CN117701691A - Primer combination constructed based on illumina sequencing library of human genome amplicon - Google Patents
Primer combination constructed based on illumina sequencing library of human genome amplicon Download PDFInfo
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- 238000012163 sequencing technique Methods 0.000 title claims abstract description 27
- 108091093088 Amplicon Proteins 0.000 title claims abstract description 20
- 238000012986 modification Methods 0.000 claims abstract description 6
- 230000004048 modification Effects 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims description 53
- 125000003729 nucleotide group Chemical group 0.000 claims description 53
- 238000011144 upstream manufacturing Methods 0.000 claims description 32
- 230000003321 amplification Effects 0.000 abstract description 22
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 22
- 108020004414 DNA Proteins 0.000 description 13
- 238000001962 electrophoresis Methods 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- 239000012634 fragment Substances 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000007838 multiplex ligation-dependent probe amplification Methods 0.000 description 3
- 210000003917 human chromosome Anatomy 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 238000011451 sequencing strategy Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
Abstract
The invention discloses a primer combination constructed based on an illuminea sequencing library of a human genome amplicon, which is obtained by changing the base sequence of the 3' end of a primer or introducing phosphorothioate bond modification. The primer combination constructed on the illuminea sequencing library based on the human genome amplicon can reduce the amplification of non-target bands.
Description
Technical Field
The invention relates to the field of nucleic acid amplification, in particular to a primer combination constructed based on an illuminea sequencing library of human genome amplicon.
Background
Strategies based on high-throughput sequencing are widely used, which have penetrated aspects of biological research and clinical diagnosis [1, 2]. For the construction of DNA library for Illumina sequencing, the objective of constructing the library is achieved by directly adding a linker [3] according to different conditions, and the DNA library is obtained by PCR amplification through specially designed universal upstream and downstream primers. The second library construction method is commonly found in amplicon strategy-based library construction, such as molecular inversion probe capture (molecular inversion probe, MIP) [4-6], multiplex ligation dependent probe amplification (multiplex ligation-dependent probe amplification, MLPA) [7], etc., and therefore amplification specificity (i.e., minimizing amplification of non-target bands) by using upstream and downstream primers is of paramount importance.
Disclosure of Invention
It is an object of the present invention to provide primer combinations constructed based on illuminea sequencing libraries of human genomic amplicons.
The primer combination constructed based on the illuminea sequencing library of the human genome amplicon is obtained by changing the base sequence of the 3' end of the primer or introducing phosphorothioate bond modification.
Preferably, the primer combination includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is one of a sequence shown as SEQ ID NO.2, a sequence shown as SEQ ID NO.3 and a sequence shown as SEQ ID NO. 4; the nucleotide sequence of the downstream primer is one of the sequence shown in SEQ ID NO.8, the sequence shown in SEQ ID NO.10 and the sequence shown in SEQ ID NO. 14.
Preferably, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown as SEQ ID NO. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 8.
Preferably, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown as SEQ ID NO. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
Preferably, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown as SEQ ID NO. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 14.
Preferably, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID NO. 3; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
Preferably, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID NO. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 8.
Preferably, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID NO. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
Preferably, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID NO. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 14.
The primer combination constructed based on the illuminea sequencing library of the human genome amplicon provided by the invention achieves the purpose of reducing non-target band amplification by changing the base sequence of the 3' end of the primer and/or introducing phosphorothioate bond modification.
Drawings
FIG. 1 is a schematic diagram of a DNA library for illuminea platform sequencing according to an embodiment of the present invention;
FIGS. 2 and 3 are different contrast electrophoresis gel patterns after template-free amplification of related primers according to examples 1-5 of the present invention;
FIG. 4 is a plot of different contrast electrophoresis gels for PCR amplification of genomic DNA (gDNA) of human 293T cells according to examples 6-10 of the present invention;
FIG. 5 is an alignment of the 38 bps DNA fragment of example 10 of the present invention with respect to human gDNA sequence;
FIG. 6 is an alignment chart of a DNA sequence comprising a 38 bps DNA fragment and an Up-Fp/Up-R3p sequence according to example 10 of the present invention;
FIGS. 7 and 8 are different contrast electrophoresis gel diagrams after PCR amplification of gDNA of human 293T cells according to example 10 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in fig. 1 to 8, the primer combination constructed based on the illuminea sequencing library of the human genome amplicon provided in this example is obtained by changing the base sequence of the 3' end of the primer or introducing phosphorothioate bond modification.
Further, the primer combination includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is one of a sequence shown as SEQ ID NO.2, a sequence shown as SEQ ID NO.3 and a sequence shown as SEQ ID NO. 4; the nucleotide sequence of the downstream primer is one of the sequence shown in SEQ ID NO.8, the sequence shown in SEQ ID NO.10 and the sequence shown in SEQ ID NO. 14.
Further, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown as SEQ ID NO. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 8.
Further, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown as SEQ ID NO. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
Further, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown as SEQ ID NO. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 14.
Further, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown as SEQ ID NO. 3; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
Further, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID NO. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 8.
Further, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID NO. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
Further, the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID NO. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 14.
As shown in fig. 1, the DNA library structure for illuminea platform sequencing, wherein P5, P7: sequences that allow library binding and can generate clusters on flow cells; i5, i7: an index sequence; read1, read2: sequencing primer binding sites for read1 and read 2; tsL, tsR: target-specific primer binding sites.
The primer sequences used in this example are shown in Table 1:
TABLE 1 primer sequences
Wherein "+" represents a phosphorothioate bond (phosphorothioate bond), i.e., a sulfur atom replaces one of the oxygen atoms in the phosphoester bond between the sugar groups of adjacent nucleotides; nucleotide sequence "aatgatacggcgaccaccgagatctacac" is a P5 sequence; nucleotide sequence "caagcagaagacggcatacgagat" is a P7 sequence; the sequence including "acggatacccacgaca" up to the last base of primer 3' is the customized read1 sequencing primer (i.e., binding site); the sequence including "ccagaggcaaaacga" up to the last base of primer 3' is the customized read2 sequencing primer (i.e., binding site); the nucleotide sequence "agcgctag" is the i5 sequence; the nucleotide sequence "aaacgcgg" is the i7 sequence; the nucleotide sequences "tgtaaaacgacggccagt", "tgtaaaacgacggc ×c×a×g×t", "tgtaaaacgacggagtcc" and "tgtaaaacgacgga ×g×c×c" are potential universal upstream primer sequences; the nucleotide sequences "cgctagttattgctcagcgg", "cgctagttattgctca g", "cgctagttattgctcagcta", "cgctagttattgctca g c t a", "cgctagttattgctcagctt", "cgctagttattgctca g c t", "cgctagttattgctcagctc", "cgctagttattgctca g c t c", "cgctagttattgctcgatct" and "cgctagttattgctcg a t c t" are potential universal downstream primer sequences.
Example 1: as shown in FIG. 2, lane C in the gel chart shows a white band, i.e., nonspecific amplification, by electrophoresis after PCR using the upstream primer named Up-F and the downstream primer of Up-R in Table 1 without template and with Q5 High-fidelity DNA Polymerase, Q5 polymerase NEB for short.
Example 2: as shown in FIG. 2, after the PCR amplification by the downstream primer Up-Rp and the upstream primer UP-F after the 4 phosphodiester bonds at the 3' end of Up-R were replaced with phosphorothioate bonds, the white bands in lanes G and C were more apparent as compared with lanes C, indicating that the non-specific amplification was significantly reduced when PCR was performed using Up-F and Up-Rp.
Example 3: as shown in FIG. 2, when two bases ("GG") at the 3' -end of Up-R were changed to the base "TA", i.e., (Up-R1), it was revealed by the electrophoresis gel diagram that the non-specific amplification in lane D was also reduced as compared with that in lane C.
Example 4: as shown in FIG. 2, after attempting to change the 4 phosphodiester bonds at the 3' -end of Up-R1 to phosphorothioate bonds (i.e., up-R1 p), it was revealed by the electrophoresis gel diagram that the nonspecific amplification of lane H was further reduced as compared with that of lane D.
Example 5: as shown in FIG. 2, after attempting to change the 4 phosphodiester bonds at the 3' end of Up-F to phosphorothioate bonds (i.e., up-Fp), it was shown by the electrophoresis gel diagram that after PCR, up-Fp and Up-R had non-specific amplification, but less than Up-F and Up-R (lane E vs. lane C), and less than Up-Fp and Up-R1 (lane E vs. lane F); non-specific amplification of Up-Fp and Up-Rp was very weak (lane A); electrophoresis gel images after PCR of Up-Fp and Up-R1p showed no macroscopic non-specific amplification (lane B).
As described in example 1-example 5, up-Fp and Up-R1p were free of non-specific amplification under the conditions tested.
Example 6: as shown in FIG. 3, without template and with Q5 Hot-start High-fidelity DNA Polymerase (Q5 Hot-start polymerase. NEB, USA) there was no non-specific amplification of Up-Fp and Up-R1p (lane A), and this result was the same as that with Q5 High-fidelity DNA polymerase by comparison with FIG. 2. When the last base at the 3' end of Up-R1 is changed from "A" to "T" (i.e., up-R2), there is non-specific amplification (lane B).
Example 7: further, after the 4 phosphodiester bonds at the 3' -end of Up-R2 were replaced with phosphorothioate bonds (i.e., up-R2 p), no non-specific amplification was observed (lane C).
Example 8: when the last base at the 3' -end of Up-R1 is changed from "A" to "C" (i.e., up-R3), there is non-specific amplification (lane D).
Example 9: further, after the 4 phosphodiester bonds at the 3' -end of Up-R3 were replaced with phosphorothioate bonds (i.e., up-R3 p), no non-specific amplification was observed (lane E).
The results from examples 6-9 show that Up-Fp and Up-R1p, up-Fp and Up-R2p, up-Fp and Up-R3p are devoid of non-specific amplification under the conditions tested.
Example 10: as shown in FIG. 3, since the 3 phosphorothioate modified primer pairs tested (i.e., up-Fp/Up-R1p, up-Fp/Up-R2p and Up-Fp/Up-R3 p) were free of non-specific amplification under template-free conditions, we further tested their performance against human gDNA.
As shown in FIG. 4, up-Fp/Up-R1p and Up-Fp/Up-R2p were not non-specifically amplified against human gDNA (lanes A and B); as can also be seen from FIG. 4, up-Fp/Up-R3p has an amplified band against human gDNA (lane C).
Cloning and sequencing of the amplified band of human gDNA with respect to Up-Fp/Up-R3p revealed that the band was 176 bps (base pairs), i.e., a 38 bps DNA fragment was included between Up-Fp and Up-R3 p. The kit used for cloning was the Hieff Clone zero background TOPO-Blunt Blunt end cloning kit (next holy, china). The 38 bps DNA fragment was used for alignment at NCBI (against human gDNA sequence). As shown in FIG. 5, it was found to be identical to a sequence of human chromosome 18 (62609601-62609564). As shown in FIG. 6, by downloading the DNA segment of human chromosome 18 (62609464-62609701, 238 bps) containing the 38 bps and aligning with the sequence of Up-Fp/Up-R3p, it was revealed that the 10 bases at the 3 'end of Up-Fp and the 10 bases at the 3' end of Up-R3p were identical to the sequences at both ends of the 38 bps DNA segment. These alignment results indicate that Up-Fp/Up-R3p, because the 10 bases at the 3' end of each and the 38 bps DNA fragment were identical and opposite in sequence, resulted in an amplified band as shown in lane C of FIG. 4.
As shown in FIGS. 4-6, since the 10 bases at the 3 'end of the Up-Fp and Up-R3p sequences are identical and opposite in sequence to a short segment of human gDNA, resulting in the amplification of a DNA fragment, we attempted to alter the sequences of the last 5 bases at the 3' end of the two primers, up-F1 and Up-R4, respectively. As shown in Table 1, the primers whose corresponding 3' -terminal 4 phosphodiester bonds were replaced with phosphorothioate bonds were designated as Up-F1p and Up-R4p, respectively.
As shown in FIG. 7, up-Fp and Up-R4 have one amplified band for human gDNA; up-Fp/Up-R4p was not amplified against human gDNA.
As shown in FIG. 8, up-F1/Up-R1p and Up-F1/Up-F4p have amplified bands for human gDNA, and Up-F1/Up-R2p has no amplified band for human gDNA. In addition, the Up-F1p/Up-R1p, up-F1p/Up-R2p and Up-F1p/Up-R4p also had no amplified bands for human gDNA.
In summary, the seven primer pair combinations, up-Fp and Up-R1p, up-Fp and Up-R2p, up-Fp and Up-R4p, up-F1 and Up-R2p, up-F1p and Up-R1p, up-F1p and Up-R2p, and Up-F1p and Up-R4p, all had no amplified bands for human gDNA under the conditions tested. Similar to the application of references [4-6], these primer pair combinations can be used for illuminea sequencing library construction based on human genomic amplicons.
It should be noted that: (1) The index sequences in table 1, which may vary from sample to sample, i.e., different (sequenced) samples have different index sequences; (2) The number of phosphorothioate bonds at the 3' -end of the primer is not limited to 4, and may be 4 or more.
Reference:
1. Rego SM, Snyder MP: High Throughput Sequencing and Assessing Disease Risk. CSH PERSPECT MED 2019, 9(1).
2. Reuter JA, Spacek DV, Snyder MP: High-throughput sequencing technologies. MOL CELL 2015, 58(4):586-597.
3. Park YS, Kim S, Park DG, Kim DH, Yoon KW, Shin W, Han K: Comparison of library construction kits for mRNA sequencing in the Illumina platform. GENES GENOM 2019, 41(10):1233-1240.
4. Biezuner T, Brilon Y, Arye AB, Oron B, Kadam A, Danin A, Furer N, Minden MD, Hwan KD, Shapira S et al: An improved molecular inversion probe based targeted sequencing approach for low variant allele frequency. NAR GENOM BIOINFORM 2022, 4(1):b125.
5. Hernandez-Neuta I, Magoulopoulou A, Pineiro F, Lisby JG, Gulberg M, Nilsson M: Highly multiplexed targeted sequencing strategy for infectious disease surveillance. BMC BIOTECHNOL 2023, 23(1):31.
6. Hiatt JB, Pritchard CC, Salipante SJ, O'Roak BJ, Shendure J: Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation. GENOME RES 2013, 23(5):843-854.
7. Moelans CB, Atanesyan L, Savola SP, van Diest PJ: Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA). Methods Mol Biol 2018, 1708:537-549.
finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; 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 technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A primer combination constructed based on an illuminea sequencing library of human genome amplicon, characterized in that the primer combination is obtained by changing the base sequence of the 3' end of the primer or introducing phosphorothioate bond modification.
2. The primer combination constructed based on an illuminea sequencing library of human genomic amplicons according to claim 1, wherein said primer combination comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is one of a sequence shown as SEQ ID NO.2, a sequence shown as SEQ ID NO.3 and a sequence shown as SEQ ID NO. 4; the nucleotide sequence of the downstream primer is one of the sequence shown in SEQ ID NO.8, the sequence shown in SEQ ID NO.10 and the sequence shown in SEQ ID NO. 14.
3. The primer combination constructed based on the illuminea sequencing library of human genomic amplicons according to claim 2, wherein the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID No. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 8.
4. The primer combination constructed based on the illuminea sequencing library of human genomic amplicons according to claim 2, wherein the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID No. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
5. The primer combination constructed based on the illuminea sequencing library of human genomic amplicons according to claim 2, wherein the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID No. 2; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 14.
6. The primer combination constructed based on the illuminea sequencing library of human genomic amplicons according to claim 2, wherein the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID No. 3; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
7. The primer combination constructed based on the illuminea sequencing library of human genomic amplicons according to claim 2, wherein the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID No. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 8.
8. The primer combination constructed based on the illuminea sequencing library of human genomic amplicons according to claim 2, wherein the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID No. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 10.
9. The primer combination constructed based on the illuminea sequencing library of human genomic amplicons according to claim 2, wherein the nucleotide sequence of the upstream primer in the primer combination is the sequence shown in SEQ ID No. 4; the nucleotide sequence of the downstream primer in the primer combination is shown as SEQ ID NO. 14.
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