Methods for the extraction of bacterial DNA

Choosing an appropriate DNA extraction method for determination of bacterial diversity is crucial and can be a challenge in case that preliminary knowledge on the sample matrix to be analyzed is scarce or lacking. The aim of the present study was to determine a DNA extraction kit suitable for 16S rRNA gene amplicon sequencing of chicken caecum taking into account criterion such as high technical reproducibility and bacterial diversity as well as easy handling. The protocols tested differed strongly with respect to DNA concentrations, its purity, technical reproducibility and furthermore alpha and beta diversity. Differences in relative taxa abundances due to the DNA extraction conditions applied were detected. Some protocols showed under-represented amount of Ruminococcaceae, Ruminococcaceae insertae sedis, Defluviitaleaceae and Erysipelotrichaceae sequences in their taxonomic profiles, which belong to the common caecal community members. Furthermore, protocols consisting of a cleaning step to remove PCR inhibitors led to significantly increased technical reproducibility of the DNA extraction as well as alpha diversity. The removal of PCR inhibitors prior to 16S rRNA gene amplicon PCR proved to be one of the main drivers for high technical reproducibility as well as a high bacterial diversity within the chicken caecum bacterial community.

选取适配的DNA提取方法以测定细菌多样性，此项工作至关重要，但倘若待分析样本基质缺乏先验研究背景，该流程往往极具挑战。本研究旨在筛选适配鸡盲肠样本16S rRNA基因扩增子测序（16S rRNA gene amplicon sequencing）的DNA提取试剂盒（DNA extraction kit），并以高技术重现性（technical reproducibility）、优异的细菌多样性保留能力及操作便捷性作为核心评价标准。本次测试的多款提取方案在DNA浓度、纯度、技术重现性以及α多样性（alpha diversity）、β多样性（beta diversity）维度上均存在显著差异。研究表明，不同DNA提取条件会导致类群相对丰度（relative taxa abundances）产生显著差异。部分提取方案的分类学谱中，属于鸡盲肠常见菌群的瘤胃球菌科（Ruminococcaceae）、瘤胃球菌科地位未定类群（Ruminococcaceae insertae sedis）、脱氟杆菌科（Defluviitaleaceae）及丹毒丝菌科（Erysipelotrichaceae）的序列占比偏低。此外，包含去除PCR抑制剂（PCR inhibitors）清洗步骤的提取方案，可显著提升DNA提取的技术重现性与样本的α多样性。在16S rRNA基因扩增子PCR（amplicon PCR）前去除PCR抑制剂，被证实是提升鸡盲肠细菌群落技术重现性与群落细菌多样性的关键因素之一。