测序技术的发展-CCS测序

三代测序

测序仪器：

“The PacBio Sequel is the newest sequencing platform released by Pacific Biosciences. The PacBio Sequel, aptly named, follows the release of the PacBio RS II System; one of the first to offer read lengths greater than 20Kb, and the PacBio Sequel is no different. In fact, while the Sequel has a much smaller footprint than its predecessor, the Sequel System is able to provide 7x the amount of reads as the RS II.  Where the RS II was able to generate up to 1 Gb of data, the Sequel is able to generate 3-8Gb of data. The driving force behind this long read sequencing technology is what PacBio calls SMRT, or Single Molecule, Real Time technology. This SMRT technology combined with Zero-Mode Waveguides (ZMWs) allow the PacBio Sequel to offer some of the longest read lengths the scientific community has ever seen. Read lengths ranging from 10Kb all the way to 60kb make the PacBio Sequel the ideal system for whole genome sequencing and de novo sequencing. With flexible run times and an increased amount of data generation, the PacBio Sequel provides long read sequencing at an affordable cost.”

1，PacBio RS II System

2，PacBio Sequel

PacBio Sequel 是Pacific Biosciences继PacBio RS II推出的最新的测序平台，他们在测序长度上没有区别，但PacBio Sequel体积更小，测序量确是PacBio RS II的七倍。

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2022/01/01更新

现在很多基因组文章已经用CCS数据进行组装了，我也再来了解一下现在的组装技术。

知乎上贝瑞基因科技服务写的“HiFi Reads基因组组装：快、准、狠”的文章挺好的。

我在Pacbio官网截图了现在的测序仪器介绍，之前写文章方法，我实在分不清使用什么仪器测的，这些有什么区别，看了这个图明白多了。之前上面也提到了Pacbio Sequel System 比 PacBio RS II System的优势。Sequel System 也不断升级，出了Sequel II System 和 Sequel IIe System。

测序原理：Single Molecule, Real-Time (SMRT) Sequencing

Original Publication: Eid, J., et al. (2009) Real-time DNA sequencing from single polymerase molecules. Science, 323(5910), 133–138.

单分子测序的模式升级了：

Wenger, Aaron M et al. (2019) Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nature biotechnology

“The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We

report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT)

sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilo bases (kb).”

之前的DNA测序技术，要么是准确的短reads，要么是低准确性的长reads。这篇文章报道了CCS，提高了SMRT测序的准确性。

CCS测序原理图：

An opportunity to produce long CCS reads was suggested by a 16-fold increase in the fraction of polymerase reads longer than 100 kb for a control Escherichia coli 10-kb amplicon library compared to a long-insert (>30 kb) library of sheared E. coli genomic DNA sequenced under identical conditions with 10-h collections (Supplementary Fig. 1a).

CCS之所以能够提高准确性，是通过将一条DNA序列循环多测几遍。多测几遍，那么产生的单分子序列肯定要长。而之所以能产生这么长的CCS reads,是通过在大肠杆菌测序发现，将文库插入片段控制在10kb,比30kb产生的大于100kb polymerase reads占比提高了16倍。

CLR 和 CCS 两种不同模式如下图：