forward specific primer 1 and a reverse specific primer 1, each of which is 17 to 25 bp in length, and the head and tail are completely complementary to the genome, or are degenerate primers.

13. The library construction method according to claim 9, characterized in that: The adapters in step 1.2 and step 2.2 are respectively universal short adapters without labels, comprising two oligonucleotide chains S1 and S2, wherein the S1 and S2 parts are reversely complementary to form a double strand, and the 3' end of S1 protrudes one more T base than the 5' end of S2, which is used for complementary pairing with the DNA fragments after the end is repaired and A is added to complete the adapter connection.

14. The library construction method according to claim 9, characterized in that: The full-length adapter primer with a label described in step 1.3 comprises a forward full-length adapter primer F1 and a reverse full-length adapter primer F2, wherein the 3' end of F1 is an S1 sequence, the 5' end of F1 is a P5 sequence, the 5' end of F2 is a P7 sequence, and the 3' end of F2 is a reverse complementary sequence of S2.

15. The library construction method according to claim 9, characterized in that: The specific primer 2 comprises a forward specific primer 2 and a reverse specific primer 2, wherein the 5' end of the forward specific primer 2 carries a universal linker sequence 1, and the 5' end of the reverse specific primer 2 carries a universal linker sequence 2.

16. The library construction method according to claim 9, characterized in that: The full-length adapter primers with tags described in step 2.3 include a forward full-length adapter primer F1, a reverse full-length adapter primer F2, and a reverse full-length adapter primer F3. The 3' end of F1 is the S1 sequence, the 5' end of F1 is the P5 sequence, the 5' end of F2 is the P7 sequence, the 3' end of F2 is the reverse complementary sequence of S2, the 5' end of F3 has the P7 sequence, and the 3' end of F3 is the same as the universal adapter sequence 2 carried at the 5' end of the reverse specific primer 2.

17. The library construction method according to claim 9, characterized in that: The specific amplification product described in step 1.2 is mixed with the original genomic DNA at a mass ratio of 1:1 to 20.

18. The library construction method according to claim 17, characterized in that: The specific amplification product described in step 1.2 is mixed with the original genomic DNA at a mass ratio of 1:5 to 20.

19. The library construction method according to claim 9, characterized in that: The genome fragmentation plus adapter product and the specific amplification product described in step 2.2 are mixed in a mass ratio of 1 to 20:

1.

20. The library construction method according to claim 19, characterized in that: The genome fragmentation plus adapter product and the specific amplification product described in step 2.2 are mixed in a mass ratio of 5 to 20:1.

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