16S 全長(zhǎng)測(cè)序
【重磅】11月6日在Nature Communication 發(fā)表的一篇有關(guān)16S 全長(zhǎng)測(cè)序的文章值得一讀。https://www.nature.com/articles/s41467-019-13036-1。通訊作者是來自Jackson Lab 的George Weinstock，其本人為Human Microbiome Project (HMP) 的領(lǐng)導(dǎo)人之一。在此文中作者闡述了用PacBio 測(cè)序全長(zhǎng)16S 基因的重要意義。
作者用數(shù)據(jù)揭示了整個(gè) 16S 測(cè)序領(lǐng)域現(xiàn)有的常用方法 – 即采用短讀取 NGS 測(cè)序局部16S variable region 的思路– 需要被新的方法和理念取代 （PacBio CCS模式作全長(zhǎng)16S 測(cè)序的方法）。文章以腸道 Microbiome 為例作了通篇的研究。
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 高通量平臺(tái)的通量和準(zhǔn)確率已經(jīng)大大超過了本文所采用的 RSII 平臺(tái)。比如不久前 馬里蘭大學(xué)的 Luke Tallon 教授的數(shù)據(jù)證實(shí) Sequel II 測(cè)序16S 全長(zhǎng)的base call accuracy 可以達(dá)到QV80 (相比 NGS 的QV40). 這勢(shì)必對(duì)達(dá)到strain level 分辨率的奢求提供一個(gè)強(qiáng)有力的技術(shù)支撐。
16S 全長(zhǎng)測(cè)序
【重磅】11月6日在Nature Communication 發(fā)表的一篇有關(guān)16S 全長(zhǎng)測(cè)序的文章值得一讀。https://www.nature.com/articles/s41467-019-13036-1。通訊作者是來自Jackson Lab 的George Weinstock，其本人為Human Microbiome Project (HMP) 的領(lǐng)導(dǎo)人之一。在此文中作者闡述了用PacBio 測(cè)序全長(zhǎng)16S 基因的重要意義。
作者用數(shù)據(jù)揭示了整個(gè) 16S 測(cè)序領(lǐng)域現(xiàn)有的常用方法 – 即采用短讀取 NGS 測(cè)序局部16S variable region 的思路– 需要被新的方法和理念取代 （PacBio CCS模式作全長(zhǎng)16S 測(cè)序的方法）。文章以腸道 Microbiome 為例作了通篇的研究。
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 高通量平臺(tái)的通量和準(zhǔn)確率已經(jīng)大大超過了本文所采用的 RSII 平臺(tái)。比如不久前 馬里蘭大學(xué)的 Luke Tallon 教授的數(shù)據(jù)證實(shí) Sequel II 測(cè)序16S 全長(zhǎng)的base call accuracy 可以達(dá)到QV80 (相比 NGS 的QV40). 這勢(shì)必對(duì)達(dá)到strain level 分辨率的奢求提供一個(gè)強(qiáng)有力的技術(shù)支撐。
