Influence of PCR cycle number on 16S rRNA gene amplicon sequencing of low biomass samples

Abstract

The objective of this study was to evaluate the effects of increased PCR cycle number on sequencing results from samples with low microbial biomass, including bovine milk, and murine pelage and blood. We hypothesized that subjecting DNA from such samples to higher PCR cycle numbers would increase 16S rRNA sequencing coverage. DNA was extracted from matched samples of each type and multiple PCR cycle numbers were evaluated to generate a total of 96 libraries from 24 milk samples, 46 libraries from 23 pelage samples, and 170 libraries from 85 blood samples. 16S rRNA sequencing was performed on the Illumina MiSeq platform, and the coverage per sample, detected richness, and beta-diversity were evaluated. Across all sample types, higher PCR cycle numbers were associated with increased coverage. Surprisingly however, while higher PCR cycle numbers resulted in greater number of useable datapoints, no differences were detected in metrics of richness or beta-diversity. While reagent controls amplified for 40 cycles yielded similarly increased coverage, control and experimental samples were clearly differentiated based on beta-diversity. The results from this study support the use of higher PCR cycle numbers to evaluate samples with low microbial biomass.

Keywords: Blood; Dairy cattle; Illumina sequencing; Low biomass; Microbiome; Milk; Mouse; Pelage.