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

Single nucleotide polymorphisms (SNPs) in homologous regions play a critical role in the field of genetics. However, genotyping these SNPs is challenging due to the presence of repetitive sequences within genome, which demand specific method. We introduce a new, mid-throughput method that simplifies SNP genotyping in homologous DNA sequences by utilizing a combination of multiplex kb level PCR (PCR size 2.5k-3.5 kb) for capturing targeted regions and multiplex nested PCR library construction for next-generation sequencing (Multi-kb level capture-seq). First of all, we randomly selected 7 SNPs in homologous regions and successfully captured 6-plex kb level amplicons (one of segments contains 2 SNPs, while the remaining segments each have only one SNP) in a single tube. And then, the amplification products were subjected to multiplex nested PCR for library construction and sequenced on Illumina platform. We tested this strategy using 600 amplicons from 100 samples and accurately genotyped 96.8% of target SNPs with a coverage depth of ≥ 15×. For the uniformity within the samples, over 66.7% (4/6) of the amplicons had a coverage depth above 0.2-fold of average sequencing depth. To validate the accuracy of this approach, we performed Ligase detection reaction PCR for genotyping the 100 samples, and found that the genotyping data was 97.71% consistent with our NGS results. In conclusion, we have developed a highly efficient and accurate method for SNP genotyping in homologous regions, which offers researchers a new strategy to explore the complex regions of genome.