CN114990194A - Method for introducing extra index in NGS library building process - Google Patents
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Abstract
The invention belongs to the technical field of NGS sequencing, and particularly relates to a method for introducing an additional index in a NGS library building process. In the method for introducing the extra index in the NGS library building process, the extra sample index is introduced into the primer or the adapter, so that the number of the index in the final library is more than 2, after the extra index is introduced into the normal double-end-index library, the result of multi-sample sequencing can be subjected to data splitting by using three to four indexes, the problem of index cross talk in multi-sample on-machine sequencing can be further reduced, the potential read pollution in ultra-high-depth sequencing is reduced, and the sensitivity of the result is improved.
Description
Technical Field
The invention belongs to the technical field of NGS sequencing, and particularly relates to a method for introducing an additional index in a NGS library building process, which is used for reducing index cross in NGS sequencing.
Background
Next Generation Sequencing (NGS), also known as deep sequencing or massively parallel sequencing, can sequence millions of small fragments simultaneously. NGS has been widely used in many fields, the most common of which are genomic DNA mutation analysis and RNA expression analysis. These analytical applications can be extended to whole genomes and whole exomes, and can also specifically sequence specific regions and gene combinations. Currently, the library construction method of the next generation sequencing targeting technology based on multiplex PCR can be divided into 2 types: 1) capturing and enriching a target DNA region by using paired specific primers to carry out conventional PCR amplification; where index can be included in the primer, integrated into the amplicon in the first 2 cycles of PCR. 2) Capturing and enriching a target DNA region by using a single-ended specific primer to carry out anchor PCR amplification; wherein the index may be contained in the universal primer. In addition to PCR-based targeted banking methods, there are also probe capture-based targeted banking. Before probe capture, a full genomic library was first performed. The library building method generally adopts a link method: the DNA fragments are first added with a universal adaptor with a ligase, wherein an index may be included within the adaptor, and then target DNA enrichment is performed using specific primers and universal primers.
Index cross talk (Index cross talk) is an internal defect in multiplex sample sequencing for NGS technology applications, which may be caused by Index hopping (Index hopping), contamination when adapter primers are synthesized, or contamination of adapters when libraries are prepared. Index crosstalk may cause a read from one sample to be incorrectly allocated to the other sample when the read split is performed. If the reads contain mutation, the false distribution may result in that negative samples without mutation are judged to be positive, thus causing false positive, and influencing the application of the NGS technology in molecular diagnosis and the like.
In order to reduce the influence degree of index crosstalk, in the past few years, library construction is mostly carried out by using double-index joints in the process of sequencing multiple samples. Many data indicate that it can indeed effectively reduce the read contamination between different samples caused by index crosstalk, but cannot eliminate it. Therefore, there is a need for an improved NGS library-building method to further reduce the index cross-talk in the sequencing of multiple samples.
Disclosure of Invention
To overcome the above-mentioned deficiencies of the prior art, it is an object of the present invention to provide a method for introducing extra index in NGS banking, which method can reduce the influence of index cross-talk during multiple sequencing.
In order to realize the purpose, the invention is realized by the following technical scheme:
the invention provides a method for introducing an extra index in a NGS library building process, which comprises the following steps: in the NGS library building process, extra sample indexes are introduced into primers or joints, so that the number of the indexes in a final library is more than 2, and the index cross problem when multiple samples are subjected to post-machine read splitting is further reduced.
The core of the invention is that: additional indexes are introduced during construction of the NGS library, namely a third index and even a fourth index are introduced on the basis of double indexes, so that the sample-to-sample read pollution caused by index crosstalk is further reduced.
Preferably, the index is an index sequence of 4-12bp in length. The design of the additional index sequence only conforms to the basic principle of index selection (Panu S, Patick K, Peng M, et al. BARCOSEL: a tool for selecting an optimal barcode set for high-throughput sequencing [ J ]. Bmc Bioinformatics,2018,19(1):257.), and the length is generally 4-12 bp. The Index sequence may be selected from the Index sequences published by Illumina, Agilent, IDT, NEB, etc., or designed by the user.
Preferably, the method for construction of the NGS library comprises a multiple PCR library construction method and a ligase library construction method. Additional index sequences can be introduced in the corresponding primers or adapters depending on NGS banking methodology.
Further, when the NGS library is constructed by adopting a multiplex PCR library construction method, the introduction position of the index is in the middle position of the primer sequence; and when the NGS library building is carried out by adopting a ligase library building method, the introduction position of the index is at the 3' end of the adaptor sequence.
Further, when NGS library construction is carried out by adopting a ligase library construction method, a joint for chaining can be a Y-type joint or a hairpin type joint; the adopted link mode can be flat end link and T/A link.
Preferably, the NGS library building platform comprises an Illumina sequencing platform, an ion torrent sequencing platform and an MGI sequencing platform. The preferred embodiment of the present invention is designed based on Illumina sequencing platform, and it will be apparent to those skilled in the art that additional index can be introduced into NGS library construction based on the same principle in the use process of other NGS sequencing platforms (such as ion torrent, wara (MGI) sequencing platform) without departing from the basic structure of the present invention.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a method for introducing extra index in an NGS library building process, which can reduce index cross talk in an NGS sequencing, and introduce extra sample index in a primer or a joint in the NGS library building process, so that the number of the index in a final library is more than 2, and after introducing the extra index in a normal double-end-index library, a result of multi-sample sequencing can be subjected to data splitting by using three to four indexes, thereby further reducing the index cross talk problem in the multi-sample on-board sequencing, reducing potential read pollution in ultra-high depth sequencing and improving the sensitivity of the result.
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FIG. 1 is a flow chart of construction of an NGS library by introducing an additional index based on the library construction method of issued patent (publication No. CN111826421B) (index 3 is introduced in random primers based on the library construction method of issued patent. after construction of an NGS library using double index adaptor primers, an NGS library with 3 indices is formed);
FIG. 2 is a flow chart of construction of an NGS library by introducing additional indices using a universal multiplex PCR method (primer additional index in the target library construction. target NGS library construction is performed using paired end specific PCR primers. third and 4 index are introduced in the specific primers. after two rounds of PCR, the NGS library contains 4 indices);
FIG. 3 is a flow chart of construction of an NGS library by introducing an additional index using a general-purpose ligase library construction method (additional index is introduced in the construction of an NGS library using linker ligation. in linker design, the 3' end of the linker is added with index. thus, after PCR enrichment of the library by ligation is completed, the 3 rd index is introduced into the NGS library).
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
The primers required by the invention are all synthesized by the Kingsler company.
Interpretation of terms:
NGS (Next-Generation-Sequencing): i.e., next generation sequencing, also known as high throughput sequencing.
Reads-the sequence generated in the second generation sequencing, consisting of A, T, G, C (representing different bases).
An NGS library: the collection of DNA molecules generally has linker sequences at both ends, including a linker sequence, an index sequence, and a sequencing primer binding sequence.
Index, the sequence of bases contained in the NGS library, typically consisting of 6-12 bases, is contained in the adaptor sequence for assignment to the Reads generated after sequencing the pooled samples.
Index Cross talk (index Cross talk) in second generation sequencing, when multiple samples are mixed and sequenced simultaneously and then the Reads are split, the Reads belonging to one sample are wrongly assigned to other samples.
Index hopping (Index hopping) in second generation sequencing, residual adaptor primers in one library amplify DNA molecules in other libraries after mixing samples, during PCR prior to sequencing, resulting in Index cross.
The present invention introduces additional indexing during NGS library preparation to reduce the effect of index cross-talk during multiple sequencing, i.e., in addition to double indexing, additional one or two indices are introduced into the library. According to the differences of the NGS library building method, the method and the position of introducing the extra index also have differences. The introduction of the additional index at the time of library construction can be performed by one skilled in the art according to the design shown in the present invention according to the selected NGS library construction method. The following are three design examples, taking the library construction method of the Illumina sequencing platform as an example, and show the method design for introducing additional indexes.
This example introduces additional indexes into random primers (exemplified by the Illumina library), and performs the construction of the NGS library using the improved multiplex PCR library construction method described in the patent (publication No.: CN111826421B) (see specifically the example 1 section of the reference patent):
as shown in FIG. 1, in the first round of amplification, index3(GTCGTGAT) was incorporated into the amplicon as part of the primer (i.e., PCR random primer, shown in SEQ ID NO: 1). In the second round of amplification, index1(CAATAGAC) and index2(GCATATCG) were integrated into the library using specific primers (identical to those in CN 111826421) and double-index Illumina adapter primers (shown in SEQ ID NO:2 and SEQ ID NO: 3). Thus, the final library contained 3 indices.
The nucleotide sequence of the random primer (SEQ ID NO:1) is as follows:
5’-GACGTGTGCTCTTCCGATCTGTCGTGATNNNNNNGTNNNNNN-3' (index 3 is underlined);
the nucleotide sequence of the forward adaptor primer (SEQ ID NO:2) is as follows:
5’-AATGATACGGCGACCACCGAGATCTACACCAATAGACACACTCTTTCCCTACACGACGCTCTTCCGA-3' (the underlined part is index 1);
the nucleotide sequence of the downstream adapter primer (SEQ ID NO:3) is as follows:
5’-CAAGCAGAAGACGGCATACGAGATGCATATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' (the underlined part is index 2).
To test whether data splitting using additional indices can reduce the extent of index cross in multiple sample simultaneous machine sequencing. Using a DNA sample containing a known mutation site, wherein the sample source is tumor tissue DNA of a patient with advanced lung adenocarcinoma, the mutation from T to A at the chr7:55242506 position is detected by a third-party medical laboratory, the mutation frequency is 90.2 percent (positive sample), using another DNA sample without mutation at the site as a negative sample, the sample source is tumor tissue DNA of a patient with advanced lung adenocarcinoma, the mutation is not detected at the site by the third-party medical laboratory, performing NGS (2-time repetition of the negative sample) after constructing a database by using the method of example 1, and performing on-machine sequencing after mixing the samples.
Data splitting was performed on negative samples using double and triple indices, respectively. The results were analyzed for the presence and number of reads with positive mutations. The method specifically comprises the following steps: the constructed library was sequenced using MGI2000, the off-line data was split using fastp software, BWA software was matched to the human genome (Hg19), and after GenCore was deduplicated, Vardict identified mutations in the negative samples to confirm whether they contained mutations in the positive samples. The results are shown in Table 1.
As can be seen from the results in table 1, the number of positive reads in the negative samples (2 experimental repetitions) is significantly reduced after the triple resolution is used, which indicates that the index cross problem can be significantly reduced by performing library construction using an additional index in the NGS library and performing resolution on the data after the multiple samples are processed.
TABLE 1 number of reads containing positive mutations in test samples
This example uses the general Multiplex PCR library construction method, by introducing additional index for NGS library construction (see "Franken A, Rivandi M, Yang L, et al. A Multiplex PCR-Based Next Generation Sequencing-Panel to Identify variants for Targeted Therapy in Breast Cancer circulation Cells [ J ]. Applied Sciences,2020,10(10): 3364."):
as shown in FIG. 2, index3(GTCGAAGT) and index4(TTGCGTTC) were introduced into the primers specific to the first round of PCR. In the design, index3 and index4 were placed between the specific and universal sequences. Such as: a forward primer:
' (double underlined section: Illumina partial linker sequence; underlined section: Index 3; underlined wavy line section: specific primer sequence). Reverse primer:
(double underlined linear portions: Illumina partial linker sequence; underlined linear portions: Index 4; underlined wavy line portions: specific primer sequence). After a first round of PCR amplification, two indices were introduced into the amplicon. In the second round of PCR, amplification was performed using Illumina adapter primers with double indices (SEQ ID NO:2 and SEQ ID NO:3 as in example 1) and indices 1(CAATAGAC) and index2 (GCATCGG) were integrated into the library. Thus, the final library carries 4 indices.
As with example 1, additional indices were introduced into the NGS library using the method of example 2.
This example uses the general method of ligase pooling for NGS library construction by introducing additional indices (see "qual M a, Kozarewa I, Smith F, et al. a large genome centers' improvements to the illumination sequencing system [ J ]. NATURE METHODS, 2008."):
as shown in FIG. 3, Index3(GTCGAAGT) was introduced at the 3' end of the linker. To clarify where the linker is introduced, the linker sequence is now exemplified as follows: the positive strand sequence: 5' -CTTTCCCTACACGACGCTCTTCCGATCTGTCGAAGTT-3' (underlined: index 3; remainder Illumina partial linker sequence); a minus strand sequence: 5' -ACTTCGACAGATCGGAAGAGCACACGTCTGAACTCC-3' (in the sense: Illumina linker sequence; underlined: index 3).
After the ligation reaction, the linker with Index3 was ligated to the template DNA, and then amplified using double-Index Illumina linker primers (SEQ ID NO:2 and SEQ ID NO:3 as in example 1) to integrate Index1(CAATAGAC) and Index2(GCATATCG) into the library. Thus, the final library contained 3 indices.
As with example 1, additional indices were also introduced into NGS libraries using the method of example 3.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
<110> Guangzhou Mijing Gene medicine science and technology Co., Ltd
<120> method for introducing extra index in NGS library building process
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> DNA/RNA
<213> random primer (Artificial Sequence) of example 1
<400> 1
gacgtgtgct cttccgatct gtcgtgatnn nnnngtnnnn nn 42
<210> 2
<211> 67
<212> DNA/RNA
<213> upstream junction of example 1 (Artificial Sequence)
<400> 2
aatgatacgg cgaccaccga gatctacacc aatagacaca ctctttccct acacgacgct 60
cttccga 67
<210> 3
<211> 66
<212> DNA/RNA
<213> downstream adapter (Artificial Sequence) of example 1
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caagcagaag acggcatacg agatgcatat cggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 4
<211> 56
<212> DNA/RNA
<213> Forward primer for specific primer of example 2 (Artificial Sequence)
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cctacacgac gctcttccga tcgtcgaagt gaaagttaaa attcccgtcg ctatca 56
<210> 5
<211> 52
<212> DNA/RNA
<213> reverse primer of specific primer of example 2 (Artificial Sequence)
<400> 5
tcagacgtgt gctcttccga tctttgcgtt ctccttgttg gctttcggag at 52
<210> 6
<211> 37
<212> DNA/RNA
<213> linker forward Sequence (Artificial Sequence) of example 3
<400> 6
ctttccctac acgacgctct tccgatctgt cgaagtt 37
<210> 7
<211> 36
<212> DNA/RNA
<213> linker minus strand Sequence (Artificial Sequence) of example 3
<400> 7
acttcgacag atcggaagag cacacgtctg aactcc 36
Claims (6)
1. A method for introducing extra indexes in an NGS library building process is characterized in that extra sample indexes are introduced into primers or joints in the NGS library building process, so that the number of the indexes in a final library is larger than 2, and the index cross talk problem in the process of separating a read after a multiple sample is installed on a machine is further reduced.
2. The method for introducing the extra index in the NGS library construction process according to claim 1, wherein the index is an index sequence with a length of 4-12 bp.
3. The method for introducing extra index in the NGS banking process according to claim 1, wherein the NGS banking method comprises a multiple PCR banking method and a ligase banking method.
4. The method for introducing the additional index in the NGS banking process according to claim 3, wherein when the NGS banking is performed by adopting a multiplex PCR banking method, the introduction position of the index is in the middle of a primer; and when the NGS library building is carried out by adopting a ligase library building method, the introduction position of the index is at the 3' end of the adaptor sequence.
5. The method for introducing extra index in the NGS library construction process according to claim 3, wherein when the NGS library construction is carried out by a ligase library construction method, the linker for ligation can be a Y-type linker or a hairpin-type linker; the adopted link mode can be flat end link or T/A link.
6. The method for introducing extra index in the NGS database building process of claim 1, wherein the NGS database building platform comprises an Illumina sequencing platform, an ion torrent sequencing platform and an MGI sequencing platform.
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| CN117343929A (en) * | 2023-12-06 | 2024-01-05 | 广州迈景基因医学科技有限公司 | PCR random primer and method for enhancing targeted enrichment by using same |
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| CN117343929A (en) * | 2023-12-06 | 2024-01-05 | 广州迈景基因医学科技有限公司 | PCR random primer and method for enhancing targeted enrichment by using same |
| CN117343929B (en) * | 2023-12-06 | 2024-04-05 | 广州迈景基因医学科技有限公司 | PCR random primer and method for enhancing targeted enrichment by using same |
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