Linker sequence and its design method for multiplex PCR library construction
A technology for linking sequences and library construction, applied in the field of high-throughput sequencing, can solve problems affecting the quality of library construction, affecting the uniformity of amplicons, data utilization, etc., to achieve cost savings in sequencing, good market application prospects, and mutual interference small effect
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Embodiment 1
[0054] Embodiment 1, connection sequence
[0055] Using the linker sequence design method provided by the present invention, human distant species refer to the following species Exophialamesophila, Photobacterium profundum, Borrelia miyamotoi, Neomicrococcusaetuarii; the human reference genome is hg19; the sequencing linker is the A linker and the P linker provided by the Ion torrent sequencing platform; finally Seven connecting sequences were screened out, the nucleotide sequences of which are shown in SEQ ID NO:1-SEQ ID NO:7.
[0056] U1: 5'-GGGCTGGCAAGCCACGTT-3' (SEQ ID NO: 1);
[0057] U2: 5'-GTCTCGTGGGCTCGGAGA-3' (SEQ ID NO: 2);
[0058] U3: 5'-CGTCGCCGTCCAGCTCGA-3' (SEQ ID NO: 3);
[0059] U4: 5'-AAATGGGCGGTAGGCTTG-3' (SEQ ID NO: 4);
[0060] U5: 5'-CGTGACGCGGTGCAGGGC-3' (SEQ ID NO: 5);
[0061] U6: 5'-CGCAAATGGGCGGTAGGC-3' (SEQ ID NO: 6);
[0062] U7: 5'-CCGGGAGCTGCATGTGTC-3' (SEQ ID NO: 7).
Embodiment 2
[0063] Example 2. Application of different junction sequences in the construction of genetic deafness pathogenic locus sequencing library
[0064] Taking the detection of genetic deafness pathogenic sites as an example, a nucleic acid sequencing library was constructed based on the junction sequence of U1-U7 and the ideas of the present invention; Genomic DNA 20ng, PCR reaction mix 12.5μL, make up to 25μL with enzyme-free water, one-step amplification to obtain the sequencing library, the amplification condition is 95°C pre-denaturation for 3min; 35 cycle amplification (95°C denaturation for 15s, 60°C annealing for 90s , 72°C for 30s); 72°C for 1 min, 16°C forever; the amplified product was purified by magnetic beads, and high-throughput sequencing was performed using the Ion Torrent sequencing platform.
[0065] Agilent 2100 analyzes the fragments of the sequencing library, the results are as follows figure 1 As shown, all the linking sequences can amplify the target fragmen...
Embodiment 3
[0069] Example 3, the application of linker sequence U4 in the construction of sequencing library of pathogenic gene of phenylketonuria
[0070] Using the connection sequence U4, taking the detection of the pathogenic gene of phenylketonuria as an example, according to the idea of the present invention, a two-step amplification method is adopted: 2 μL of 61 pairs of connection primer mixture, 200 ng of sample genomic DNA, 10 μL of PCR reaction solution mix, and 1 μL of DMSO , Make up to 20 μL with enzyme-free water, and carry out the first step of PCR amplification to realize the connection of the junction sequence and the target fragment; the reaction conditions of the first step of PCR amplification are set as follows: 95°C pre-denaturation for 5 minutes; 15 cycles of amplification (95°C Denaturation for 35 s, annealing at 60°C for 90 s, extension at 72°C for 30 s), extension at 72°C for 1 min, and forever at 16°C; then 10.5 μL of the first step PCR amplification reaction p...
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