Most commonly, next generation sequencing‐based microbiome studies are performed on the total DNA (totDNA) pool; however, this consists of extracellular‐ (exDNA) and intracellular (iDNA) DNA fractions. By investigating the microbiomes of different anaerobic digesters over time, we found that totDNA suggested lower species richness considering all and/or only common species and yielded fewer unique reads as compared to iDNA. Additionally, exDNA‐derived sequences were more similar to those from totDNA than from iDNA and, finally, iDNA showed the best performance in tracking temporal changes in microbial communities. We postulate that abundant sequences present within the exDNA fraction mask the overall results of totDNA and provide evidence that exDNA has the potential to qualitatively bias microbiome studies at least in the anaerobic digester environment as it contains information about cells that were lysed hours or days ago. iDNA, however, was found to be more appropriate in providing reliable genetic information about potentially alive as well as rare microbes within the target habitat.

中文翻译：

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厌氧消化器 NGS 研究中细胞外 DNA 的掩蔽效应和细胞内 DNA 的稳健性：总 DNA 库的判别性研究

最常见的是，基于下一代测序的微生物组研究是在总 DNA (totDNA) 池上进行的；然而，这由细胞外 (exDNA) 和细胞内 (iDNA) DNA 部分组成。通过随着时间的推移研究不同厌氧消化器的微生物组，我们发现与 iDNA 相比，totDNA 表明考虑到所有和/或仅常见物种的物种丰富度较低，并且产生的独特读数较少。此外，exDNA 衍生的序列与来自 totDNA 的序列比来自 iDNA 的更相似，最后，iDNA 在跟踪微生物群落的时间变化方面表现出最佳性能。我们假设 exDNA 部分中存在的丰富序列掩盖了 totDNA 的总体结果，并提供证据表明 exDNA 至少在厌氧消化器环境中具有定性偏差微生物组研究的潜力，因为它包含有关数小时或数天前裂解的细胞的信息。然而，人们发现 iDNA 更适合提供有关目标栖息地内潜在存活微生物和稀有微生物的可靠遗传信息。