Pacific Biosciences of California, provider of the PacBio RS II DNA Sequencing System, has announced noteworthy new product enhancements to support researchers who are working on complex genome projects.
The new P5 polymerase and C3 chemistry combination (P5-C3) extends the industry-leading sequence read lengths to an average of around 8,500 bases, with the longest exceeding 30,000 bases. The inclusion of new photo-protected dyes – used to distinguish the four DNA bases using four colors of fluorescence – is a key feature of this performance enhancement. The P5 polymerase is shielded from laser-induced damage by these photo-protected dyes, which allows the polymerase to provide exceptionally long sequencing reads.
A genome sequence that is complete is extremely valuable in various types of scientific research, yet most organisms, including humans, do not have their complete genome assembled. Complex genomes are, instead, partially completed with difficult repetitive sections and longer structural variants excluded from the assembly, which results in highly fragmented, incomplete genomes. In order to sequence the complete genome of an organism, very long sequencing reads are needed to span these regions, which can be critical in understanding gene function. Pacific Biosciences’ PacBio RS II Single Molecule, Real-time (SMRT) sequencing system provides very long sequence reads, which enables researchers to fill gaps or reduce the number of fragments in assemblies of moderate and large genomes.
“With the P5-C3 release, we saw another step change in the evolution of PacBio data,” said Dr. Paul Coupland, research scientist at the Wellcome Trust Sanger Institute, which served as a test site. “We obtained polymerase mean read lengths of greater than 8,500 bases, enabling us to span longer repeats, phase SNPs and discover structural variants that were previously hidden. Our first data set was used for hybrid de novo assembly, and when combined with existing P4-C2 data the assembly was considerably improved.”
“Following our steady stream of system and chemistry improvements during 2013, including our v3 SMRT Cells, the PacBio RS II launch and the highly accurate polymerase P4, this release is our next-generation chemistry upon which future developments and read length improvements will be built,” added Kevin Corcoran, senior vice president of market development for Pacific Biosciences. “Now 50 percent of the bases come from reads over 10,000 bases in length, nearly doubling the per-SMRT Cell throughput. As our customers continue to sequence larger and more complex genomes, the long reads generated from P5-C3 chemistry make it an ideal choice for scaffolding and hybrid de novo assemblies.”
Also included in the latest release are 20,000-base size-selected library protocols that are optimized for use with the Sage Science BluePippin DNA size selection system, as well as an upgraded version of SMRT Analysis, which introduces an update to the popular HGAP assembler. The time required to perform de novo genome assembly is substantially decreased by the new HGAP 2 assembler. The range of genome sizes that can be assembled is also extended by these HGAP performance enhancements. Other added software features include diploid-aware SNP detection and haplotype phasing of amplicons, which enables users to utilize the platform for applications like HLA analysis. The new reagents are available in addition to the previously released P4-C2 chemistry, which is still recommended for PacBio-only de novo assembly, minor-variant detection, targeted sequencing and cDNA applications.
To learn more about the PacBio RS II and hear from customers who are using the system, researchers attending the 2013 American Society of Human Genetics annual meeting in Boston can attend a workshop on Oct. 24 from 12:30-2 p.m.
For more information about Pacific Biosciences and the PacBio RS II, visit www.pacb.com
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