Pacific Biosciences of California Inc (PACB)

[Paulieme]

Long,but a good read on PACB-1) CCS for sequencing full-length 16S rRNA
Before the days of Next Generation Sequencing, people used to amplify 16S regions of samples and make bacterial clone libraries, sequencing a set of clones using Sanger sequencing. NGS allowed for many more reads per sample, but restricted the length of the sequenced part to a few hundred bases at best. NGS therefore yielded much deeper sequenced datasets, but at a lower discriminatory power (less phylogenetic/taxonomic signal).
The long CCS reads now possible with PacBio imply that one could consider going back to full-length 16S sequencing. The throughput (40 000 reads per SMRTCell) will be much higher than doable using Sanger sequencing, and the quality potentially even better than Sanger. However, the price per read will be significantly higher than using short-read technology. I think that for certain diversity studies, using PacBio CCS with full-length 16S amplicons will be very beneficial.

2) CCS for shotgun metagenomics
Similarly, people interested in whole-sample shotgun metagenomics (as opposed to PCR-based diversity studies) could consider using the 1-2 kbp CCS reads. The long reads could yield much more useful information for gene mining, for example. Some may suggest that using the Roche/454 GS FLX+, which now seems to be working – at least in our lab it does – will yield much more reads (1 to 1.2 million) around that length (we have seen 1kb mode read lengths with GS FLX+!), making this technology more cost-effective. Some back-of-the-envelope calculations using prices for the Norwegian Sequencing Centre show that the comparison actually is in favour of PacBio. Given one library preparation, and 1 million required reads, the 454 is currently out-priced by PacBio, while the latter has the potential to give better quality (no homopolymer errors) and longer reads. However, first, generating 1 million CCS reads (25 SMRTCells) takes more time (several days, not counting library preparation) than a full 454 run (about a day) [Note that lab teams will like not having to do emulsion PCR for PacBio CCS!]. Secondly, the pricing situation may be different for other centres (pricing structures I think really differ from centre to centre).

3) CCS to replace Sanger capillary plate sequencing
Sanger sequencing is far from dead. Its main attraction is that it allows very small sample sizes (down to a single sample can be submitted to a facility), and long reads with high quality. A key difference between Sanger and NGS is the fact that each read can be traced back to a well on a plate. I think that, if a suitable and cost-effective barcoding scheme could be designed for multiplexed PacBio CCS sequencing, PacBio CCS could potentially replace Sanger plate sequencing. To keep the costs down, one would need to massively multiplex, perhaps dozens of 96 well plates with fragments that need to be tracked back to their original plate and well. But a laboratory with good automation experience might pull it off. At the same time, the scheme requires a steady flow of Sanger samples. It wouldn’t work for a facility that sequences less then, say, a dozen plates per week. Commercial providers may actually already consider doing this switch. The benefits could be longer reads than one can get with Sanger, with higher per-base qualities.

[Technical note: the per-SMRTCell throughput of 40 000 reads may allow for adding the same template multiple times, increasing the final consensus accuracy. The fraction of raw reads too short to give a consensus call (see above) may actually contribute to quality as well as they are barcoded]
In summary: PacBio CCS may be an alternative for short read sequencing, or even Sanger Capillary sequencing. However, there will be a trade-off in information content, versus price per read.

For a technical, but very readable paper describing CCS (from the PacBio researchers themselves), see this paper in Nucleic Acids Research.

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