What is the rationale of the third generation of gene sequencing?

The third generation of gene sequencing technology is “Single Molecule Real Time (SMRT) DNA sequencing “. The method is based on single molecules reading technology of nanopores, and can quickly read the sequence without amplification.

Application areas

Whole de novo genome sequencing

Compared with the second generation sequencing, PacBio RS II long read length will effectively solve the problem of short sequence data splicing. At the same time, pacbio RS II can be sequenced directly to single molecules without amplification, effectively avoiding the PCR amplification preference and preference for GC. PacBio RS II can easily cross the GC content anomaly (too high or too low) and a high sequence-repeated region, covering the sequence integrity and uniformity.

Organelle genome sequencing

Chloroplast and mitochondrial genome sequences contain complex structure. PacBio RS II long read length can directly cross these areas to get these organelles of the whole genome sequence information. Genome assembly does not rely on whether there is a closely related species of mitochondrial and chloroplast genome information etc.

Epigenomics

PacBio RS II uses the polymerase reaction kinetics in the sequencing process, the first time to achieve a direct sequencing of the base modification. When the base is modified, the synthesis rate of DNA polymerase slows down, and the corresponding signal is detected.

Each base modification event makes a small difference in the polymerase’s “PacBio RS II pause mode”, which is ultimately reflected in the interval of the fluorescence pulse signal. In addition to the methylation modification, it can also detect 5-hC, 5-hmU, 5-hU, 1-mA, 6-mA, 8-oxoA, BPDE, 6-mT, 6-mG etc. and even can detect methylation and hydroxymethylation modification that traditional bisulfite sequencing can not distinguish. PacBio platform can detect epigenetic modification information while sequencing at the same time. It just analyzes the base modification information for the sequencing data to select the appropriate software.