Customization of a DADA2-based pipeline for fungal internal transcribed spacer 1 (ITS1) amplicon data sets.
Customization of a DADA2-based pipeline for fungal internal transcribed spacer 1 (ITS1) amplicon data sets.
复制标题
DOI：
10.1172/jci.insight.151663
复制
发表时间：
2022-01-11
期刊：
影响因子：
8
通讯作者：
Taur Y
中科院分区：
文献类型：
--
作者：
Rolling T;Zhai B;Frame J;Hohl TM;Taur Y
真菌群落的鉴定和分析通常依赖于基于内部转录间隔的扩增子测序。没有金标准用于推断和分类真菌成分，因为方法已经从细菌群落的分析改编。为了实现真菌成分的高分辨率推断，我们使用11种医学相关真菌的混合物定制了基于dada2的管道。虽然DADA2允许区分单核苷酸不同的ITS1序列，但质量过滤、测序偏差和数据库选择被认为是决定样本推断准确性的关键变量。由于测序质量的物种特异性差异，默认过滤设置删除了大多数来自曲霉、酿酒酵母和光假丝酵母的reads。通过微调质量过滤过程，我们实现了真菌群落的改进表示。通过在ITS1前引物区引入一个摆动核苷酸，我们进一步提高了酿酒葡萄球菌和C. glabrata序列的产量。最后，我们发现基于UNITE+INSD或NCBI NT数据库的blast算法在种级分类标注上比在DADA2中实现的朴素贝叶斯分类器具有更高的可靠性。这些步骤优化了一个强大的真菌ITS1测序管道，在大多数情况下，使群落成员的物种水平分配成为可能。
Identification and analysis of fungal communities commonly rely on internal transcribed spacer–based (ITS-based) amplicon sequencing. There is no gold standard used to infer and classify fungal constituents since methodologies have been adapted from analyses of bacterial communities. To achieve high-resolution inference of fungal constituents, we customized a DADA2-based pipeline using a mix of 11 medically relevant fungi. While DADA2 allowed the discrimination of ITS1 sequences differing by single nucleotides, quality filtering, sequencing bias, and database selection were identified as key variables determining the accuracy of sample inference. Due to species-specific differences in sequencing quality, default filtering settings removed most reads that originated from Aspergillus species, Saccharomyces cerevisiae, and Candida glabrata. By fine-tuning the quality filtering process, we achieved an improved representation of the fungal communities. By adapting a wobble nucleotide in the ITS1 forward primer region, we further increased the yield of S. cerevisiae and C. glabrata sequences. Finally, we showed that a BLAST-based algorithm based on the UNITE+INSD or the NCBI NT database achieved a higher reliability in species-level taxonomic annotation compared with the naive Bayesian classifier implemented in DADA2. These steps optimized a robust fungal ITS1 sequencing pipeline that, in most instances, enabled species-level assignment of community members.
登录
查看更多内容
影响因子：
6.4
作者：
Bokulich, Nicholas A.;Rideout, Jai Ram;Caporaso, J. Gregory
通讯作者：
Caporaso, J. Gregory
影响因子：
14.9
作者：
Clark K;Karsch-Mizrachi I;Lipman DJ;Ostell J;Sayers EW
通讯作者：
Sayers EW
影响因子：
4
作者：
Hartmann P;Lang S;Zeng S;Duan Y;Zhang X;Wang Y;Bondareva M;Kruglov A;Fouts DE;Stärkel P;Schnabl B
通讯作者：
Schnabl B
影响因子：
2.7
作者：
Edgar RC
通讯作者：
Edgar RC
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