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首页> 外文期刊>Applied Microbiology and Biotechnology >Impact of DNA extraction, sample dilution, and reagent contamination on 16S rRNA gene sequencing of human feces
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Impact of DNA extraction, sample dilution, and reagent contamination on 16S rRNA gene sequencing of human feces

机译：DNA提取，样品稀释和试剂污染对16S rRNA基因序列的影响

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Culture-independent methods have granted the possibility to study microbial diversity in great detail, but technical issues pose a threat to the accuracy of new findings. Biases introduced during DNA extraction can result in erroneous representations of the microbial community, particularly in samples with low microbial biomass. We evaluated the DNA extraction method, initial sample biomass, and reagent contamination on the assessment of the human gut microbiota. Fecal samples of 200 mg were subjected to 1:10 serial dilutions; total DNA was obtained using two commercial kits and the microbiota assessed by 16S ribosomal RNA (rRNA) gene sequencing. In addition, we sequenced multiple technical controls. The two kits were efficient in extracting DNA from samples with as low as 2 mg of feces. However, in instances of lower biomass, only one kit performed well. The number of reads from negative controls was negligible. Both DNA extraction kits allowed inferring microbial consortia with similar membership but different abundances. Furthermore, we found differences in the taxonomic profile of the microbial community. Unexpectedly, the effect of sample dilution was moderate and did not introduce severe bias into the microbial inference. Indeed, the microbiota inferred from fecal samples was distinguishable from that of negative controls. In most cases, samples as low as 2 mg did not result in a dissimilar representation of the microbial community compared with the undiluted sample. Our results indicate that the gut microbiota inference is not much affected by contamination with laboratory reagents but largely impacted by the protocol to extract DNA.
机译：独立于文化的方法已经赋予了详细研究微生物多样性的可能性，但技术问题对新发现的准确性构成了威胁。在DNA提取期间引入的偏差可以导致微生物群落的错误表示，特别是在具有低微生物生物量的样品中。我们评估了DNA提取方法，初始样品生物质和试剂污染对人体肠道微生物的评估。对200mg的粪便样品进行1:10连续稀释液;使用两种商业套件和16S核糖体RNA（RRNA）基因测序评估的微生物瘤获得总DNA。此外，我们对多种技术控制进行了测序。两种试剂盒有效地从用低至2mg粪便中从样品中提取DNA。然而，在低生物量的情况下，只表现出一个套件。来自负控的读数的数量可以忽略不计。 DNA提取套件均可允许推断使用相似的成员但不同的丰富子系统。此外，我们发现了微生物群落的分类学概况的差异。出乎意料的是，样品稀释的效果中等，并未将严重的偏置引入微生物推理。实际上，从粪便样品推断的微生物群与阴性对照的可区分。在大多数情况下，与未稀释的样品相比，低至2mg的样品不会导致微生物群落的不同表示。我们的结果表明，肠道微生物群推断不受实验室试剂污染的影响，但在很大程度上受到提取DNA的方案的影响。

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