Pacific Biosciences (often abbreviated PacBio; previously named Nanofluidics), rather than taking the NGS field head on, has carved out a niche by focusing on how they can complement and supplement other sequencing platforms. Rather than generating a large number of relatively short reads, PacBio generates a relatively small number of very long reads. These long reads are what makes PacBio a good fit for a number of applications not well served by other sequencing platforms: de novo genome assembly, genome finishing, structural variations, HLA typing, full length transcriptomes, etc.
The RS II is an upgrade of the original PacBio sequencer, the RS. While this platform is still available as of early 2016, the more capable and less expensive Sequel will likely rapidly replace it.
The Sequel is the aptly named smaller, less expensive follow-on to the RS II. Based on the fruits of a collaboration with Roche, it is able to generate ~7X more data than its predecessor (and performance will likely improve over time).
PacBio calls their technology SMRT sequencing – single molecule, real-time. Unlike most other sequencing technologies, it doesn’t require clonal amplification of DNA – it sequences single molecules. Also, while it is a form of ‘sequencing by synthesis’, it operates in real-time rather than incorporating a single base (or type of base) at a time. The optical system is essentially taking a movie as the polymerase incorporates fluorescent nucleotides. While unincorporated nucleotides would normally cause too much background signal for this to work, PacBio has solved this problem with the use of ‘zero mode waveguides’ (ZMWs). ZMWs are microwells, at the bottom of which are placed individual polymerases that are designed to capture signal only from nucleotides as they are being incorporated (while filtering out the signal from all of the unincorporated nucleotides). The real-time nature of PacBio leads to three distinct advantages. First, the reads are quite fast, with runs generally lasting from 30 minutes to three hours (rather than days). Second, the reads are substantially longer than most other commercially available sequencing platforms (including Sanger-based sequencers), with a mean of ~15 kb. Third, the movie captures information about the rate of nucleotide incorporation, which can be used to determine the modification status of the template nucleotide (e.g. 5-mC, 5-hmC, etc.)
The other way in which the PacBio differs from other commercial sequencing platforms is their error model. The raw read error rate is substantially higher at around 14% compared with the 0.1 to 1% error rate of other leading systems. However, unlike the others, the error model is stochastic, so very high quality reads across all bases can be achieved in the consensus sequence. Additionally, the SMRT sequencing chemistry is able to sequence regions of high GC content, leading to much more uniform coverage of the genome.
These advantages do come at a cost of low output compared to other commercially available platforms, with the PacBio Sequel generating 5-10 Gb across ~500k reads per chip. For comparison, Illumina’s flagship sequencer, the HiSeq X, generates 1.8 Tb across 6B reads per run.
Given this unique mix of strengths and weaknesses, PacBio tends to be used for a particular set of applications, such as sequencing small genomes (e.g., viral and bacterial), difficult to sequence regions or genomes (e.g., high GC content), native detection of modified bases or any application where ultra long reads are important (e.g., full length cDNAs). However, given their steadily improving specs, the high quality consensus sequence, and the recent launch of the Sequel, PacBio is starting to be used to generate high quality whole human genome sequences.
PacBio has been making steady improvements and will likely continue to do so in the future. They have plans for longer reads, and improving the loading and density of the ZMWs on the Sequel platform.
PacBio Specifications Table
|RS II (P6-C4)||Sequel|
|Run time||up to 240 min||up to 240 min|
|Total output||~500 Mb - 1 Gb||5 Gb - 10 Gb|
|Output/day||~2 Gb||20 Gb|
|Mean read length||10 -15 kb||10 -15 kb|
|Single pass accuracy||~86%||~86%|
|Consensus (30X) accuracy||>99.999%||>99.999%|
|# of reads||~50k||~500k|
PacBio Applications Table
|RS II (P6-C4)||Sequel|
|Human Whole Genome|
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(Images Courtesy of Pacific Biosciences of California, Inc., Menlo Park, CA, USA)