Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing

Abstract

Background: In addition to shotgun sequencing, next generation sequencing has been shown to be suitable for deep sequencing of many specific PCR-amplified target genes in parallel. However, unspecific product formation is a common problem in amplicon sequencing since these fragments are difficult to fully remove by gel purification, and their presence inevitably reduces the number of mappable sequence reads that can be obtained in each sequencing run.Results: We have used a novel flow cytometric sorting approach to specifically enrich Roche/454 DNA Capture beads carrying target DNA sequences on their surface, and reject beads carrying unspecific sequences. This procedure gives a nearly three-fold increase in the fraction of informative sequences obtained. Presented results also show that there are no significant differences in the distribution or presence of different genotypes between a FACS-enriched sample and a standard-enriched control sample.Conclusions: Target-specific FACS enrichment prior to Roche/454 sequencing provides a quick, inexpensive way of increasing the amount of high quality data obtained in a single sequencing run, without introducing any sequence bias. © 2010 Sandberg et al; licensee BioMed Central Ltd.