Amplicon Sequence Variants Artificially Split Bacterial Genomes into Separate Clusters.
Amplicon Sequence Variants Artificially Split Bacterial Genomes into Separate Clusters.
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DOI：
10.1128/msphere.00191-21
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发表时间：
2021-08-25
期刊：
影响因子：
4.8
通讯作者：
Schloss PD
中科院分区：
文献类型：
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作者：
Schloss PD
扩增子测序变异体（asv）已被提出作为操作分类单位（OTUs）的替代方法来分析微生物群落。asv越来越受欢迎，部分原因是人们希望反映更精细的分类水平，因为它们不基于基于距离的阈值对序列进行聚类。然而，asv和使用过窄的阈值来识别otu增加了将单个基因组分裂成单独簇的风险。为了评估这种风险，我分析了rrn拷贝数数据库中细菌基因组中16S rRNA基因的基因组内变异，该数据库包含来自5,972个物种的20,427个基因组。随着基因组中16S rRNA基因拷贝数的增加，asv的数量也随之增加。对于全长16S rRNA基因，平均每个拷贝有0.58个asv。对于含有7个16S rRNA拷贝的基因组（如大肠杆菌），需要使用5.25%的距离阈值将来自同一基因组的全长asv聚类为单个OTU，置信度为95%。这项研究强调了当asv用于分析16S rRNA基因序列数据时，将单个细菌基因组分裂成单独簇的风险。虽然当使用宽距离阈值时，也存在将不同物种的asv聚类到同一OTU中的风险，但与人为地将基因组拆分为单独的asv和OTU相比，这些风险较少受到关注。16S rRNA基因测序引起了人们对微生物群落研究的极大兴趣。试图将16S rRNA基因序列分类到越来越低的分类水平，与使用比16S rRNA基因片段更多的序列和生理信息来定义这些水平的现实之间一直存在紧张关系。此外，细菌分类群的命名反映了命名者的偏见。最近推动在微生物群落分析中采用asv代替otu的一个动机是允许研究人员在反映物种水平分类学的尽可能高的水平上进行分析。目前的研究意义重大，因为它量化了人工将细菌基因组分成不同簇的风险。ASV方法非但不能更好地代表细菌的分类和生物学，还可能导致对来自同一基因组的不同ASV的生态学的相互矛盾的推断。
Amplicon sequencing variants (ASVs) have been proposed as an alternative to operational taxonomic units (OTUs) for analyzing microbial communities. ASVs have grown in popularity, in part because of a desire to reflect a more refined level of taxonomy since they do not cluster sequences based on a distance-based threshold. However, ASVs and the use of overly narrow thresholds to identify OTUs increase the risk of splitting a single genome into separate clusters. To assess this risk, I analyzed the intragenomic variation of 16S rRNA genes from the bacterial genomes represented in an rrn copy number database, which contained 20,427 genomes from 5,972 species. As the number of copies of the 16S rRNA gene increased in a genome, the number of ASVs also increased. There was an average of 0.58 ASVs per copy of the 16S rRNA gene for full-length 16S rRNA genes. It was necessary to use a distance threshold of 5.25% to cluster full-length ASVs from the same genome into a single OTU with 95% confidence for genomes with 7 copies of the 16S rRNA, such as Escherichia coli. This research highlights the risk of splitting a single bacterial genome into separate clusters when ASVs are used to analyze 16S rRNA gene sequence data. Although there is also a risk of clustering ASVs from different species into the same OTU when using broad distance thresholds, these risks are of less concern than artificially splitting a genome into separate ASVs and OTUs. IMPORTANCE 16S rRNA gene sequencing has engendered significant interest in studying microbial communities. There has been tension between trying to classify 16S rRNA gene sequences to increasingly lower taxonomic levels and the reality that those levels were defined using more sequence and physiological information than is available from a fragment of the 16S rRNA gene. Furthermore, the naming of bacterial taxa reflects the biases of those who name them. One motivation for the recent push to adopt ASVs in place of OTUs in microbial community analyses is to allow researchers to perform their analyses at the finest possible level that reflects species-level taxonomy. The current research is significant because it quantifies the risk of artificially splitting bacterial genomes into separate clusters. Far from providing a better representation of bacterial taxonomy and biology, the ASV approach can lead to conflicting inferences about the ecology of different ASVs from the same genome.