Great Advantages of PicBio SMRT Sequencing
Posted by kiko on January 24th, 2019
Single Molecular Real-Time (SMRT) sequencing is one of several next-generation sequencing technologies that are currently in use. In the past, it has been a little overlooked because of its lower throughput compared with methods such as Illumina and Ion Torrent, and some rumors that it is inaccurate. Here, it is necessary to dispel these misconceptions and show that SMRT is indeed a highly accurate method with many advantages when used to sequence small genomes, including the possibility of facile closure of bacterial genomes without additional experimentation. Meanwhile, we also highlight its value in being able to detect modified bases in DNA.
The SMRT approach to sequencing has three advantages. First, considering the impact of the longer reads lengths, especially for de novo assemblies. While typical next-generation sequencing such as single-cell sequencing can provide abundant coverage of a genome, the short read lengths and amplification biases of those sequencing technologies lead to fragmented assemblies whenever poorly amplified region and a complex repeat is encountered. As a result, GC-rich and GC-poor regions which tend to be poorly amplified, are susceptible to poor quality sequencing. Resolving fragmented assemblies requires further sequencing and additional costly bench work. By including the longer reads of SMRT sequencing runs, the read set will span more repeats and missing bases, thereby closing many of the gaps automatically and simplifying, or even eliminating, the finishing time. It is becoming routine for bacterial genomes to be assembled entirely with this approach, and we expect this practice will translate to larger genomes in the future.
Second, considering DNA methyltransferases. These can exist as solitary entities or parts of restriction-modification systems. In both cases, they methylate relatively short sequence motifs that can easily be recognized from SMRT sequencing data because of the change in DNA polymerase kinetics, as it moves along the template molecule, that results from the presence of epigenetic modifications. Moreover, SMRT sequencing is also able to identify RNA base modifications through the same approach as DNA base modifications, but using an RNA transcriptase in place of the DNA polymerase. In fact, SMRT sequencing represents an essential step toward uncovering the biology that happens between proteins and DNA, including both the study of mRNA sequences and the regulation of translation. Therefore, functional information emerges directly from the SMRT sequencing approach.
Third, considering the persistent rumor that SMRT sequencing is less accurate than other next-generation sequencing platforms, which has now been demonstrated to be unreal in several ways. Firstly, a direct comparison of several approaches to determining genetic polymorphisms has shown that SMRT sequencing has comparable performance to other sequencing techniques. Secondly, the accuracy of assembling a complete genome using SMRT sequencing in combination with other technologies has proved to be as reliable and accurate as more traditional approaches. Conclusions
The considerations above make a strong case for combining the more conventional sequence-dense data from other technologies with at least moderate coverage of SMRT data so that genomes can be improved, their methylation patterns obtained, and the functional activity of their methyltransferase genes deduced. We would especially urge all groups currently sequencing bacterial genomes to adopt this policy. That said, SMRT sequencing has also substantially improved eukaryotic genome assemblies, and we hope that it will be more widely applied in this context over time.
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About the Authorkiko
Joined: November 27th, 2018
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