PacBio RSII

The Pacific Biosciences RSII is a single-molecule, real-time (SMRT) sequencing machine that uses sequencing-by-synthesis methods to generate data. With 150,000 ZMW’s (zero mode waveguides) on a SMRT cell, the machine is a complex piece of optical engineering capable of resolving the incorporation of a single nucleotide on a single strand of DNA by a single polymerase. The platform produces high quality (with sufficient coverage), very long reads.

You can find out more about the PacBio RSII on the Pacific Biosciences website.

PacBio RSII

The applications.

The PacBio RSII can be used to sequence genomes of all sizes, however if you are interested in microbial genomes then a ‘PacBio only’ genome assembly may be all the sequencing you ever need to do to get a high-quality finished genome. PacBio sequencing for larger genomes is often combined with short-read sequencing data (e.g. from the Illumina HiSeq) in a ‘hybrid’ assembly which will give you greater contiguity than the HiSeq data alone.

It is possible to do target capture and sequencing on the PacBio RSII, this allows you to capture long DNA molecules and sequence them, particularly useful for targeting genes which are highly polymorphic, and hard to sequence on short read platforms, like MHC or HLA genes.

If you’re interested in transcriptomics, the Iso-Seq protocol gives you the ability to sequence full length transcripts. The PacBio RSII is also capable of doing modified base detection natively, which means you can identify methylated bases for epigenetic studies.

The throughput.

Each SMRT cell can produce more than 1Gb of raw data. A run can consist of 16 SMRT cells at a time, so each run is capable of producing more than 16Gb of sequence data.

What we use it for.

At EI we use the PacBio RSII for supporting genome assembly and annotation work. We have used the PacBio RSII to help provide additional genome sequence for our work on the ash dieback disease - including sequencing the ash dieback pathogen as well as the tree genomes.

It also forms an important part of our annotation pipelines, data from Iso-Seq is used to build gene models and discover alternatively spliced isoforms.

Related reading.