16S 全长测序
【重磅】11月6日在Nature Communication 发表的一篇有关16S 全长测序的文章值得一读。https://www.nature.com/articles/s41467-019-13036-1。通讯作者是来自Jackson Lab 的George Weinstock，其本人为Human Microbiome Project (HMP) 的领导人之一。在此文中作者阐述了用PacBio 测序全长16S 基因的重要意义。
作者用数据揭示了整个 16S 测序领域现有的常用方法 – 即采用短读取 NGS 测序局部16S variable region 的思路– 需要被新的方法和理念取代 （PacBio CCS模式作全长16S 测序的方法）。文章以肠道 Microbiome 为例作了通篇的研究。
Based on their extensive database from the Human Microbiome Project, they demonstrate that the ability to re-identify species from 16S sequences varies widely depending on what sub-region is used. In contrast, full-length sequences allowed them to re-assign all sequences to the species of origin in their database. They then show that each 16S sub-region had significant biases in which bacterial clades they were able to identify at the species level.
• V1–V3: good results for Escherichia / Shigella
• V3–V5: good results for Klebsiella,
• V6–V9: good results for Clostridium and Staphylococcus
Since all of the above clades can be present in the human gut, they note that only full length16S (V1-V9) will yield a comprehensive, strain level picture of the human gut microbiome.
The authors then make the case that now that the technology is available, new 16S analysis tools will inevitably arise to enable to strain level resolution, which currently can only be done with shotgun sequencing. This will be accomplished by leveraging the ability of PacBio SMRT Sequencing to resolve all copies of the 16S gene in each bacteria to do strain-level assignment. Bacteria have between 1-15 copies of the 16S genes. The number of copies is consistent within a species, but intragenomic variation among the copies is strain specific. Using his catalogue of isolates collected during the HMP, Weistock and team show that most species found in the gut have enough intragenomic 16S variation to do strain-level assignment, and that furthermore PacBio 16S sequencing is able to resolve all the variants present within each strain.
值得一提的是目前PacBio Sequel II 高通量平台的通量和准确率已经大大超过了本文所采用的 RSII 平台。比如不久前 马里兰大学的 Luke Tallon 教授的数据证实 Sequel II 测序16S 全长的base call accuracy 可以达到QV80 (相比 NGS 的QV40). 这势必对达到strain level 分辨率的奢求提供一个强有力的技术支撑。
16S 全长测序
【重磅】11月6日在Nature Communication 发表的一篇有关16S 全长测序的文章值得一读。https://www.nature.com/articles/s41467-019-13036-1。通讯作者是来自Jackson Lab 的George Weinstock，其本人为Human Microbiome Project (HMP) 的领导人之一。在此文中作者阐述了用PacBio 测序全长16S 基因的重要意义。
作者用数据揭示了整个 16S 测序领域现有的常用方法 – 即采用短读取 NGS 测序局部16S variable region 的思路– 需要被新的方法和理念取代 （PacBio CCS模式作全长16S 测序的方法）。文章以肠道 Microbiome 为例作了通篇的研究。
Based on their extensive database from the Human Microbiome Project, they demonstrate that the ability to re-identify species from 16S sequences varies widely depending on what sub-region is used. In contrast, full-length sequences allowed them to re-assign all sequences to the species of origin in their database. They then show that each 16S sub-region had significant biases in which bacterial clades they were able to identify at the species level.
• V1–V3: good results for Escherichia / Shigella
• V3–V5: good results for Klebsiella,
• V6–V9: good results for Clostridium and Staphylococcus
Since all of the above clades can be present in the human gut, they note that only full length16S (V1-V9) will yield a comprehensive, strain level picture of the human gut microbiome.
The authors then make the case that now that the technology is available, new 16S analysis tools will inevitably arise to enable to strain level resolution, which currently can only be done with shotgun sequencing. This will be accomplished by leveraging the ability of PacBio SMRT Sequencing to resolve all copies of the 16S gene in each bacteria to do strain-level assignment. Bacteria have between 1-15 copies of the 16S genes. The number of copies is consistent within a species, but intragenomic variation among the copies is strain specific. Using his catalogue of isolates collected during the HMP, Weistock and team show that most species found in the gut have enough intragenomic 16S variation to do strain-level assignment, and that furthermore PacBio 16S sequencing is able to resolve all the variants present within each strain.
值得一提的是目前PacBio Sequel II 高通量平台的通量和准确率已经大大超过了本文所采用的 RSII 平台。比如不久前 马里兰大学的 Luke Tallon 教授的数据证实 Sequel II 测序16S 全长的base call accuracy 可以达到QV80 (相比 NGS 的QV40). 这势必对达到strain level 分辨率的奢求提供一个强有力的技术支撑。
