부산대학교 도서관

AbstractIllumina-based amplicon sequencing suffers from the deleterious effects of highly homogenous nucleotide composition, limiting the number of high-quality reads generated per run. We attempted to alleviate this limitation by comparing the results obtained from 16S ribosomal DNA (16S rDNA) sequencing of mouse gut microbiomes using Illumina V3–V4 primers (Run 1) and custom primers that incorporate a heterogeneity spacer (0–7 nucleotides) upstream of the 16S priming region (Run 2). Overall, Run 2 had higher quality sequences, a more diverse microbial profile, and higher precision within, and variation between, experimental groups than Run 1. Our primer design offers a simple way to increase the quality of 16S rDNA sequencing and increases the number of useable reads generated per Illumina run.METHOD SUMMARYThe standard Illumina primers for 16S rDNA sequencing of the V3–V4 region were modified by adding heterogeneity spacer regions (0–7 nucleotides) upstream of the 16S rDNA specificity binding region, which improved the effectiveness of the sequencing reaction as measured by sequence quality, increased sensitivity and increased observed variation in microbial abundance and diversity of the samples.