PDF(Pubmed)
Abstract：
Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared different sequencing-based methods to assess their relative accuracy with respect to distinguishing between viable and non-viable cells, using a live and heat-inactivated model community spiked into bovine milk. The methods used were shotgun metagenomics with and without propidium monoazide (PMA) treatment, RNA-based 16S rRNA sequencing and metatranscriptomics. The results showed that methods were generally accurate, though significant differences were found depending on the library types and sequencing technologies. Different molecular targets were the basis for variations in the results generated using different library types, while differences in the derived composition data from Oxford Nanopore Technologies-and Illumina-based sequencing likely reflect a combination of different sequencing depths, error rates and bioinformatics pipelines. Although PMA was successfully applied in this study, further optimisation is required before it can be applied in a more universal context for complex microbiomes. Overall, these methods show promise and represent another important step towards the ultimate establishment of approaches that can be applied to accurately identify live microorganisms in milk and other food niches.
摘要：
尽管基于高通量DNA测序的方法对于确定各种环境中微生物群落的组成具有重要价值，死微生物的DNA测序可能会导致不准确。在这项试点研究中，我们比较了不同的基于测序的方法，以评估它们在区分活细胞和非活细胞方面的相对准确性，使用添加到牛乳中的活热灭活模型社区。使用的方法是有和没有单叠氮化物丙啶（PMA）处理的shot弹枪宏基因组学，基于RNA的16SrRNA测序和超转录组学。结果表明,该方法总体准确,尽管根据文库类型和测序技术发现了显着差异。不同的分子靶标是使用不同文库类型产生的结果变化的基础。虽然来自牛津纳米孔技术和基于Illumina的测序的衍生组成数据的差异可能反映了不同测序深度的组合，错误率和生物信息学管道。虽然PMA在本研究中成功应用，在将其应用于更普遍的复杂微生物组之前，需要进一步优化。总的来说,这些方法显示出希望，并代表了朝着最终建立可用于准确识别牛奶和其他食品利基中的活微生物的方法迈出的又一重要一步。
