Genotyping single nucleotide polymorphisms in homologous regions using multiplex kb level amplicon capture sequencing

Area of Science:

• Genetics
• Genomics
• Molecular Biology

Background:

• Single nucleotide polymorphisms (SNPs) in homologous regions are crucial in genetics.
• Genotyping these SNPs is challenging due to repetitive genomic sequences.
• Existing methods require specialized techniques for accurate SNP identification.

Purpose of the Study:

• To develop a novel, mid-throughput method for simplifying SNP genotyping in homologous DNA sequences.
• To introduce a strategy combining multiplex kb-level PCR and nested PCR for next-generation sequencing (Multi-kb level capture-seq).

Main Methods:

• Utilized multiplex kb-level PCR (2.5k-3.5kb amplicons) to capture targeted homologous regions.
• Employed multiplex nested PCR for library construction followed by Illumina sequencing.
• Tested the strategy on 600 amplicons from 100 samples, targeting 7 SNPs in homologous regions.

Main Results:

• Successfully captured 6-plex kb-level amplicons in a single tube.
• Achieved accurate genotyping of 96.8% of target SNPs with coverage depth ≥15×.
• Demonstrated 97.71% consistency with Ligase detection reaction PCR results.

Conclusions:

• Developed a highly efficient and accurate method for SNP genotyping in homologous regions.
• The Multi-kb level capture-seq strategy provides a new approach for exploring complex genomic regions.
• This method simplifies SNP genotyping, overcoming challenges posed by repetitive DNA sequences.