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