This week, our CSO Jonas Korlach hosted a webinar entitled “Gain New Insights in Genome and Transcriptome Research with Greater than 10,000 bp Reads.” He spoke to attendees about the PacBio® technology, elements of sequencing, and applications of the ultra-long reads generated by Single Molecule, Real-Time (SMRT®) Sequencing. Here’s a quick recap.
Jonas offered a look at how PacBio’s technology performs in the four key sequencing characteristics that one should consider for any sequencing work: contiguity, accuracy, uniformity, and originality. For contiguity, or how much of a DNA fragment can be sequenced in a single pass, the PacBio platform outperforms all other technologies. Half of data generated in a SMRT Sequencing run is captured in reads longer than 10 kb, and many reads exceed 30 kb. Jonas added that the PacBio RS II also has industry-leading consensus accuracy, routinely at QV50 (>99.999%). Regarding uniformity, or the ability to uniformly sequence all of the DNA present in your sample, the PacBio platform has been shown in an independent study to have the least bias of any sequencing technology. Finally, originality refers to the ability to sequence native, unamplified DNA to avoid amplification bias and allow access to detect epigenetic marks as part of the sequencing, which Jonas noted is a unique attribute of SMRT Sequencing.
Jonas then turned to examples of how scientists are using the PacBio technology for a wide range of applications, starting with high-quality de novo assemblies and finishing of genomes ranging from microbial to large and complex genomes like the human genome. With plant genomes, such as Arabidopsis, SMRT Sequencing assemblies have been shown to include hundreds of thousands of SNPs that were missed by short-read assemblies of the same organism. A scientist working on the spinach genome saw his assembly go from 1.3 million contigs with short-read sequencing to fewer than 10,000 contigs with PacBio. In a human data set we released, the contig N50 of 4.4 Mb represented a 30-fold improvement over the previous best de novo human assembly with a contig N50 of 144 Kb.
The long reads produced by SMRT Sequencing have also been particularly useful for detecting various forms of structural genomic variation, thereby resolving complex or even previously “unsequenceable” genomic regions, and elucidating the complex landscape of alternative splice isoforms in transcriptomes, Jonas noted. Other applications include genome editing, demonstrating SMRT Sequencing as a powerful tool to quantify outcomes of genome-editing experiments, as well as epigenomics since PacBio technology can directly detect base modifications.
Finally, Jonas spoke about upcoming improvements in sample preparation, read length, and data analysis. To see full details, you can view the recorded webinar.