人粪便和阴道菌群16S rRNA基因Illumina MiSeq平台和NovaSeq平台测序结果的比较

Comparison of Sequencing Results of Human Feces and Vaginal Microbiota 16S rRNA Genes on Illumina MiSeq and NovaSeq Platforms

Illumina MiSeq是最常用的人体共生菌群16S rRNA基因高通量测序平台之一,NovaSeq是Illumina最新推出的、通量更高的测序平台。虽然两个平台的测序原理相同,但软硬件和试剂有差异,目前尚不清楚在两个平台进行16S rRNA基因测序得到的人体共生菌群alpha和beta多样性结果的异同。本研究分别在两个平台对10个人粪便样本和8个人阴道拭子样本的16S rRNA基因V3-V4区进行PCR扩增、建库和测序,用统一的生物信息学流程处理数据。在相同测序深度下,NovaSeq检测到更多的低丰度细菌;在门、纲、目、科、属和扩增子序列变体(amplicon sequence variant,ASV)水平上,两个平台检测到的优势细菌的种类一致,且相对丰度呈显著正相关,同一样本中若干优势细菌的丰度在两个平台之间有显著差异。在两个平台之间,所有样本的alpha多样性指数Shannon、Pielou′s evenness、观测到的ASV数以及粪便样本的Faith′s PD指数呈显著正相关,前3个alpha多样性指数的绝对数值有显著差异。在beta多样性方面,所有样本的菌群整体结构在两个平台之间没有显著差异,两个平台鉴定的粪便菌群和阴道菌群之间的差异细菌大多数(23个ASV中的19个)是相同的。以上结果表明,对于人粪便样本和阴道拭子样本,MiSeq和NovaSeq平台不改变不同样本之间优势细菌丰度、alpha多样性指数和beta多样性的相对差异,但相同样本在两个平台测定的细菌的种类数目和丰度、alpha多样性指数的绝对数值会有显著差异。

Illumina MiSeq is one of the most widely used platforms for human commensal microbiota 16S rRNA gene high throughput sequencing.NovaSeq is the newest Illumina sequencing platform with higher throughput.Although the sequencing principles of MiSeq and NovaSeq are similar,they differ in hardware,software,and reagents,and the similarities and differences in alpha and beta diversity of human commensal microbiota characterized with 16S rRNA gene sequencing at the two platforms remain unclear.In this study,the 16S rRNA gene V3-V4 regions of 10 human fecal samples and 8 human vaginal swab samples were amplified and library was constructed and sequenced at MiSeq and NovaSeq platform,respectively,and identical bioinformatic pipeline was used to process the sequencing data generated by the two platforms.At the same sequencing depth,NovaSeq detected more bacteria taxa of low abundance than MiSeq.At the Phylum,Class,Order,Family,Genus,and amplicon sequence variant(ASV)levels,MiSeq and NovaSeq detected same dominant bacterial taxa,and their relative abundances were correlated significantly,and positively.The abundances of some dominant bacteria in the same sample differed significantly between the two platforms.Alpha diversity indexes at two platforms,including index,Pielou′s evenness,and number of observed ASVs for all samples,and Faith′s PD for fecal samples were significantly,strongly,and positively correlated,the absolute values of the first three alpha diversity indexes significantly differed between the two platforms.At the aspect of beta diversity,the overall microbiota structure of all samples showed no significant difference between the two platforms,and the majority of the differential bacteria ASV between the fecal and vaginal microbiota identified by the two platforms(19 out of 23 ASV)were identical.In conclusion,for human fecal samples and vaginal swab samples,the MiSeq and NovaSeq platforms do not change the relative differences in dominant bacterial abundances,alpha diversity indexes,and beta diversity among varied samples,but indivi-dual samples differ in the numbers and abundances of detected bacterial taxa,and absolute values of alpha diversity indexes between the two sequencing platforms.