Pacific Biosciences of California Inc (PACB)

[Paulieme]

Longing for the longest reads: PacBio and BluePippin
Posted on June 19, 2013 by flxlex
PacBio sequencing is all about looong reads, especially in relation to de novo sequencing. A few things are needed to get the longest reads possible:

•the longer the enzyme is active on the template, the longer the raw read will be – PacBio calls these ‘Polymerase reads’
•a library for pacBio sequencing consist of circular molecules, with the target insert between two hairpin adaptors, allowing the enzyme to ‘go around’ and sequence the opposite strand once it reaches the end of the insert. See my previous post on this here. It then follows that the longer the template used for library preparation, the smaller the chance the polymerase goes around the hairpin, leading to longer uniquely sequenced template – PacBio calles these ‘reads of insert’ – and these represent the most useful reads for de novo sequencing applications
•finally, the distribution of sizes of the library has an influence: any high-throughput sequencing technology, as well as PCR, has problems with ‘preferential treatment’ of smaller fragments. With PacBio sequencing, shorter molecules tend to load preferentially into the wells of the SMRTCell (‘chip’). It then makes sense to try to reduce the shoulder of shorter fragments for the final library preparation.
Recently, PacBio and Sage Science announced a co-marketing partnership for the BluePippin. This instrument allows for tight size selection of DNA samples, effectively making the peak of the size distribution much more narrow. With regard to PacBio sequencing, a narrow peak lessens the problem of preferential loading of short fragments, leading to much longer ‘reads of insert’. A demonstration can be seen on this poster (I think I know which fish they used for that one plot…).


It’s nice when a company demonstrates a new improvement to their technology. But, the proof is always in the pudding, in this case, can the sequencing centres out there actually show the same results? Based on recent tweets from the PacBio USA User group meeting, one would believe so:
The Norwegian Sequencing Centre, with which I am affiliated, recently bought a BluePippin instrument for the reasons outlined above. Our first test was on a good, 10 kb insert PacBio (SMRTBell) library that we already had used for sequencing. The (excellent!) lab team ran a (large) sample of the library on the BluePippin instrument and showed a significant reduction of the small fragments:
(see link for chart) Note that we didn’t try to remove the larger fragments (which you would probably do for mate pair libraries) because in the case of PacBio library preparation, length is more important than a tight distribution per se. The next question was what the effect is of such a cleanup has on sequencing. We ran both libraries on our PacBio instrument with C2XL chemistry and 120 minute movies. First some numbers. Please note that the regular library data was obtained from our PacBio RS before maintenance and upgrade to RSII (doubling of the laser capacity), and this may have had a lsight negative effect on the read lengths. There were also more reads included for the ‘before’ data, and these were analysed with version 1.4 of the PacBio software versus 2.0 for the BluePippin library.

Library # of reads Ave. length Longest subread N50* Longest
Regular library 693,275 3,009 bp 4,041 bp 22,298 bp
BluePippin library 325,407 6,045 bp 8,820 bp 25,931 bp

*N50 is a metric often used for genome assemblies, and here translates to ‘the length at which half the bases of the entire dataset is in reads of at least length N50'.

The following graph shows the distribution of the longest subreads (or, reads of insert) for each raw read produced, for the library before, and after BluePippin cleanup.

FOR MORE GO TO THIS LINK http://flxlexblog.wordpress.com/2013/06/19/longing-for-the-longest-reads-pacbio-and-bluepippin/