CN116064754A

CN116064754A - Method for constructing amplicon sequencing library with reduced cross contamination

Info

Publication number: CN116064754A
Application number: CN202211026830.2A
Authority: CN
Inventors: 郭永超; 王艳平; 雷湘华; 谭爱女
Original assignee: Shenzhen Uni Medica Technology Co ltd
Current assignee: Shenzhen Uni Medica Technology Co ltd
Priority date: 2018-08-28
Filing date: 2018-08-28
Publication date: 2023-05-05
Also published as: CN108998508A; CN116064753A; CN108998508B

Abstract

The invention describes a method for constructing an amplicon sequencing library with reduced cross contamination, comprising the following steps: obtaining sample DNA; performing first PCR amplification on sample DNA by using the first primer, the second primer and the third primer to obtain an amplification product of a sample; purifying; performing second PCR amplification on the purified product by using the fourth primer and the fifth primer to obtain an amplicon sequencing library; the first primer sequentially comprises a first sequencing tag sequence and an amplicon specific forward primer from a5 'end to a 3' end; the second primer is a second sequencing tag sequence and an amplicon specific reverse primer in sequence from the 5 'end to the 3' end; the third primer sequentially comprises a second sequencing joint sequence, a sample tag sequence and a second sequencing tag sequence from the 5 'end to the 3' end; the fourth primer is sequentially provided with a first sequencing joint sequence and a first sequencing tag sequence from the 5 'end to the 3' end; the fifth primer is a second sequencing adapter sequence. According to the invention, a method for constructing a sequencing library with reduced cross contamination can be provided.

Description

Method for constructing amplicon sequencing library with reduced cross contamination

The application is filing date2018, 08, 28Application number is201810986265.1The invention is named asAmplification of Construction method of sequencing library, primer set and kitIs a divisional application of the patent application of (2).

Technical Field

The invention belongs to the technical field of gene sequencing, and particularly relates to a method for constructing an amplicon sequencing library for reducing cross contamination.

Background

Along with the rapid increase of information flux in recent years, the high-flux sequencing technology has wider research and application in the fields of life science and medicine, particularly plays an important role in disease diagnosis and prevention, and is mainly applied to the aspects of prenatal screening, tumor diagnosis, major disease prevention, health-related metagenomic analysis and the like. Although human whole genome sequencing has a huge leap in terms of sequencing time and capital cost, huge data analysis and genetic information extraction are time-consuming and labor-consuming, compared with the whole exon sequencing, gene Panel sequencing and direct impact on most of gene sequences possibly causing diseases, the advantages of stronger purpose of amplicon sequencing, rapid data analysis, small capital cost and the like are more beneficial to clinical disease detection, such as two-line detection for screening newborns, and the use of an illuminea second-generation high-throughput sequencing platform for deep sequencing of certain congenital metabolic diseases genes of newborns can sensitively detect newborns carrying congenital diseases before clinical symptoms are expressed or slightly expressed, and early diagnosis and early treatment can be realized through screening, so that irreversible damage of organism tissues and organs can be prevented.

Polymerase chain reaction (i.e., PCR) is a widely used technique for molecular genetics and diagnostics, for amplifying specific DNA fragments, which can be regarded as specific DNA replication in vitro. The PCR is characterized in that the trace DNA in the sample is greatly increased to reach the detectable level. Amplicon sequencing is the sequencing of PCR products or captured fragments of specific length, and multiplex PCR is an important means of target segment enrichment technology due to its low cost and simple operation. In the face of capturing target sections of a large number of complex genome samples, the multiplex PCR technology has become the first technology due to the advantages of strong specificity, low price, good repeatability and the like.

At present, the amplicon library establishment based on the Illumina sequencing platform mainly adopts a traditional two-step amplification library establishment method, namely, two rounds of PCR are carried out by using two pairs of primers to establish the library. Although the two-step amplification method of the two pairs of primers can directly use the reagent matched with the Illumina for sequencing, when the sample amount is large in the process of library establishment, the sample label is not added in the first round of amplification, so that the subsequent operation is complicated and cross contamination among samples is easy to cause. On one hand, the common two-round PCR amplicon library construction method needs to construct a library for each sample independently, and cannot combine and uniformly process a large number of samples, so that the operation is complicated, the library construction efficiency is low, and cross contamination among samples is easy to cause; on the other hand, in the multiplex PCR reaction, as the primer pair number increases, primer dimer and non-specific amplification product increase sharply, so that the copy number difference of different target fragments is huge, and uniform product cannot be obtained; and the adopted library-building primers incorporate the public sequences, so that a large number of specific primers and an equal amount of public sequences are synthesized during primer synthesis, the cost is high, and the PCR amplification efficiency is reduced due to the fact that the primer sequences are too long, and the coverage and uniformity of the library are affected.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides a construction method of an amplicon sequencing library, a primer group and a kit, and aims to solve the technical problems of low efficiency, high cost and poor coverage and uniformity of the library of the existing two-step amplicon library construction method.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a method for constructing an amplicon sequencing library, comprising the steps of:

obtaining sample DNA;

performing first PCR amplification on the sample DNA by using a first primer, a second primer and a third primer to obtain an amplification product;

performing second PCR amplification on the amplification product by using a fourth primer and a fifth primer to obtain an amplicon sequencing library;

wherein, the liquid crystal display device comprises a liquid crystal display device,

the first primer sequentially comprises a first sequencing tag sequence and an amplicon specific forward primer from a5 'end to a 3' end;

the second primer is sequentially a second sequencing tag sequence and an amplicon specific reverse primer from a5 'end to a 3' end;

the third primer sequentially comprises a second sequencing joint sequence, a sample tag sequence and the second sequencing tag sequence from the 5 'end to the 3' end;

the fourth primer sequentially comprises a first sequencing joint sequence and the first sequencing tag sequence from the 5 'end to the 3' end;

the fifth primer is the second sequencing adapter sequence.

In the method for constructing the amplicon sequencing library, firstly, sample DNA is extracted, and then, the primer set designed by the method is utilized to carry out two-round PCR amplification to obtain the amplicon; in two rounds of PCR amplification: the method comprises the steps of firstly carrying out first round PCR by using a first primer, a second primer and a third primer to obtain fragments of all amplicons, adding a first sequencing tag sequence to the 5' end of the fragments of all amplicons of different sample sources in the process, adding a second sequencing tag sequence, a sample tag sequence and a second sequencing joint sequence which are sequentially connected to the 3' end, and combining amplified products of all samples into a tube after the first round PCR amplification is finished because the sample tag sequences can mark different samples, then carrying out second round PCR by using purified products of the first round PCR as templates by using a fourth primer and a fifth primer, and adding the first sequencing joint sequence to the 5' end of amplified products of the first round PCR, thereby completing the construction of an amplicon sequencing library. The construction method can greatly save reagents and workload, reduce cost, has high amplification efficiency of two rounds of PCR, and has better coverage and uniformity of the finally constructed amplicon sequencing library.

In another aspect, the invention provides a primer set for amplicon sequencing library construction, the primer set comprising: a first primer, a second primer and a third primer for a first PCR amplification, and a fourth primer and a fifth primer for a second PCR amplification; wherein, the liquid crystal display device comprises a liquid crystal display device,

the fifth primer is the second sequencing adapter sequence.

The primer group for constructing the amplicon sequencing library provided by the invention is characterized in that a first primer, a second primer and a third primer are used for first PCR amplification, and a fourth primer and a fifth primer are used for second PCR amplification; when the primer group is used for constructing an amplicon sequencing library, in the first round of PCR amplification, the 5 'end of all fragments of the amplicons can be added with a first sequencing tag sequence through three primers (a first primer, a second primer and a third primer) with special structures, the 3' end of all fragments of the amplicons is added with a second sequencing tag, a sample tag sequence and a second sequencing joint sequence which are sequentially connected, and as different samples can be marked by the sample tag sequences, amplified products of all samples can be combined into one tube after the first PCR amplification is finished, and then the second PCR amplification is directly carried out by a fourth primer and a fifth primer, so that the construction of the sequencing library is completed, the reagents can be greatly saved, and the workload can be reduced; and the second sequencing joint sequence, the sample tag sequence and the second sequencing tag sequence in the third primer are separated from the amplicon primers (namely the amplicon specific forward primer and the amplicon specific reverse primer), so that the third primer is more flexible to use, when one sample detects N amplicons, N pairs of specific primers, namely the first primer and the second primer, are needed, but only one third primer is needed, namely only 2N+1 primers are needed to be synthesized, therefore, when the quantity and the sample quantity of the amplicons are large, the primer set for library construction greatly reduces the cost of primer synthesis, and the primers in the primer set can be shorter compared with the existing primers, so that the PCR amplification efficiency can be further improved, and the amplicon sequencing library with better coverage and uniformity can be constructed.

Finally, the invention also provides a kit for constructing an amplicon sequencing library, which comprises the primer set.

The kit contains the special primer group, so that when the kit is used for constructing the amplicon sequencing library, the kit can save reagents and workload, reduce cost, further improve PCR amplification efficiency and construct the amplicon sequencing library with better coverage and uniformity.

Drawings

FIG. 1 is a schematic diagram of the construction flow of an amplicon sequencing library of the present invention;

FIG. 2 is a graph showing the results of amplicon sequencing depth according to example 1 of the present invention, wherein the abscissa represents the homogenized sequencing depth (the actual sequencing depth divided by the median of the amplicon depth) and the ordinate represents the proportion of amplicons at or above the sequencing depth.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

It should be noted that the terms "first," "second," and "second" are used merely for descriptive purposes and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.

In one aspect, embodiments of the present invention provide a method of constructing an amplicon sequencing library, comprising the steps of:

s01: obtaining sample DNA;

s02: performing first PCR amplification on the sample DNA by using a first primer, a second primer and a third primer to obtain an amplification product;

s03: performing second PCR amplification on the amplification product by using a fourth primer and a fifth primer to obtain an amplicon sequencing library;

the fifth primer is the second sequencing adapter sequence.

In the method for constructing the amplicon sequencing library, firstly, sample DNA is extracted, and then, two rounds of PCR amplification are carried out by utilizing the primer set designed in the embodiment of the invention; in two rounds of PCR amplification: the method comprises the steps of firstly carrying out first round PCR by using a first primer, a second primer and a third primer to obtain fragments of all amplicons, adding a first sequencing tag sequence to the 5' end of the fragments of all amplicons of different sample sources in the process, adding a second sequencing tag sequence, a sample tag sequence and a second sequencing joint sequence which are sequentially connected to the 3' end, and combining amplified products of all samples into a tube after the first PCR amplification is finished because the sample tag sequences can mark different samples, then carrying out second round PCR by using purified products of the first round PCR as templates by using a fourth primer and a fifth primer, and adding the first sequencing joint sequence to the 5' end of amplified products of the first round PCR amplification, thereby completing the construction of an amplicon sequencing library. The construction method can greatly save reagents and workload, reduce cost, has high amplification efficiency of two rounds of PCR, and has better coverage and uniformity of the finally constructed amplicon sequencing library.

When the prior amplicon sequencing library construction is carried out, two pairs of primers are used, the first round of PCR amplification is carried out, the primers consist of sequencing tag sequences and amplicon specific primers, when a plurality of samples, such as 96 samples, are required to be detected simultaneously, after the first round of PCR amplification is finished, each sample is required to be subjected to independent magnetic bead purification, 96 reaction systems are required to be prepared when the purified products are used for the second round of PCR, templates are required to be added one by one, namely, the subsequent magnetic bead purification and the second round of PCR are required to process the plurality of samples, which is equivalent to the independent library construction of each sample, a large amount of repeated work is required, and the operation is complicated and the reagent is consumed. In the embodiment of the invention, as shown in fig. 1, the same first sequencing tag sequence, second sequencing tag sequence and second sequencing joint sequence are added to all samples, meanwhile, different sample tag sequences are added to the 3' ends of different sample target fragments, the same first sequencing tag sequence and second sequencing tag sequence facilitate the second round of PCR to add the sequencing joint through a pair of primers (a fourth primer and a fifth primer), after the first round of PCR amplification is finished, the PCR products of all samples can be combined into one tube and then purified, thus avoiding artificial operation errors in experiments, hole site differences of a PCR instrument and possible cross contamination among samples, being capable of more truly and effectively reflecting the test results of the samples, saving the consumption of subsequent magnetic beads, simplifying the original purification of 96 samples into one sample, and greatly reducing the workload; meanwhile, the products purified after combination can be used as one sample for the second round of PCR, namely, the reaction system for preparing one sample by the second round of PCR can be completed, so that reagents are saved and the workload is reduced.

Further, in the above step S01, the sample DNA may be a genomic DNA sample from various organisms, such as blood, saliva, tissue, etc. from a person, and the weight of each sample DNA may be 200 to 300ng.

Further, in the above step S03, the molar ratio of the first primer, the second primer, and the third primer is 3:1:3, performing a first PCR amplification of the sample DNA, under which conditions the amplification efficiency can be improved even better. In the embodiment of the invention, the first PCR amplification is multiplex PCR amplification, and the first round of PCR is performed by using the three primers to obtain a specific amplicon fragment with a first sequencing tag sequence at the 5 'end and a second sequencing tag sequence, a sample tag sequence (Index) and a second sequencing adaptor sequence at the 3' end. The amplification process is as follows: firstly, a first primer and a second primer are amplified by taking sample DNA as a template to obtain a specific amplicon intermediate product (the intermediate product is not a target product) with a first sequencing tag sequence at the 5 'end and a second sequencing tag sequence at the 3' end; and then the first primer and the third primer are amplified by taking the specific amplicon intermediate product as a template to obtain a target fragment. It can be seen that the first primer is consumed maximally during the reaction, and that only the intermediate amplified by the first primer and the second primer is generated during the random reaction of the PCR amplification, so that the concentration of the first primer and the third primer should be increased to promote the reaction in order to ensure that the intermediate is converted into the target fragment as much as possible. On the other hand, we verified that the primer concentration ratios of the first primer, the second primer and the third primer (e.g. "2:1:2", "3:1:3", "5:1:5" and "10:1:10") which were different were compared, and the result showed that "the molar ratio of the first primer, the second primer and the third primer was 3:1:3, when the first PCR amplification is carried out on the sample DNA, the target fragment obtained by amplification has the largest proportion and the largest amplification efficiency.

Further, after the step of first PCR amplification, a step of first magnetic bead purification is further included. Preferably, the purification is performed 2 times using AMPure XP Beads. And the step of second PCR amplification is followed by a step of second bead purification, preferably 1 purification using AMPure XP Beads.

Further, in the step of the first PCR amplification, annealing and extension are combined into one step, and three annealing temperatures are set in each cycle, so that the coverage of the amplicon sequencing library can be further improved.

According to the number of the amplicons, a plurality of pairs of forward primers and reverse primers specific to the amplicons can be designed, so that the first PCR can be an ultra-high multiplex PCR reaction aiming at a plurality of amplicons (up to hundreds, such as more than six hundred amplicons), namely, a reaction system has more than six hundred pairs of specific primers, annealing temperatures of different primers are different, and three gradient annealing temperatures are set, so that each primer can find a template at the optimal annealing temperature, and the amplification efficiency can be increased; meanwhile, annealing and extension are combined in one step, so that the annealing/extension time is prolonged, more primers can be matched with corresponding templates, and meanwhile, effective extension is obtained. Preferably, in a preferred embodiment, three annealing temperatures in parallel gradients in the range of 58-62℃may be selected, and in an embodiment of the present invention, three annealing temperatures of 62℃60℃and 58℃are preferred, resulting in optimal coverage of the amplicon sequencing library.

In another aspect, the present embodiment further provides a primer set for amplicon sequencing library construction, the primer set comprising: a first primer, a second primer and a third primer for a first PCR amplification, and a fourth primer and a fifth primer for a second PCR amplification; wherein, the liquid crystal display device comprises a liquid crystal display device,

the fifth primer is the second sequencing adapter sequence.

The primer group for constructing the amplicon sequencing library provided by the embodiment of the invention comprises a first primer, a second primer and a third primer, wherein the first primer, the second primer and the third primer are used for first PCR amplification, and the fourth primer and the fifth primer are used for second PCR amplification; when the primer group is used for constructing an amplicon sequencing library, in the first round of PCR amplification, the 5 'end of all fragments of the amplicons can be added with a first sequencing tag sequence through three primers (a first primer, a second primer and a third primer) with special structures, the 3' end of all fragments of the amplicons is added with a second sequencing tag, a sample tag sequence and a second sequencing joint sequence which are sequentially connected, and as different samples can be marked by the sample tag sequences, amplified products of all samples can be combined into one tube after the first PCR amplification is finished, and then the second PCR amplification is directly carried out by a fourth primer and a fifth primer, so that the construction of the sequencing library is completed, the reagents can be greatly saved, and the workload can be reduced; the second sequencing joint sequence, the sample tag sequence and the second sequencing tag sequence in the third primer are separated from the amplicon primers (namely the amplicon specific forward primer and the amplicon specific reverse primer), so that the third primer is more flexible to use, when one sample detects N amplicons, N pairs of specific primers, namely the first primer and the second primer, are needed, but only one third primer is needed, namely only 2N+1 primers are needed to be synthesized, and therefore, when the number of amplicons and the sample size are large, the primer set for library construction greatly reduces the cost of primer synthesis, and the primers in the primer set can be shorter than the existing primers, so that the PCR amplification efficiency can be further improved, and an amplicon sequencing library with better coverage and uniformity can be constructed.

Specifically, in the primer set of the embodiment of the invention, the amplicon specific forward primer and the amplicon specific reverse primer are specific primers designed according to the amplicon sequence and can be specifically combined with the amplicon; the sample tag sequences (i.e., index) are formed by combining bases in different arrangements, and the sample tag sequences corresponding to different samples are different, i.e., the sample tag sequences have unique correspondence with the samples. And a first sequencing tag sequence and a second sequencing tag sequence, the first sequencing linker sequence and the second sequencing linker sequence being sequences used in sequencing.

Preferably, the first sequencing linker sequence and the second sequencing linker sequence in the embodiment of the invention are linker sequences P5 and P7 of an Illumina sequencing platform, the specific first sequencing linker sequence (P5) is shown as SEQ ID No.1, and the second sequencing linker sequence (P7) is shown as SEQ ID No. 2. More preferably, due to the large synthesis amount of the primer and high synthesis cost, the first sequencing tag sequence and the second sequencing tag sequence introduced in the embodiment of the invention are not sequencing tags of the Illumina sequencing platform, but CS1 and CS2 with fewer bases than the first sequencing tag sequence, wherein the specific first sequencing tag sequence (CS 1) is shown as SEQ ID NO.3, and the specific second sequencing tag sequence (CS 2) is shown as SEQ ID NO. 4.

Still further, in the primer set of the embodiment of the present invention, the molar ratio of the first primer to the third primer is 3:1:3. under the condition of the molar ratio, the first primer and the third primer have the highest efficiency of the first PCR amplification.

Finally, the embodiment of the invention also provides a kit for constructing the amplicon sequencing library, which comprises the primer set.

The kit provided by the embodiment of the invention contains the primer group special for the embodiment of the invention, so that the kit can save reagents and workload, reduce cost, further improve PCR amplification efficiency and construct an amplicon sequencing library with better coverage and uniformity when used for constructing the amplicon sequencing library.

Further, the kit also comprises PCR buffer solution and DNA polymerase.

The invention has been tested several times in succession, and the invention will now be described in further detail with reference to a few test results, which are described in detail below in connection with specific examples.

Example 1

1. Primer design and synthesis

Designing specific primers (namely an amplicon specific forward primer and an amplicon specific reverse primer) according to the exon regions of target genes PAH, PTS, SLC A13, SLC22A5, MMACHC, MUT, PCCA, PCCB, MCCC1, MCCC2, IVD and OTC by using Ion AmpliSeqTM Designer software, and adding a first sequencing tag sequence (namely CS 1) to the 5' end of the amplicon specific forward primer to obtain a first primer; the second sequencing tag sequence (CS 2) is added to the 5' end of the amplicon specific reverse primer to obtain a second primer; combining the second sequencing adapter (i.e., P7), the sample tag sequence (i.e., index), and the second sequencing tag sequence into a third primer; the fourth primer consists of a first sequencing joint (namely P5) and a first sequencing tag sequence; the fifth primer is a second sequencing adapter sequence. Because 275 amplicons were synthesized in this example, 275 first and second primers were synthesized, 96 third primer 3 was synthesized temporarily, 1 fourth primer and 1 fifth primer were synthesized, and 1 CS1 sequence, CS1 reverse complement sequence, CS2 sequence and CS2 reverse complement sequence were synthesized. After the primer design was completed, it was synthesized by Invitrogen corporation.

The first sequencing joint sequence P5 and the second sequencing joint sequence P7 are joint sequences of an Illumina sequencing platform; in addition, because of large primer synthesis amount and high synthesis cost, the sequence tag introduced in this embodiment is not a sequence tag of the Illumina sequencing platform, but a first sequence tag sequence CS1 and a second sequence tag sequence CS2 with fewer bases than the sequence tag sequence CS1 and the second sequence tag sequence CS2, and the sequences are as follows:

p5 sequence (SEQ ID NO. 1): AATGATACGGCGACCACCGAGATCT;

p7 sequence (SEQ ID NO. 2): CAAGCAGAAGACGGCATACGAGAT;

CS1 sequence (SEQ ID NO. 3): ACACTGACGACATGGTTCTACA;

CS2 sequence (SEQ ID NO. 4): TACGGTAGCAGAGACTTGGTCT.

2. Sample DNA extraction

Sample DNA selected from human blood, saliva, and tissue can be used for amplification. After extracting the sample DNA with an automatic nucleic acid extractor, the sample DNA was quantified using a Qubit Flurometer 3.0, and about 200-300ng of DNA template was required for detection of each sample.

3. First round PCR reaction

Multiplex PCR amplification was performed on sample DNAs of each obtained genome using the following PCR reaction systems of tables 1 and 2 and the PCR program of Table 3. The round of multiplex PCR was performed in 2 tubes with 275 amplicons in total, and with 139 amplicons in multiplex PCR reaction system 1 (Table 1: total reaction system volume 25 ul) and 136 amplicons in multiplex PCR reaction system 2 (Table 2: total reaction system volume 25 ul). Preparing an upstream primer (first primer) mixing pool and a downstream primer (second primer) mixing pool of amplicons required by 96 samples, wherein the final concentration of each primer is 1 mu M; the final concentration of the corresponding third primer for 96 different sample tags was set at 50. Mu.M. The concentration ratio of the three primers of each amplicon in the reaction system is that the first primer: and (2) a second primer: third primer = 3:1:3, adding a third primer of the same sample label into 2 multiplex PCR reaction systems of the same sample, wherein the specific reaction system is as follows:

TABLE 1

TABLE 2

The components of the above reaction system were added to a PCR tube, which was then placed in a PCR apparatus, and the PCR procedure of Table 3 below was performed:

TABLE 3 Table 3

4. First round PCR product purification

After the first round of PCR reaction was completed, the PCR products of 96 samples were pooled into 1 tube in an amount of 10. Mu.l each, and after shaking and mixing, 500. Mu.l each was purified 2 times with 0.8-fold volume of AMPure XP Beads, and then dissolved in 50. Mu.l of TE.

5. Second round PCR reaction

The second round of PCR reaction was performed using the first round of PCR purified product described above, with only two primers: a fourth primer and a fifth primer. 50ul of PCR reaction system, as shown in Table 4 below:

TABLE 4 Table 4

The components of the reaction system were added to a PCR tube, and then placed in a PCR instrument to perform the PCR procedure shown in Table 5 below.

TABLE 5

6. Second round PCR product purification

After completion of the second round of PCR reaction, the PCR product was purified 1 time with 0.8-fold volume of AMPure XP Beads, dissolved in 50. Mu.l of TE, and library construction for sequencing was completed.

7. Library quantification and on-machine sequencing

The amplicon sequencing library established after two rounds of PCR was accurately quantified with reference to the Qubit fluorometer 3.0 instructions. And after the product is qualified through the library detection, performing on-machine sequencing on PE150 by using an Illumina sequencing platform, wherein the steps are strictly used according to the requirements of suppliers.

8. Sequencing data information analysis

After low quality sequence and linker sequence filtering are carried out on the data obtained by sequencing, the data is compared to a reference genome (GRCh 37/hg 19) by using comparison software BWA, and the sequencing depth (reads) of different amplicons is analyzed to judge the performance and uniformity of multiplex PCR library establishment.

9. Sequencing results

Library construction by the method of this example, sequencing analysis results were as follows:

the sequencing depth of the different amplicons is shown in FIG. 2, 275 amplicons of 96 samples have at least 1 sequence number (reads), 95.3% of the amplicons have a sequencing depth of 30X or more and 100% of the amplicons have repeatability, and most of the sequence differences measured in the range of 1-2 orders of magnitude are concentrated in all the amplicon depth profiles detected in this example.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. A method of constructing a cross-contamination reducing amplicon sequencing library, comprising the steps of:

obtaining DNA of a sample;

performing first PCR amplification on the DNA of the sample by using a first primer, a second primer and a third primer to obtain a first PCR amplification product;

purifying the first PCR amplification product to obtain a first PCR amplification purified product;

taking the first PCR amplified and purified product as a template, and performing second PCR amplification by using a fourth primer and a fifth primer to obtain the amplicon sequencing library;

the first primer sequentially comprises a first sequencing tag sequence and an amplicon specific forward primer from a5 'end to a 3' end; the second primer is sequentially a second sequencing tag sequence and an amplicon specific reverse primer from a5 'end to a 3' end; the third primer sequentially comprises a second sequencing joint sequence, a sample tag sequence and the second sequencing tag sequence from the 5 'end to the 3' end; the sample tag sequence is used for marking different samples; the fourth primer sequentially comprises a first sequencing joint sequence and the first sequencing tag sequence from the 5 'end to the 3' end; the fifth primer is the second sequencing adapter sequence.

2. The method of claim 1, wherein the DNA of the sample is from any one or more of human blood, saliva, and tissue samples.

3. The construction method of claim 1, wherein the amplicon-specific forward primer and the amplicon-specific reverse primer are specifically designed based on the exon regions of the genes of interest PAH, PTS, SLC a13, SLC22A5, MMACHC, MUT, PCCA, PCCB, MCCC1, MCCC2, IVD and OTC.

4. The method of construction of claim 3, wherein the first PCR amplification comprises 275 amplicons and in the step of first PCR amplification each sample is PCR amplified in two multiplex PCR reaction systems comprising a first multiplex PCR reaction system and a second multiplex PCR reaction system, wherein the first multiplex PCR reaction system comprises 139 amplicons and the second multiplex PCR reaction system comprises 136 amplicons.

5. The construction method according to claim 4, wherein the first multiplex PCR reaction system is composed of 15.5. Mu.l of PCR buffer, 0.5. Mu.l of the first primer mixing tank, 0.17. Mu.l of the second primer mixing tank, 1.39. Mu.l of the third primer, 0.6. Mu.l of DNA polymerase, 3. Mu.l of DNA of the sample and 3.84. Mu.l of water.

6. The construction method according to claim 4, wherein the second multiplex PCR reaction system is composed of 15.5. Mu.l of PCR buffer, 0.5. Mu.l of the mixing tank for the first primer, 0.17. Mu.l of the mixing tank for the second primer, 1.36. Mu.l of the third primer, 0.6. Mu.l of DNA polymerase, 3. Mu.l of DNA of the sample and 3.87. Mu.l of water.

7. The method of construction according to claim 1 or 4, wherein in the first PCR amplification step, three annealing temperatures of parallel gradients of 62 ℃, 60 ℃ and 58 ℃ are selected.

8. The method of construction of claim 7, wherein the first PCR amplification is performed at 95 ℃ for 3 minutes followed by 17 cycles of 95 ℃ for 30 seconds, 62 ℃ for 2 minutes, 60 ℃ for 2 minutes, and 58 ℃ for 2 minutes, followed by 72 ℃ for 3 minutes, and finally 4 ℃ is maintained.

9. The construction method according to claim 4, wherein the reaction system for the second PCR amplification consists of 25. Mu.l of 2X KAPA HiFi Hotstart Ready MIX, 1.5. Mu.l of the fourth primer, 1.5. Mu.l of the fifth primer, 10. Mu.l of the amplification product of the first PCR amplification and 12. Mu.l of water.

10. The method of construction of claim 9, wherein the second PCR amplification is performed at 95 ℃ for 3 minutes followed by 10 cycles of 95 ℃ for 20 seconds, 60 ℃ for 15 seconds, 72 ℃ for 30 seconds, 72 ℃ for 3 minutes, and finally 4 ℃.