CN114686580B - Compositions, kits, methods and systems for nucleic acid sample amplification - Google Patents
Compositions, kits, methods and systems for nucleic acid sample amplification Download PDFInfo
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Abstract
The invention relates to a composition, a kit, a nucleic acid amplification method, a library construction method and a system for nucleic acid sample amplification, which can be used for detecting monogenic disease related variation sites, and can obtain high uniformity and/or high coverage while avoiding primer dimer and nonspecific amplification.
Description
Technical Field
The present invention relates to the field of molecular detection, and in particular, to compositions, kits, methods and systems for nucleic acid sample amplification.
Background
A monogenic genetic disease (abbreviated as monogenic disease) is a disease caused by a single gene defect, conforming to the mendelian genetic pattern, and is therefore also called mendelian genetic disease. Is a disease which causes serious injury (death, disability or teratogenesis) to human health and lacks effective diagnosis and treatment means, and is also one of main reasons for causing birth defects. The single-gene diseases which are found at present are more than 10000, and the comprehensive incidence rate is up to 1/100 according to the World Health Organization (WHO) statistics. Monogenic diseases often do not exhibit structural deformities during fetal development, are difficult to find in routine assays, and many recessive genetic diseases often have no obvious family history. It was found that on average each individual carries 2.8 pathogenic mutations of recessive genetic disease, and if parents all carry the same pathogenic mutation, their offspring have a 1/4 chance of suffering. Monogenic diseases not only cause serious harm to the health of patients, but also bring heavy mental and economic burden to families and society. Therefore, an effective gene detection method is needed to prevent and intervene in advance, so that people can know the health condition of the people, and the birth of the children suffering from serious defects can be effectively avoided, and the prenatal and postnatal care can be realized.
The existing gene detection technology for monogenic diseases mainly comprises conventional PCR, sanger sequencing, second-generation high-throughput sequencing and the like. Conventional PCR and Sanger sequencing can only realize detection of single disease species, and the cost is low. And the second-generation high-throughput sequencing can realize the one-time detection of multiple diseases, so that the detection efficiency is greatly improved. The targeted sequencing technology can enrich and sequence the genomic region of interest, single sample sequencing data is less in output and high in analysis speed, so that the advantages of the second-generation high-throughput sequencing technology can be more economically and efficiently exerted. The method is widely applied to various fields such as clinical detection, health screening and the like.
The methods of targeted sequencing are largely divided into two types: hybrid capture sequencing and multiplex PCR amplicon sequencing. The main application of hybridization capture sequencing is liquid phase hybridization capture sequencing at present, namely, based on the base complementary pairing principle, designing a synthetic nucleic acid probe, carrying out hybridization enrichment on a target area of a DNA library based on a liquid phase environment, and sequencing. Multiplex PCR amplicon sequencing is a technique in which multiple PCR primers are designed for amplification enrichment and sequencing of a target region of interest.
The hybridization capture sequencing can involve multiple technical clamping points such as probe sequence design, probe synthesis, liquid phase hybridization capture and the like, the whole experimental process is complex, the speed is low, the requirement on the initial quantity of a sample is higher than that of multiplex PCR amplicon sequencing, and the probe synthesis cost is high.
Compared with hybrid capture sequencing, the method for capturing the target sequence by using multiple PCR has the advantages of simple operation, low cost and low requirement on the initial quantity of samples, and greatly shortens the detection flow and the detection time. However, the more target regions to be captured by multiplex PCR, the coverage and uniformity of primer amplification will be affected, and the difficulty in implementing multiplex PCR capture techniques will be greater.
In view of this, the present invention has been made.
Disclosure of Invention
The present invention creatively developed a composition for nucleic acid sample amplification, comprising primer set 9 for detecting at least 212 pathogenic sites on PAH gene, wherein the primer set 9 uses the nucleic acid sample as a template to amplify the amplicons shown in table 9, and unexpectedly achieves high uniformity and/or high coverage.
The invention also provides other compositions, kits, nucleic acid amplification methods, library building methods and systems based on the compositions.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a gel diagram after multiplex PCR amplification in one embodiment of the present invention;
FIG. 2 is a diagram of an amplicon sequencing library according to one embodiment of the present invention.
Detailed Description
When multiple sites on a gene are obtained in parallel, two difficulties exist, namely, how to design a set of combinations with small interaction among different primers, and how to design a set of primer combinations covering multiple sites as much as possible; breaking through any one of the difficulties requires the inventor to perform creative work.
The inventors have creatively designed primers that can be used to detect the mutation sites of a target gene, not only avoid primer dimer, reduce specific amplification, but also use a variety of primer composition for multiplex PCR amplification and/or amplicon sequencing library construction, unexpectedly achieving high uniformity and/or high coverage, overcoming the difficulties of the prior art.
The present invention relates to a composition for nucleic acid sample amplification, comprising primer set 9; primer set 9 amplified amplicons shown in table 9 using the nucleic acid sample as a template;
In some embodiments, the above composition for nucleic acid sample amplification further comprises at least one of primer sets 1-8, 10-11; primer set 1 uses a nucleic acid sample as a template to amplify the amplicons shown in table 1; primer set 2 uses the nucleic acid sample as a template to amplify the amplicons shown in table 2; primer set 3 amplified amplicons shown in table 3 using the nucleic acid sample as a template; primer set 4 amplified amplicons shown in table 4 using the nucleic acid sample as a template; primer set 5 amplified amplicons shown in table 5 using the nucleic acid sample as a template; primer set 6 amplified amplicons shown in table 6 using the nucleic acid sample as a template; primer set 7 amplified amplicons shown in table 7 using the nucleic acid sample as a template; primer set 8 amplified amplicons shown in table 8 using the nucleic acid sample as a template; primer set 10 uses the nucleic acid sample as a template to amplify the amplicons shown in table 10; the primer set 11 amplified the amplicons shown in Table 11 using the nucleic acid sample as a template.
The invention also relates to a composition for amplifying a nucleic acid sample, comprising a primer group 9 with a nucleotide sequence shown as SEQ ID NO. 163-SEQ ID NO. 190.
In some embodiments, the composition further comprises at least one of primer sets 1-8, 10-11; wherein, the primer group 1 has a nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 46; the primer group 2 has a nucleotide sequence shown as SEQ ID NO. 47-SEQ ID NO. 60; the primer group 3 has a nucleotide sequence shown as SEQ ID NO. 61-SEQ ID NO. 86; the primer group 4 has a nucleotide sequence shown as SEQ ID NO. 87-SEQ ID NO. 96; the primer group 5 has a nucleotide sequence shown as SEQ ID NO. 97-SEQ ID NO. 138; the primer group 6 has a nucleotide sequence shown as SEQ ID NO. 139-SEQ ID NO. 144; primer group 7 has a nucleotide sequence shown as SEQ ID NO. 145-SEQ ID NO. 150; primer group 8 has a nucleotide sequence shown as SEQ ID NO. 151-SEQ ID NO. 162; primer group 10 has a nucleotide sequence shown as SEQ ID NO. 191-SEQ ID NO. 202; primer set 11 has a nucleotide sequence as shown in SEQ ID NO. 203-SEQ ID NO. 212.
In some embodiments, the composition includes other primer sets in addition to primer set 9. For example: in some embodiments, the composition comprises primer set 9 and primer set 2. In some embodiments, the composition comprises primer set 9 and primer set 5. In some embodiments, the composition comprises primer set 9, primer set 10, and primer set 6. In some embodiments, the composition comprises primer set 9, primer set 7, and primer set 8. In some embodiments, the composition comprises primer set 9 and primer set 11. In some embodiments, the composition comprises primer set 9, primer set 3, and primer set 4. In some embodiments, the composition comprises primer set 9 and primer set 1. In some embodiments, the composition comprises primer sets 9, 1,2, and 5. In some embodiments, the composition comprises primer set 1-primer set 11.
In some embodiments, the above composition is used to detect a mutation site on a PAH gene.
In some embodiments, the above composition may also be used to detect a mutation site on at least one of the following genes: ATP7B, CYP A2, F8, F9, GAA, GJB2, HBA1, HBA2, HBB, SLC26A4, SMN1.
In some embodiments, primer set 9 is used to detect at least 212 pathogenic sites on the PAH gene. In some embodiments, primer set 1 is used to detect at least 98 pathogenic sites on the ATP7B gene. In some embodiments, primer set 2 is used to detect at least 10 pathogenic sites on the CYP21A2 gene. In some embodiments, primer set 3 is used to detect at least 16 pathogenic sites on the F8 gene. In some embodiments, primer set 4 is used to detect at least 9 pathogenic sites on the F9 gene. In some embodiments, primer set 5 is used to detect at least 75 pathogenic sites on the GAA gene. In some embodiments, primer set 6 is used to detect at least 11 pathogenic sites on the GJB2 gene. In some embodiments, primer set 7 is used to detect at least 8 pathogenic sites on the HBA1, HBA2 genes. In some embodiments, primer set 8 is used to detect at least 85 pathogenic sites on the HBB gene. In some embodiments, primer set 10 is used to detect at least 9 pathogenic sites on the SLC26A4 gene. In some embodiments, primer set 11 is used to detect at least 9 pathogenic sites on the SMN1 gene.
The pathogenic site is usually a mutation site causing diseases, and mutation forms include SNV (single nucleotide variant, single nucleotide mutation), short repeat (Short sequence repeat), inversion, insertion, deletion, duplication (repeat) and the like.
In some embodiments, the primer sequences in the primer set described in any of the embodiments above are allowed to be modified; or ligating universal sequences required for library-building sequencing.
In some embodiments, the type of modification includes thio, 2 fluoro ribonucleic acid, 2' -O-methyl ribonucleic acid, 5-methyl deoxycytosine, deoxyinosine, deoxyuracil, 2-aminopurine, 5-bromo-deoxyuracil, inverted dT, dideoxycytidine, a spacer, amino modification, carboxyl modification, phosphorylation modification, digoxin modification, biotin modification, and the like, and one skilled in the art can modify the primer according to the purpose, e.g., 2-5 bases on both ends of the primer can be thio modified in order to prevent degradation of the primer by nucleases.
In some embodiments, the 5' end of the primer sequences in the pair primer set is allowed to ligate to a universal sequence.
In some embodiments, the universal sequence comprises a library primer. Library primers are used to achieve ligation of amplicons to sequencing primers, tags or adaptors. It will be appreciated by those skilled in the art that library primers will vary from sequencing platform to sequencing platform, and from one library approach to another. In some embodiments, the pool-building primer can be a portion of the nucleotide sequence of a sequencing primer or adaptor, in which embodiment ligation of the amplicon to the sequencing primer or adaptor can be accomplished by the pool-building primer for pool-building sequencing purposes. In some embodiments, the pool-building primer can be a sequencing primer or a portion of the nucleotide sequence near the 3' end of the linker. In some embodiments, the pool-building primer can be a nucleotide sequence that is different from a sequencing primer, linker, or tag. Ligation of the amplicon to the sequencing primer is accomplished, for example, by introducing a pool-building primer into the sequencing primer, adaptor, or tag, forming a new sequence, e.g., ligating the pool-building primer to the 3' end of the sequencing primer; for example, ligation of the pool-building primer to the 3' end of the adaptor, allowing ligation of the amplicon to the adaptor; for example, the ligation of the amplicon to the tag is achieved by ligating a pool primer to the 3' end of the tag linker. The adaptor is understood in a broad sense, and the nucleotide sequence of the adaptor includes a sequencing primer sequence, and further may include a tag sequence or a library primer sequence, etc. In some embodiments, the linker (also known as a sequencing linker) of the illumine platform is 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', which includes sequencing primers, which may be part of the linker; for example, in some embodiments, pool-building primer 1 (corresponding to general sequence 1 in example 11) is: 5'-CTACACGACGCTCTTCCGATCT-3'. In some embodiments, the linker of the illuminea platform (also known as a tag linker) is 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3', which includes sequencing primers and tag sequences, of which the library primer may be a part; for example, in some embodiments, pool-building primer 2 (corresponding to general sequence 2 in example 11) is: 5'-CAGACGTGTGCTCTTCCGATCT-3'.
In some embodiments, the universal sequence comprises a sequencing primer. In some embodiments, the sequencing primer is ligated to the 5' end of the primer, in which case the amplicon comprising the sequencing primer may be obtained directly.
In some embodiments, the universal sequence comprises a tag (index). The label is used for realizing sample marking, and in the construction process of the nucleic acid sequencing library, the differentiation of different samples can be realized by adding the label, so that high-throughput detection is realized. In some embodiments, the tag is a 4-18 nucleotide composition, e.g., a random NNNNNN, e.g., a sequencing platform recommended tag sequence, such as an illumine platform recommended tag sequence. In some embodiments, the tag may be attached to the 5' end of the primer sequence. In this case, the amplicon comprising the tag can be obtained directly.
In some embodiments, the universal sequence comprises a linker. In some embodiments, the adaptor is attached to the 5' end of the primer, in which case the amplicon comprising the adaptor can be obtained directly.
In some embodiments, a universal sequence may be ligated to only one of the primer pairs. In some embodiments, the universal sequences may be separately linked to both primers in a primer pair. In some embodiments, the two universal sequences separately attached to the primer pair may be the same or different.
The invention also relates to a kit comprising a composition according to any of the embodiments described above. In some embodiments, the kit further comprises a PCR buffer, DNA polymerase to effect PCR amplification. In some embodiments, the kit further comprises linker primers and/or tag primers to effect amplicon library construction. In some embodiments, the kit further comprises nucleic acid extraction reagents to effect the obtaining of a nucleic acid sample. In some embodiments, the kit further comprises a purification reagent, such as magnetic beads or the like.
The invention also relates to a method of nucleic acid amplification comprising: the nucleic acid sample is amplified using the composition of any of the above embodiments. In some embodiments, amplifying the nucleic acid sample is performed under conditions that allow amplification to occur. In some embodiments, the nucleic acid sample is amplified according to conventional amplification reaction conditions, e.g., in a system with PCR buffer, DNA polymerase. In some embodiments, a fluorescent probe or fluorescent dye may also be added to the amplification reaction, indicating a PCR reaction.
The invention also relates to a method for building the library, which comprises the following steps: an amplicon sequencing library was constructed for nucleic acid sequencing using the composition of any of the above embodiments. In some embodiments, the composition is used to amplify the amplicon, the amplicon is ligated or tagged according to sequencing platform requirements, and an amplicon sequencing library is enriched for sequencing.
In some embodiments of the above nucleic acid amplification method and/or the above library building method, the 10 μl multiplex PCR amplification system is: 2. Mu.L of 5 XHyperPCR Buffer, 1. Mu.L of 0.5-10. Mu.M primer pool working solution, 0.1-3. Mu.L of CA DNA polymerase, X. Mu.L of gDNA (. Gtoreq.10 ng), (6.5-X) mu.L of nucleic acid-FREE WATER.
In some embodiments, the conditions for multiplex PCR amplification are ：94-96℃5-10min;3-5cycles(94-96℃25-35s,58-63℃1.5-2.5min,63-67℃2.5-3.5min,65-68℃0.5-1.5min,70-74℃0.5-1.5min);12-25cycles(94-96℃30s,64-68℃2.5-3.5min).
The invention also relates to a system comprising: a library building unit; the library building unit is used for building an amplicon sequencing library by using the composition according to any embodiment.
In some embodiments, the system further comprises a sequencing unit for sequencing the amplicon sequencing library output by the pooling unit; in some embodiments, the sequencing unit sequences the amplicon library, e.g., a common sequencing module can be nested, and nucleic acid information of the amplicon sequencing library can be obtained.
In some embodiments, the system further comprises an analysis unit for performing bioinformatic analysis on the off-machine data output by the sequencing unit. In some embodiments, the bioinformatic analysis includes analysis of tag identification, sequence alignment, data de-drying, data correction, or mutation site identification, among others.
In some embodiments, the nucleic acid sample is DNA.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
A composition for nucleic acid sample amplification comprising primer set 9. Wherein, phenylketonuria (Phenylketonuria) is a disease caused by PAH gene defect, the primers are creatively designed aiming at 212 pathogenic sites on PAH, the primer dimer and specific amplification are avoided, the high uniformity and the high coverage are both considered, and the primer group 9 (the nucleotide sequence shown as SEQ ID NO:163-SEQ ID NO: 190) is obtained through optimization.
Example 2
A composition for nucleic acid sample amplification comprising primer set 9 and primer set 2. Wherein, 21-hydroxylase deficiency type congenital adrenocortical hyperplasia (ADRENAL HYPERPLASIA, congenital, due to21-hydroxylase deficiency) is a disease caused by CYP21A2 gene deficiency, primers are creatively designed aiming at 10 pathogenic sites on CYP21A2, so that primer dimer and specific amplification are avoided, high uniformity and high coverage are considered, and primer group 2 (nucleotide sequence shown as SEQ ID NO:47-SEQ ID NO: 60) is obtained through optimization; primer set 9 is the same as in example 1.
Example 3
A composition for nucleic acid sample amplification comprising primer set 9 and primer set 5. Wherein Glycogen Storage disease type 2 (Glycogen Storage DISEASE II) is a disease caused by a GAA gene defect, primers are creatively designed aiming at 75 pathogenic sites on GAA, primer dimer and specific amplification are avoided, high uniformity and high coverage are considered, and primer group 5 (nucleotide sequence shown as SEQ ID NO:97-SEQ ID NO: 138) is obtained through optimization; primer set 9 is the same as in example 1.
Example 4
A composition for nucleic acid sample amplification comprising primer set 9, primer set 10 and primer set 6. Wherein autosomal recessive deafness 1A type (Deafness, autosomal Recessive a) is a disease caused by GJB2 gene defect, creatively designs primers aiming at 11 pathogenic sites on GJB2, avoids primer dimer and specific amplification, combines high uniformity and high coverage, and obtains a primer group 6 (nucleotide sequence shown as SEQ ID NO:139-SEQ ID NO: 144) through optimization; autosomal recessive deafness with vestibular aqueduct expansion type 4 (Deafness, autosomal recessive 4,with enlarged vestibular aqueduc) is a disease caused by SLC26A4 gene defect, primers are creatively designed aiming at 9 pathogenic sites on SLC26A4, primer dimer and specific amplification are avoided, high uniformity and high coverage are considered, and primer group 10 (nucleotide sequence shown as SEQ ID NO:191-SEQ ID NO: 202) is obtained through optimization; primer set 9 is the same as in example 1.
Example 5
A composition for nucleic acid sample amplification comprising primer set 9, primer set 7 and primer set 8. Wherein, alpha-thalassemia (Alpha-thalassemia) is a disease caused by a globin gene defect, primers are creatively designed aiming at 8 pathogenic sites on HBA1 and HBA2, so that primer dimer and specific amplification are avoided, high uniformity and high coverage are considered, and a primer group 7 (a nucleotide sequence shown as SEQ ID NO:145-SEQ ID NO: 150) is obtained through optimization; beta-thalassemia (Beta-thalassemia) is a disease caused by a defect of an HBB gene, primers are creatively designed aiming at 85 pathogenic sites on the HBB, primer dimer and specific amplification are avoided, high uniformity and high coverage are considered, and a primer group 8 (a nucleotide sequence shown as SEQ ID NO:151-SEQ ID NO: 162) is obtained through optimization; primer set 9 is the same as in example 1.
Example 6
A composition for nucleic acid sample amplification comprising primer set 9 and primer set 11. Wherein spinal muscular atrophy (Spinal muscular atrophy) is a disease caused by SMN1 gene defect, primers are creatively designed aiming at 9 pathogenic sites on SMN1, so that primer dimer and specific amplification are avoided, high uniformity and high coverage are considered, and a primer group 11 (a nucleotide sequence shown as SEQ ID NO:203-SEQ ID NO: 212) is obtained through optimization; primer set 9 is the same as in example 1.
Example 7
A composition for nucleic acid sample amplification comprising primer set 9, primer set 3 and primer set 4. Wherein hemophilia A (Hemophilia A) is a disease caused by F8 gene defect, creatively designs primers aiming at 16 pathogenic sites on F8, avoids primer dimer and specific amplification, combines high uniformity and high coverage, and obtains a primer group 3 (nucleotide sequence shown as SEQ ID NO:61-SEQ ID NO: 86) through optimization; hemophilia B (Hemophilia B) is a disease caused by F9 gene defect, creatively designs primers aiming at 9 pathogenic sites on F9, avoids primer dimer and specific amplification, combines high uniformity and high coverage, and obtains primer group 4 (nucleotide sequence shown as SEQ ID NO:87-SEQ ID NO: 96) through optimization; primer set 9 is the same as in example 1.
Example 8
A composition for nucleic acid sample amplification comprising primer set 9 and primer set 1. Wherein, hepatolenticular degeneration (Wilson Disease) is a Disease caused by ATP7B gene defect, the invention creatively designs primers aiming at 98 pathogenic sites on ATP7B, avoids primer dimer and specific amplification, combines high uniformity and high coverage, and obtains a primer group 1 (nucleotide sequence shown as SEQ ID NO:1-SEQ ID NO: 46) through optimization; primer set 9 is the same as in example 1.
Example 9
A composition for nucleic acid sample amplification comprising primer set 9, primer set 1, primer set 2 and primer set 5. Wherein, primer set 9 is the same as example 1, primer set 1 is the same as example 8, primer set 2 is the same as example 2, and primer set 5 is the same as example 3.
Example 10
A composition for amplifying a nucleic acid sample comprises a primer set 1-a primer set 11, i.e., comprising the nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 212.
Example 11
DNA extraction
In the examples, the nucleic acid sample is DNA derived from isolated human peripheral blood. DNA sample extraction whole genome DNA (gDNA) was extracted using DNeasy Blood & Tissue Kit (QIAGEN) extraction Kit, and the extracted DNA was subjected to purity detection using Nanodrop and concentration measurement using Qubit. The DNA samples employed in the examples were of good integrity.
2. Multiplex PCR amplification
The composition of any of examples 1-10 was used. In order to achieve the aim of library construction, the 5' end of the primer is connected with a general sequence. Specifically, for example, an upstream primer or a downstream primer of the primer pair used is ligated to the general sequence 1 or the general sequence 2, respectively, specifically, if the upstream primer is ligated to the general sequence 1, the downstream primer is ligated to the general sequence 2; if the upstream primer is ligated to universal sequence 2, the downstream primer is ligated to universal sequence 1, specifically for example universal sequence 1 is 5'-CTACACGACGCTCTTCC GATCT-3'; general sequence 2 is 5'-CAGACGTGTGCTCTTCCGATCT-3'. After the primer having the universal sequence attached thereto was dissolved, 5. Mu.M of primer pool working solution was obtained by mixing at an equimolar concentration. 10. Mu.L of a multiplex PCR reaction system was prepared, which included 2. Mu.L of 5 XHyperPCR Buffer (Fapo n), 1. Mu.L of 5. Mu.M primer pool working solution, 0.5. Mu.L of CA DNA polymerase (Fapon), X. Mu.L of gDNA (20 ng), (6.5-X) mu.L of nucleic acid-FREE WATER, and the mixture was thoroughly mixed and then placed on a PC R apparatus to carry out the following reaction: 95 ℃ for 10min;5cycles (95 ℃ C. 30s,63 ℃ C. 2min,65 ℃ C. 3min,67 ℃ C. 1min,72 ℃ C. 1 min); 20cycles (95 ℃ C. 30s,66 ℃ C. 3 min).
3. Construction of amplicon sequencing library
After the multiplex PCR amplification is finished, adding 40 mu L to 50 mu L of water into 10 mu L of multiplex PCR reaction system, adding 60 mu L of Ampure XP Beads for purification, standing at room temperature for 5min after uniform mixing, separating and removing supernatant on a magnetic rack, adding 200 mu L of freshly prepared 80% ethanol for washing twice, drying the Beads at room temperature for 2-3min, adding 20 mu L of Nuclease-FREE WATER re-dissolved Beads, standing at room temperature for 2min, separating on the magnetic rack, and transferring the supernatant into a new centrifuge tube.
A 50 μl amplicon sequencing library PCR reaction system was configured comprising: 25. Mu.L of 2 XHIFI PCR Mix (Fapon), 2. Mu.L of 10. Mu.M Uni_linker, 2. Mu.L of 10. Mu.M Index_linker, 4. Mu.L of multiplex PCR amplification product purified in the previous step, 17. Mu.L of nucleic-FREE WATER; after mixing well, the following reactions were performed on a PCR instrument: 45s at 98 ℃;6cycles (98 ℃ C. 20s,60 ℃ C. 30s,72 ℃ C. 30 s); hold at 4 ℃.
Uni_Joint:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT (underlined is the overlap with universal sequence 1);
index_linker:
CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (underlined is the overlap with universal sequence 2, NNNNNN is the tag sequence that recognizes different multiplex amplification products).
After the PCR procedure is finished, adding 50 mu L of Ampure XP Beads into 50 mu L of a PCR amplification system, purifying, uniformly mixing, standing at room temperature for 5min, separating and removing the supernatant on a magnetic rack, adding 200 mu L of freshly prepared 80% ethanol for washing twice, drying the Beads at room temperature for 2-3min, adding 20 mu L of Nuclease-FREE WATER re-dissolved Beads, standing at room temperature for 2min, separating on the magnetic rack, and transferring the supernatant into a new centrifuge tube. The supernatant obtained by purification is the constructed amplicon sequencing library. 4. High throughput sequencing of amplicon sequencing libraries
And (3) carrying out equal-quantity mixing on an amplicon sequencing library meeting the quality inspection requirement according to the quantitative concentration of the Qubit, and then carrying out on-machine sequencing, wherein a sequencing platform is Illumina Hiseq 2500.
5. Bioinformatics analysis
And performing quality control and analysis of conventional parameters on the obtained sequencing off-machine data.
Detection result
1. The compositions of examples 1-10 were each tested as in example 11, with MAPPING RATE being greater than 95% and Ontarget Rate being greater than 90%. >0.2x Average Depth Rate, >30x Depth Rate and coverage for each example are shown in the table below.
Note that: "MAPPING RATE" represents the comparison rate at the time of resequencing; "Ontarget Rate" represents the mid-target rate, representing the ratio of sequencing data aligned to the targeted amplicon; ">0.2x Average Depth Rate" represents a proportion of sites of greater than 0.2x average sequencing depth, the higher the value, the better the amplification uniformity; ">30x Depth Rate" represents the proportion of greater than 30x sequencing Depth, the higher this value, indicating a higher amplicon effective coverage; "coverage" means the coverage of the target site, and the higher the value, the higher the target site coverage.
2. After multiplex PCR amplification using the composition of example 10. As a result of electrophoresis on 2% agarose gel, as shown in FIG. 1, the target amplified fragment was apparent, and no apparent primer dimer was found, thus resulting in high amplification efficiency.
3. Using the composition of example 10, a constructed amplicon sequencing library was obtained, and the library size was detected using an Agilent2100Bioanalyzer, with the results shown in fig. 2, and with a major library peak size of 300-400bp, without significant small fragments (primer dimer or adaptor dimer); the concentration of the constructed amplicon sequencing library is 40-60 ng/. Mu.L by quantitative determination of the Qubit concentration, and the amplification efficiency is high.
In summary, the composition of the invention can obtain excellent uniformity and coverage, and has high amplification efficiency. Wherein, the primer group 9 can ensure that 212 mutation sites on the PAH gene are completely covered, thereby realizing excellent detection effect.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
SEQUENCE LISTING
<110> Guangdong Fit biological Co., ltd
<120> Compositions, kits, methods, and systems for nucleic acid sample amplification
<130>
<140> 202011584398.X
<141> 2020-12-28
<160> 216
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213> synthetic construct
<400> 1
ACGCCCAAGTCCACGTACCTCTTTACAGT
<210> 2
<211> 27
<212> DNA
<213> synthetic construct
<400> 2
CAGGAGTGTGACTATGGAAGCCCCTCC
<210> 3
<211> 28
<212> DNA
<213> synthetic construct
<400> 3
TGCTAGCCACCACATCCAGCAAATCATT
<210> 4
<211> 27
<212> DNA
<213> synthetic construct
<400> 4
AAGGTCCAGGAGCTCCAGAATAAAGGG
<210> 5
<211> 33
<212> DNA
<213> synthetic construct
<400> 5
ACCTCAATAATTTTGATAATATCCCGTGGACCG
<210> 6
<211> 27
<212> DNA
<213> synthetic construct
<400> 6
CCTGGGTCTGTGGGATTCTTGCCATCC
<210> 7
<211> 29
<212> DNA
<213> synthetic construct
<400> 7
TCTGAACCTGAAGCTGCTGTTACCTTTGC
<210> 8
<211> 27
<212> DNA
<213> synthetic construct
<400> 8
TCGGTGGGTGGTACTTCTACGTTCAGG
<210> 9
<211> 34
<212> DNA
<213> synthetic construct
<400> 9
AAAGCCCAGTGAATCTAAGATATGAAAGAACAGG
<210> 10
<211> 32
<212> DNA
<213> synthetic construct
<400> 10
CATAGGTTGTAATTTCCCATGGTCTTGGTGTT
<210> 11
<211> 27
<212> DNA
<213> synthetic construct
<400> 11
TCAGGATGGGGAAAGCCGTGCTACAGG
<210> 12
<211> 27
<212> DNA
<213> synthetic construct
<400> 12
CTCAACCTGCCTCTGACTCTGTCCTGT
<210> 13
<211> 28
<212> DNA
<213> synthetic construct
<400> 13
ACAAGGGTAAAGGCAGCTAATCCAGGAG
<210> 14
<211> 29
<212> DNA
<213> synthetic construct
<400> 14
CCTCCTTTTTCCCCACCCCTCTCTTTTTA
<210> 15
<211> 31
<212> DNA
<213> synthetic construct
<400> 15
ACTCTTTTGCCTGATATCTGCAGAAAACTGT
<210> 16
<211> 33
<212> DNA
<213> synthetic construct
<400> 16
GTGAAATTGGACCATTTAGAAATAACCACAGCC
<210> 17
<211> 27
<212> DNA
<213> synthetic construct
<400> 17
ACTGGAAGACCTGTGATCTGTCCCACT
<210> 18
<211> 26
<212> DNA
<213> synthetic construct
<400> 18
CACCAAGGAAGCCATGTGGTCACCCT
<210> 19
<211> 26
<212> DNA
<213> synthetic construct
<400> 19
CTCTCCGCCTTCTCAGCCACAGCAAC
<210> 20
<211> 27
<212> DNA
<213> synthetic construct
<400> 20
GGCCCTGTGTCGCTCATTGAACTCTCC
<210> 21
<211> 29
<212> DNA
<213> synthetic construct
<400> 21
ACAGTTTGCAACATTAAAGGGCTGTACCT
<210> 22
<211> 26
<212> DNA
<213> synthetic construct
<400> 22
GCAAGTGTGGTATCTTGGTGCGGGGT
<210> 23
<211> 29
<212> DNA
<213> synthetic construct
<400> 23
ATAAATCAGTGCCAGGACCAGGTTGATGC
<210> 24
<211> 26
<212> DNA
<213> synthetic construct
<400> 24
CGAGGCAGCCGACGTCGTCCTTATCA
<210> 25
<211> 28
<212> DNA
<213> synthetic construct
<400> 25
TGAGAGGCAAGTTCCACTGTGCTAAGCA
<210> 26
<211> 27
<212> DNA
<213> synthetic construct
<400> 26
TGAGTGCGAGTTCTTTCTTCCCCAGGT
<210> 27
<211> 27
<212> DNA
<213> synthetic construct
<400> 27
TTAACCAGCTGCAGAGACAAAAGCCAG
<210> 28
<211> 28
<212> DNA
<213> synthetic construct
<400> 28
CCCCTCCCTTTCACTTCACCCCTCTTGG
<210> 29
<211> 29
<212> DNA
<213> synthetic construct
<400> 29
CCCATGTCCCCAATTTGATGGCAAACCTG
<210> 30
<211> 31
<212> DNA
<213> synthetic construct
<400> 30
GGAACCAGCAATGAAGAAGAGTTTTGCTTTT
<210> 31
<211> 28
<212> DNA
<213> synthetic construct
<400> 31
CCTGAGGGAACATGAAACAAGCCATCTC
<210> 32
<211> 25
<212> DNA
<213> synthetic construct
<400> 32
AGCTGGCCTAGAACCTGACCCGGTG
<210> 33
<211> 27
<212> DNA
<213> synthetic construct
<400> 33
TGCCCACACTCACAAGGTCTATTGCAA
<210> 34
<211> 27
<212> DNA
<213> synthetic construct
<400> 34
TTCCTAGAGCAAAACCTCAGAAGCCCT
<210> 35
<211> 29
<212> DNA
<213> synthetic construct
<400> 35
ACCAAGCTAAAGCACTATGTTTGCGCTTA
<210> 36
<211> 27
<212> DNA
<213> synthetic construct
<400> 36
TTTGGCATCCCTGTCATGGCCTTAATG
<210> 37
<211> 27
<212> DNA
<213> synthetic construct
<400> 37
TGCTCTTGATGGCAGCTTCAAATCCCA
<210> 38
<211> 27
<212> DNA
<213> synthetic construct
<400> 38
GCTTCGAGGCCAGCATTGCAGAAGGAA
<210> 39
<211> 27
<212> DNA
<213> synthetic construct
<400> 39
TCTAAAGGGTCCTTTTCTCCCACGCCA
<210> 40
<211> 28
<212> DNA
<213> synthetic construct
<400> 40
GCAGGAGAGACAGATCACAGCCAGAGAA
<210> 41
<211> 28
<212> DNA
<213> synthetic construct
<400> 41
ACGAGGTCTATACGCAGCATTCCTAAGT
<210> 42
<211> 27
<212> DNA
<213> synthetic construct
<400> 42
CTACATCTGTGCAGGAAGTGGCTCCCC
<210> 43
<211> 29
<212> DNA
<213> synthetic construct
<400> 43
ACCAGAAACGACTGAAGCCTCAAATCCCA
<210> 44
<211> 26
<212> DNA
<213> synthetic construct
<400> 44
AGTTCTCATTCCCCTGGCTCCCCACC
<210> 45
<211> 35
<212> DNA
<213> synthetic construct
<400> 45
CTTACCCATTCACTGATATCCTCCCTCAGATTATT
<210> 46
<211> 29
<212> DNA
<213> synthetic construct
<400> 46
CCAGCAAAGCCCTTGTTAAGTTTGACCCG
<210> 47
<211> 25
<212> DNA
<213> synthetic construct
<400> 47
TCAGCCTCGCCTCTCACAGTAGCCC
<210> 48
<211> 27
<212> DNA
<213> synthetic construct
<400> 48
AAGTTGTCGTCCTGCCAGAAAAGGAGG
<210> 49
<211> 26
<212> DNA
<213> synthetic construct
<400> 49
TTTCACCCTCTGCAGGAGAGCCTCGT
<210> 50
<211> 25
<212> DNA
<213> synthetic construct
<400> 50
GCCTTTTGCTTGTCCCCAGGACGCA
<210> 51
<211> 27
<212> DNA
<213> synthetic construct
<400> 51
ACTCCACCCGATCATTCCCCAGATTCA
<210> 52
<211> 27
<212> DNA
<213> synthetic construct
<400> 52
CTTTCCTCACTCATCCCCAACCCTCGG
<210> 53
<211> 27
<212> DNA
<213> synthetic construct
<400> 53
GGCCCCAAAACAGTCTACACAGCAGGA
<210> 54
<211> 27
<212> DNA
<213> synthetic construct
<400> 54
GCCTGTAGATGGGCCCGAATTTCTGAG
<210> 55
<211> 26
<212> DNA
<213> synthetic construct
<400> 55
GGCCCTCAGCTGCCTTCATCAGTTCC
<210> 56
<211> 27
<212> DNA
<213> synthetic construct
<400> 56
TCAGGAGCCCAGCCTTACCTCACAGAA
<210> 57
<211> 25
<212> DNA
<213> synthetic construct
<400> 57
GCTCTTCGTGGTGCTGACCCGACTG
<210> 58
<211> 28
<212> DNA
<213> synthetic construct
<400> 58
GCAATAAAGGAGAAACTGAGGTACCCGG
<210> 59
<211> 29
<212> DNA
<213> synthetic construct
<400> 59
GCTGAAGCAGGCCATAGAGAAGAGGGATC
<210> 60
<211> 25
<212> DNA
<213> synthetic construct
<400> 60
CCATCATGTCCCTCCACTGGCCTGC
<210> 61
<211> 26
<212> DNA
<213> synthetic construct
<400> 61
CTCGCAGCCCAGAACCTCCATCCTCA
<210> 62
<211> 29
<212> DNA
<213> synthetic construct
<400> 62
ACAACTAGAAGTGAGAAAAGCGTCTGTGC
<210> 63
<211> 27
<212> DNA
<213> synthetic construct
<400> 63
AGTCCCAGTCCTCCTCTTCAGCAGCAA
<210> 64
<211> 30
<212> DNA
<213> synthetic construct
<400> 64
AGCAAGACACTCTGACATTGTTTGGTTTGT
<210> 65
<211> 31
<212> DNA
<213> synthetic construct
<400> 65
ACTCTATAATGTCCCCTTCAGCAACACACTA
<210> 66
<211> 29
<212> DNA
<213> synthetic construct
<400> 66
AAGTGCACTCAATATTCCTCGAAGGTCAC
<210> 67
<211> 24
<212> DNA
<213> synthetic construct
<400> 67
GAGCTTGAGGTCCGGCCAACAGCT
<210> 68
<211> 34
<212> DNA
<213> synthetic construct
<400> 68
AAGAATCAAGCAAGCAGACCATATAACATCTACC
<210> 69
<211> 27
<212> DNA
<213> synthetic construct
<400> 69
AGGCAAGAACTCACCTGGTTTCCTCTT
<210> 70
<211> 34
<212> DNA
<213> synthetic construct
<400> 70
AGGATTTTCCAATTCTGCCAGGAGAAATATTCAA
<210> 71
<211> 32
<212> DNA
<213> synthetic construct
<400> 71
TTTCTTTATTCACCACCCACTGGACTTAAGTG
<210> 72
<211> 33
<212> DNA
<213> synthetic construct
<400> 72
GACAACATCAGTAGCATTTCTTTACCCCTTTCA
<210> 73
<211> 31
<212> DNA
<213> synthetic construct
<400> 73
ACCAGTGTTCTTGTCACAACTAGAAACCTTC
<210> 74
<211> 27
<212> DNA
<213> synthetic construct
<400> 74
ACAACAGCAGCAATGCAAAAACCACTT
<210> 75
<211> 35
<212> DNA
<213> synthetic construct
<400> 75
GTTCTTCATGAAATTCTTAGTGCCAGTCACTGTAT
<210> 76
<211> 31
<212> DNA
<213> synthetic construct
<400> 76
ACGTAGGACTCAAAGAGATGGTTTTTCCAAG
<210> 77
<211> 29
<212> DNA
<213> synthetic construct
<400> 77
ACCAGTCATCTCCAAGGTTAGAATGGCTA
<210> 78
<211> 27
<212> DNA
<213> synthetic construct
<400> 78
CGAGGAGTCATAGCATCCCTCAAGCAA
<210> 79
<211> 32
<212> DNA
<213> synthetic construct
<400> 79
GCCTCTCCACTGCAGCAATAAAATAGTGTCGT
<210> 80
<211> 27
<212> DNA
<213> synthetic construct
<400> 80
AAAACCCACCAGTCTTGAAACGCCATC
<210> 81
<211> 27
<212> DNA
<213> synthetic construct
<400> 81
ATCACAGCCCATCAACTCCATGCGAAG
<210> 82
<211> 27
<212> DNA
<213> synthetic construct
<400> 82
TGGATGCTGTTGAGAAACGCACAAAGC
<210> 83
<211> 30
<212> DNA
<213> synthetic construct
<400> 83
TGATTGTGTTCCCAGTGCCTAGACCATTTA
<210> 84
<211> 28
<212> DNA
<213> synthetic construct
<400> 84
AATGGCTCAGGATCAAAGGATTCGATGG
<210> 85
<211> 35
<212> DNA
<213> synthetic construct
<400> 85
CTTGACACTACTACATTTTTGCTGATATGTCCGTA
<210> 86
<211> 29
<212> DNA
<213> synthetic construct
<400> 86
GTCAGAAGTTCTCCAGCCTCTACATCTCT
<210> 87
<211> 33
<212> DNA
<213> synthetic construct
<400> 87
ACATTTCAGTTTTTCTTGATCATGAAAACGCCA
<210> 88
<211> 32
<212> DNA
<213> synthetic construct
<400> 88
TGCTCTGCATCTGAAGGGTATTATGTGGAAAT
<210> 89
<211> 28
<212> DNA
<213> synthetic construct
<400> 89
ACCGGGCATTCTAAGCAGTTTACGTGCC
<210> 90
<211> 34
<212> DNA
<213> synthetic construct
<400> 90
ACACCAATATTGCATTTTCCAGTTTCAACTTGTT
<210> 91
<211> 32
<212> DNA
<213> synthetic construct
<400> 91
TCAGTGGTCCCAAGTAGTCACTTAGAAAATCT
<210> 92
<211> 30
<212> DNA
<213> synthetic construct
<400> 92
CCAGAAGGGCAATGTCATGGTTGTACTTAT
<210> 93
<211> 28
<212> DNA
<213> synthetic construct
<400> 93
ACGAACCCTTAGTGCTAAACAGCTACGT
<210> 94
<211> 27
<212> DNA
<213> synthetic construct
<400> 94
GGGTCCCCCACTATCTCCTTGACATGA
<210> 95
<211> 30
<212> DNA
<213> synthetic construct
<400> 95
TGCATTCTGTGGAGGCTCTATCGTTAATGA
<210> 96
<211> 29
<212> DNA
<213> synthetic construct
<400> 96
TGAAATTATGACCCTTCTGCCTTTAGCCC
<210> 97
<211> 24
<212> DNA
<213> synthetic construct
<400> 97
GGGCTCTGGGTCACTTGGCCTGAG
<210> 98
<211> 25
<212> DNA
<213> synthetic construct
<400> 98
CCACCCCCACCCTACCAGACTGAGC
<210> 99
<211> 21
<212> DNA
<213> synthetic construct
<400> 99
CTTCCTTTGCCCCCGCCTGCC
<210> 100
<211> 22
<212> DNA
<213> synthetic construct
<400> 100
GGAGTTACGTGCCCCTCCCCCA
<210> 101
<211> 21
<212> DNA
<213> synthetic construct
<400> 101
GAGAGCCCCGTGAGTGCCGCC
<210> 102
<211> 27
<212> DNA
<213> synthetic construct
<400> 102
TGAGCTGGGTGAGTCTCCTCCAGGACT
<210> 103
<211> 26
<212> DNA
<213> synthetic construct
<400> 103
GTGCTTCTTCCCACCCAGCTACCCCA
<210> 104
<211> 25
<212> DNA
<213> synthetic construct
<400> 104
GCAACATGCACCCCACCCTTGTGAG
<210> 105
<211> 25
<212> DNA
<213> synthetic construct
<400> 105
CTGTACCAGCCTAGCATTCCCGGGC
<210> 106
<211> 27
<212> DNA
<213> synthetic construct
<400> 106
GGAAGATGACCTGTGTGTAGGCCCCTC
<210> 107
<211> 24
<212> DNA
<213> synthetic construct
<400> 107
CTGAAGCGCCGTCTCCTGCATGTC
<210> 108
<211> 25
<212> DNA
<213> synthetic construct
<400> 108
CCCTCATGCGGACCTCCAGTCTCCA
<210> 109
<211> 25
<212> DNA
<213> synthetic construct
<400> 109
GGTCTCCCCACTGCAGCCTCTCGTT
<210> 110
<211> 25
<212> DNA
<213> synthetic construct
<400> 110
AGCCCCTGATGAAGTTGGAAGGCTC
<210> 111
<211> 20
<212> DNA
<213> synthetic construct
<400> 111
CACCCCGGCCGTCCCAGAGC
<210> 112
<211> 27
<212> DNA
<213> synthetic construct
<400> 112
CCGTGTAGCCCATTTCAGAGGAGCTCA
<210> 113
<211> 26
<212> DNA
<213> synthetic construct
<400> 113
CACGCCCATTTGTGATCTCCCGCTCG
<210> 114
<211> 26
<212> DNA
<213> synthetic construct
<400> 114
TGACCCAGAGCCCCACCTGCTAAGTG
<210> 115
<211> 27
<212> DNA
<213> synthetic construct
<400> 115
CCCCATCCCATTCATCACCCGTATGCC
<210> 116
<211> 25
<212> DNA
<213> synthetic construct
<400> 116
CTGCTCTGGTCTCCCGTCTCCCCAG
<210> 117
<211> 30
<212> DNA
<213> synthetic construct
<400> 117
ACCCCCATCTCTTCTAGATCAAAGATCCAG
<210> 118
<211> 27
<212> DNA
<213> synthetic construct
<400> 118
AAACAAACATGTGTCGCCCTGCCCATC
<210> 119
<211> 25
<212> DNA
<213> synthetic construct
<400> 119
GTGTGGCCCCTTGGGTGTGAGCAAG
<210> 120
<211> 20
<212> DNA
<213> synthetic construct
<400> 120
GCACCTGCGCTCCCCAGCTC
<210> 121
<211> 27
<212> DNA
<213> synthetic construct
<400> 121
AGAGCTGCTTCCCTTCCAGATGTGGTC
<210> 122
<211> 23
<212> DNA
<213> synthetic construct
<400> 122
AGGTGGAAGCCCAGGCCCCAGTA
<210> 123
<211> 20
<212> DNA
<213> synthetic construct
<400> 123
CCCCAAGGCTCCCTCCTCCC
<210> 124
<211> 21
<212> DNA
<213> synthetic construct
<400> 124
CAGCCCCTTGCCTCCCCTGCC
<210> 125
<211> 27
<212> DNA
<213> synthetic construct
<400> 125
CGGAGGGACTTCACGTTCAACAAGGAT
<210> 126
<211> 27
<212> DNA
<213> synthetic construct
<400> 126
ACCCTCAGAAAACATCCTCGGCGACCA
<210> 127
<211> 27
<212> DNA
<213> synthetic construct
<400> 127
ACGCATGATGTCATCCCCAGCCTCATC
<210> 128
<211> 26
<212> DNA
<213> synthetic construct
<400> 128
CCTCTGCTTTCTAACCCCCGTCCCCT
<210> 129
<211> 25
<212> DNA
<213> synthetic construct
<400> 129
CTGCCTTCCCCGACTTCACCAACCC
<210> 130
<211> 21
<212> DNA
<213> synthetic construct
<400> 130
GCAGCCGTCCTCAGAGCCCCT
<210> 131
<211> 22
<212> DNA
<213> synthetic construct
<400> 131
GTTCCCGGGTAACGCCAGCCCC
<210> 132
<211> 27
<212> DNA
<213> synthetic construct
<400> 132
GGCCGTAGAGGTTGTGCAGGTTGTAGT
<210> 133
<211> 27
<212> DNA
<213> synthetic construct
<400> 133
CCATGATTTCCTGCTGGTTCCCCGAGA
<210> 134
<211> 27
<212> DNA
<213> synthetic construct
<400> 134
CACTGTTCCTGGGTGATGGCCTTGTCA
<210> 135
<211> 27
<212> DNA
<213> synthetic construct
<400> 135
CACCTCCCTGATGCCATCATGAGTCCC
<210> 136
<211> 27
<212> DNA
<213> synthetic construct
<400> 136
GAGACAGGGACACCGTTGGAGAGGACC
<210> 137
<211> 27
<212> DNA
<213> synthetic construct
<400> 137
GCACGGCCCAGAATCCTCAAAGCAACA
<210> 138
<211> 25
<212> DNA
<213> synthetic construct
<400> 138
CTCCCCCACCATCTCCCTGTGCCTC
<210> 139
<211> 27
<212> DNA
<213> synthetic construct
<400> 139
CAGACAAAGTCGGCCTGCTCATCTCCC
<210> 140
<211> 29
<212> DNA
<213> synthetic construct
<400> 140
TGTGCATTCGTCTTTTCCAGAGCAAACCG
<210> 141
<211> 31
<212> DNA
<213> synthetic construct
<400> 141
ACTCTTTATCTCCCCCTTGATGAACTTCCTC
<210> 142
<211> 31
<212> DNA
<213> synthetic construct
<400> 142
CCGTCCTCTTCATTTTTCGCATTATGATCCT
<210> 143
<211> 29
<212> DNA
<213> synthetic construct
<400> 143
AGGATGCAAATTCCAGACACTGCAATCAT
<210> 144
<211> 30
<212> DNA
<213> synthetic construct
<400> 144
ATCGAGGAGATCAAAACCCAGAAGGTCCGC
<210> 145
<211> 27
<212> DNA
<213> synthetic construct
<400> 145
TTCCCCACCACCAAGACCTACTTCCCG
<210> 146
<211> 22
<212> DNA
<213> synthetic construct
<400> 146
CGCGTGATCCTCTGCCCTGCGA
<210> 147
<211> 22
<212> DNA
<213> synthetic construct
<400> 147
CACCTCCCCGCCGAGTTCACCC
<210> 148
<211> 27
<212> DNA
<213> synthetic construct
<400> 148
ATTCCGGGATAGAGAGAACCCAGGCAC
<210> 149
<211> 27
<212> DNA
<213> synthetic construct
<400> 149
TGTCTCCTGCCGACAAGACCAACGTCA
<210> 150
<211> 27
<212> DNA
<213> synthetic construct
<400> 150
GTGGCTCAGGTCGAAGTGCGGGAAGTA
<210> 151
<211> 31
<212> DNA
<213> synthetic construct
<400> 151
AGAAAAGAAGGGGAAAGAAAACATCAAGCGT
<210> 152
<211> 26
<212> DNA
<213> synthetic construct
<400> 152
CACCCTTAGGCTGCTGGTGGTCTACC
<210> 153
<211> 28
<212> DNA
<213> synthetic construct
<400> 153
TCCCCCAGTTTAGTAGTTGGACTTAGGG
<210> 154
<211> 31
<212> DNA
<213> synthetic construct
<400> 154
CCAAGCTAGGCCCTTTTGCTAATCATGTTCA
<210> 155
<211> 27
<212> DNA
<213> synthetic construct
<400> 155
AAAGGACTCAAAGAACCTCTGGGTCCA
<210> 156
<211> 27
<212> DNA
<213> synthetic construct
<400> 156
TGAGGAGAAGTCTGCCGTTACTGCCCT
<210> 157
<211> 27
<212> DNA
<213> synthetic construct
<400> 157
ACTTCATCCACGTTCACCTTGCCCCAC
<210> 158
<211> 27
<212> DNA
<213> synthetic construct
<400> 158
AAGCCAGTGCCAGAAGAGCCAAGGACA
<210> 159
<211> 33
<212> DNA
<213> synthetic construct
<400> 159
AGTGTATTTTCCCAAGGTTTGAACTAGCTCTTC
<210> 160
<211> 28
<212> DNA
<213> synthetic construct
<400> 160
ACTAAGCTCGCTTTCTTGCTGTCCAATT
<210> 161
<211> 30
<212> DNA
<213> synthetic construct
<400> 161
TGGACTCAGAATAATCCAGCCTTATCCCAA
<210> 162
<211> 35
<212> DNA
<213> synthetic construct
<400> 162
GCAATAATGATACAATGTATCATGCCTCTTTGCAC
<210> 163
<211> 27
<212> DNA
<213> synthetic construct
<400> 163
CTTCGATTACTGAGAAACCGAGTGGCC
<210> 164
<211> 30
<212> DNA
<213> synthetic construct
<400> 164
CTTAAGACTACCTTTCTCCAAATGGTGCCC
<210> 165
<211> 35
<212> DNA
<213> synthetic construct
<400> 165
TGAAGACAGTGTGGAGTTACTTATGTTGCAAAATT
<210> 166
<211> 27
<212> DNA
<213> synthetic construct
<400> 166
TGACTGTCTCCTCACCCTCCCCATTCT
<210> 167
<211> 29
<212> DNA
<213> synthetic construct
<400> 167
CCAGCCAGCAATGAACCCAAACCTCATTC
<210> 168
<211> 28
<212> DNA
<213> synthetic construct
<400> 168
TCTGACTCAGTGGTGATGAGCTTTGAGT
<210> 169
<211> 27
<212> DNA
<213> synthetic construct
<400> 169
TCTCTGGAGGCCCAAATTCCCCTAACT
<210> 170
<211> 29
<212> DNA
<213> synthetic construct
<400> 170
CCACGCAAGAGACACCCTTTGTAACTCTC
<210> 171
<211> 28
<212> DNA
<213> synthetic construct
<400> 171
CCCCACACACACATACACGCATGCACAT
<210> 172
<211> 28
<212> DNA
<213> synthetic construct
<400> 172
ACCAGACCTCTTCCTATGAAGCCTTGAA
<210> 173
<211> 25
<212> DNA
<213> synthetic construct
<400> 173
GCTAGTGGCTCACCTTTGTCACCAC
<210> 174
<211> 29
<212> DNA
<213> synthetic construct
<400> 174
TGGCCTATGGGATGCAGCAGGGAATACTG
<210> 175
<211> 34
<212> DNA
<213> synthetic construct
<400> 175
ATTGAATGACTTAACTAAGGTCACACAGTTGGTT
<210> 176
<211> 34
<212> DNA
<213> synthetic construct
<400> 176
TGTAGGAAACAAGCTATATTGAAGACAACTGCAA
<210> 177
<211> 27
<212> DNA
<213> synthetic construct
<400> 177
TATAGCACTCCACCATCCACCCAGGGA
<210> 178
<211> 27
<212> DNA
<213> synthetic construct
<400> 178
ACACACTCAGGGTCTATGTGGGCTGTT
<210> 179
<211> 27
<212> DNA
<213> synthetic construct
<400> 179
TCACTGGAGAATGAGTTCCCAGGTTGC
<210> 180
<211> 29
<212> DNA
<213> synthetic construct
<400> 180
AGTGTGCTCTCAGATTGACTTTCCATTCC
<210> 181
<211> 28
<212> DNA
<213> synthetic construct
<400> 181
CCCAGCCCTCGTGTAAATAGGAACACAA
<210> 182
<211> 28
<212> DNA
<213> synthetic construct
<400> 182
TGTACTCAGGACGTTGCCTTCTCTGTGT
<210> 183
<211> 28
<212> DNA
<213> synthetic construct
<400> 183
CCTGATGTGGACTTACTCTGCAGGAACT
<210> 184
<211> 26
<212> DNA
<213> synthetic construct
<400> 184
CCCCTGCCCTGCTTGAGACACCTATT
<210> 185
<211> 26
<212> DNA
<213> synthetic construct
<400> 185
CCTCTCCTCTGCCTCAATCCTCCCCC
<210> 186
<211> 27
<212> DNA
<213> synthetic construct
<400> 186
AAAACATGGGGCACAGTGTTCAAGACT
<210> 187
<211> 29
<212> DNA
<213> synthetic construct
<400> 187
TCATAGAACTGTACCTGGTTTCCGCTCTT
<210> 188
<211> 29
<212> DNA
<213> synthetic construct
<400> 188
TGCCCATTCCTCATGTAGAAAGACTGAGT
<210> 189
<211> 34
<212> DNA
<213> synthetic construct
<400> 189
GATGACCCCAAAAGATTTACCATTATGCTCTTGA
<210> 190
<211> 27
<212> DNA
<213> synthetic construct
<400> 190
ATTGGAATCCTTTGCAGTGCCCTCCAG
<210> 191
<211> 27
<212> DNA
<213> synthetic construct
<400> 191
ACTAGCTGCAGTTCCTGTCGGATATGG
<210> 192
<211> 29
<212> DNA
<213> synthetic construct
<400> 192
ACACTGAAATCCCATTTCCCTGACAACAT
<210> 193
<211> 33
<212> DNA
<213> synthetic construct
<400> 193
ACACAAAATCCCAGTCCCTATTCCTATAGAAGT
<210> 194
<211> 27
<212> DNA
<213> synthetic construct
<400> 194
TAAGAGGAACACCACACTCACCCCCTT
<210> 195
<211> 27
<212> DNA
<213> synthetic construct
<400> 195
AGGAATTCATTGCCTTTGGGATCAGCA
<210> 196
<211> 28
<212> DNA
<213> synthetic construct
<400> 196
TGAAAGGCAAATTGTCCTGCTAAGCTCG
<210> 197
<211> 28
<212> DNA
<213> synthetic construct
<400> 197
GGAGTAGGGTAGCCTGGGAGTAGACAGA
<210> 198
<211> 33
<212> DNA
<213> synthetic construct
<400> 198
TCCTCAGCGCTTTCAGCCTCTTATTATATACTC
<210> 199
<211> 32
<212> DNA
<213> synthetic construct
<400> 199
TGCTGTTTCAACAAATAATGCTTTTGAGCCTG
<210> 200
<211> 27
<212> DNA
<213> synthetic construct
<400> 200
TGATCTCACTCCAACAACGTCCAGGAA
<210> 201
<211> 27
<212> DNA
<213> synthetic construct
<400> 201
GGCAGATCCTTTGGTTGGTGGCTTCAC
<210> 202
<211> 29
<212> DNA
<213> synthetic construct
<400> 202
CGCCTGGCCCAGACTCAGAGAATGAATAC
<210> 203
<211> 31
<212> DNA
<213> synthetic construct
<400> 203
GGGCAACATAGCAAGACCTCGTCTTTGTTTA
<210> 204
<211> 28
<212> DNA
<213> synthetic construct
<400> 204
AGCCAGTATGATAGCCACTCATGTACCA
<210> 205
<211> 28
<212> DNA
<213> synthetic construct
<400> 205
TGTCTCCAGATAATTCCCCCACCACCTC
<210> 206
<211> 35
<212> DNA
<213> synthetic construct
<400> 206
AATGCAAGAGTAATTTAAGCCTCAGACAGTTGTAT
<210> 207
<211> 27
<212> DNA
<213> synthetic construct
<400> 207
GGGCGATAACCACTCGTAGAAAGCGTG
<210> 208
<211> 23
<212> DNA
<213> synthetic construct
<400> 208
CTGCGACCTCACCTGGCCTGTGC
<210> 209
<211> 31
<212> DNA
<213> synthetic construct
<400> 209
GCCACTGCAAGAAAACCTTAACTGCAGCCTA
<210> 210
<211> 33
<212> DNA
<213> synthetic construct
<400> 210
AATGTGAGCACCTTCCTTCTTTTTGATTTTGTC
<210> 211
<211> 34
<212> DNA
<213> synthetic construct
<400> 211
AGTTGTTTTTATTTCTTACCCTTTCCAGAGCGAT
<210> 212
<211> 35
<212> DNA
<213> synthetic construct
<400> 212
GGCTATCAACTTCTAAAGGAGGATATCACCTGATT
<210> 213
<211> 58
<212> DNA
<213> synthetic construct
<400> 213
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
<210> 214
<211> 22
<212> DNA
<213> synthetic construct
<400> 214
CTACACGACGCTCTTCCGATCT
<210> 215
<211> 64
<212> DNA
<213> synthetic construct
<400> 215
CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
<210> 216
<211> 22
<212> DNA
<213> synthetic construct
<400> 216
CAGACGTGTGCTCTTCCGATCT
Claims (11)
1. A primer group for detecting mutation sites on PAH genes is characterized in that the primer group has a nucleotide sequence shown as SEQ ID NO. 163-SEQ ID NO. 190.
2. The primer set of claim 1, wherein the primer sequences in the primer set are allowed to be modified or ligated to universal sequences required for library-building sequencing.
3. The primer set of claim 2, wherein a universal sequence is allowed to ligate to the 5' end of the primer sequences in the primer set.
4. The primer set of claim 2, wherein the universal sequence comprises a pool-building primer, a sequencing primer, a tag, or a linker.
5. A kit comprising the primer set of any one of claims 1-4.
6. The kit of claim 5, further comprising at least one of primer set 2-8 or primer set 10-11; wherein, the primer group 2 has a nucleotide sequence shown as SEQ ID NO. 47-SEQ ID NO. 60; the primer group 3 has a nucleotide sequence shown as SEQ ID NO. 61-SEQ ID NO. 86; the primer group 4 has a nucleotide sequence shown as SEQ ID NO. 87-SEQ ID NO. 96; the primer group 5 has a nucleotide sequence shown as SEQ ID NO. 97-SEQ ID NO. 138; the primer group 6 has a nucleotide sequence shown as SEQ ID NO. 139-SEQ ID NO. 144; the primer group 7 has a nucleotide sequence shown as SEQ ID NO. 145-SEQ ID NO. 150; the primer group 8 has a nucleotide sequence shown as SEQ ID NO. 151-SEQ ID NO. 162; the primer group 10 has a nucleotide sequence shown as SEQ ID NO. 191-SEQ ID NO. 202; the primer group 11 has a nucleotide sequence shown as SEQ ID NO. 203-SEQ ID NO. 212.
7. The kit according to claim 6, wherein the kit consists of the nucleotide sequence shown as SEQ ID NO. 163-SEQ ID NO. 190, primer set 6 and primer set 10.
8. The kit according to claim 6, wherein the kit consists of a nucleotide sequence shown as SEQ ID NO. 163-SEQ ID NO. 190, a primer set 7 and a primer set 8.
9. The kit according to claim 6, wherein the kit consists of the nucleotide sequence shown as SEQ ID NO. 163-SEQ ID NO. 190, primer set 3 and primer set 4.
10. A method for amplifying a nucleic acid, comprising: amplifying a nucleic acid sample using the primer set of any one of claims 1-4 or the kit of any one of claims 5-9.
11. A method of creating a library, comprising: an amplicon sequencing library is constructed for nucleic acid sequencing using the primer set of any one of claims 1-4 or the kit of any one of claims 5-9.
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