Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
University of Missouri, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
J Microbiol Methods. 2020 Sep;176:106033. doi: 10.1016/j.mimet.2020.106033. Epub 2020 Aug 14.
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.
本研究的目的是评估增加 PCR 循环数对低微生物生物量样本（包括牛牛奶、鼠毛和血液）测序结果的影响。我们假设对这些样本的 DNA 进行更高的 PCR 循环数会增加 16S rRNA 测序覆盖率。从每种类型的匹配样本中提取 DNA，并评估多个 PCR 循环数，共从 24 个牛奶样本中生成 96 个文库，从 23 个毛发样本中生成 46 个文库，从 85 个血液样本中生成 170 个文库。在 Illumina MiSeq 平台上进行 16S rRNA 测序，评估每个样本的覆盖率、检测到的丰富度和 beta 多样性。在所有样本类型中，更高的 PCR 循环数与更高的覆盖率相关。然而，令人惊讶的是，尽管更高的 PCR 循环数导致了更多可用数据点的产生，但丰富度或 beta 多样性的指标没有差异。虽然扩增 40 个循环的试剂对照也产生了类似的增加覆盖率，但基于 beta 多样性，对照和实验样本可以清楚地区分。本研究的结果支持使用更高的 PCR 循环数来评估低微生物生物量样本。
