Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling.
Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling.
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DOI：
10.1038/s43705-021-00033-z
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发表时间：
2021-06-29
期刊：
影响因子：
--
通讯作者：
Wilmes, Paul
中科院分区：
文献类型：
--
作者：
Busi, Susheel Bhanu;de Nies, Laura;Habier, Janine;Wampach, Linda;Fritz, Joelle V.;Heintz-Buschart, Anna;May, Patrick;Halder, Rashi;de Beaufort, Carine;Wilmes, Paul
16S rRNA 基因扩增子测序是研究微生物组的常用方法。然而，一些基本概念尚未得到全面研究。我们使用基于文献或三个测序设施生成的数据的定义的微生物群落研究了虚假序列的出现，并通过操作分类单位（OTU）和扩增子序列变体（ASV）方法进行分析。 OTU 聚类和单例去除是一种常用的方法，可提供大约 50%（模拟群落）至 80%（无菌小鼠）的虚假类群。根据 ASV 分析，虚假类群的比例通常较低，但根据目标基因区域和所使用的条形码系统而变化。 0.25% 的相对丰度被发现是一个有效阈值，低于该阈值，在基于 OTU 和 ASV 的分析方法中，可以在很大程度上防止虚假类群的分析。使用此截止值提高了分析的再现性，即，与在七次测序运行中使用六个人类粪便样本进行单例过滤相比，丰富度估计的变化减少了 38%。人类粪便群落的β多样性分析明显受到过滤策略和用于比较的系统发育距离类型的影响，这凸显了在得出微生物组变化结论之前仔细分析数据的重要性。总之，在 16S rRNA 基因扩增子数据的生物信息学处理过程中处理伪影序列需要仔细注意，以避免产生误导性的结果。我们提出有效丰富度的概念，以方便比较不同研究中的α多样性。
16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.
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影响因子：
14.9
作者：
Klindworth A;Pruesse E;Schweer T;Peplies J;Quast C;Horn M;Glöckner FO
通讯作者：
Glöckner FO
使用通用的unifrac距离将微生物组组成与环境协变量相关联。
DOI：
10.1093/bioinformatics/bts342
发表时间：
2012-08-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Chen J;Bittinger K;Charlson ES;Hoffmann C;Lewis J;Wu GD;Collman RG;Bushman FD;Li H
通讯作者：
Li H
DOI：
10.1038/ismej.2012.8
发表时间：
2012-08
期刊：
The ISME journal
影响因子：
--
作者：
通讯作者：
--
影响因子：
48
作者：
Callahan BJ;McMurdie PJ;Rosen MJ;Han AW;Johnson AJ;Holmes SP
通讯作者：
Holmes SP
影响因子：
14.9
作者：
Alanis-Lobato G;Andrade-Navarro MA;Schaefer MH
通讯作者：
Schaefer MH