PacBio Research Topics
The read length of next-generation sequencing is relatively short, and the research on environmental metagenomic samples is greatly limited. The authors performed metagenomic sequencing of winter mixed seawater samples from the Mediterranean Sea by three-generation sequencing. Utilizing the ultra-long read length of the PacBio Sequel II platform can significantly improve the quality of metagenomic assembly, and can also significantly improve the quality of MAGs. The researchers confirmed the above conclusions by comparing the assembly indicators of the PacBio Sequel II platform CCS (HiFi) mode and the traditional illumina NGS:
The researchers assembled CCS data using different software such as SPAdes 3.14, Flye v2.7 and HiCanu v2.0, and compared it with the conventional NGS process: the contig N50 of the PacBio assembly result can reach 5.35-5.95 Mb; it is dozens of times that of NGS, Among them, the longest contig reached 2.6Mb, exactly ten times that of NGS. Looking at the average length of the genes, PacBio provided more reliable results.
The following table compares the data of short read length (SR) and long read length (LR):
Construction of MAGs based on PacBio data
Compared with NGS screening 2K contigs for MAGs generation, PacBio data can calmly select more than 5K sequences for analysis. Compared with the results of NGS sequencing, the medium-high-quality MAGs (>50% integrity, and contamination rate <5%) obtained by the third-generation sequencing found that in addition to the increase in quantity, the average length of the contig of the MAGs obtained based on the third-generation sequencing increased by 4 times , and the longest contig has increased by 2.7 times . What is important is that the accuracy of genome bases has been significantly improved, and the computing resources and time consumed by genome assembly have been greatly reduced at the same cost.
(A) Comparison of intermediate-quality MAGs (>50% intact, <5% contaminated) and NGS results in this study (LR) (blue and red histograms, respectively).
(B) Violin plot showing the average continuous size and integrity of the MAGs depicted in the pane.
in conclusion:
1) Using the long reads of the third-generation sequencing can directly obtain the complete gene, avoiding errors caused by gene assembly.
2) 三代测序的HiFi reads有助于重建更完整的元基因组（MAGs），特别是针对具有高度多样性的微生物。
3) 利用三代测序重建的MAGs完整性更高，包含更多自适应基因，可用于生物技术潜能挖掘。
4) 基于PacBio测序的CCS模式获得的序列，错误率更小，更适用于针对复杂微生态样本的MAGs研究。
doi:10.3389/fmicb.2021.708782
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