Long-read Sequencing on the PacBio RSII

Ever wanted to do long-read sequencing of genomic material?  Maybe you’ve needed a closed microbial genome or full transcript length sequences to identify isoforms.  Well never fear – now you can do all that and more.  At the beginning of November Research and Testing Laboratory brought in-house PacBio sequencing online.  Although RTL has offered PacBio Sequencing for over a year, up until now we were forced to subcontract that service to another lab.  This led to significant delays and higher costs.  However, now that we have the machine in house we expect to be able to provide high quality sequences in a fraction of the time.

If you aren’t familiar with PacBio that’s ok – it isn’t a very common sequencing technology.  However, its utility is starting to become more apparent and the number of researchers who use it is increasing.  The benefits of PacBio are it allows for long reads (10kb or more) that easily read through stretches of the genome that other technologies struggle to get through (long repeats, high CG regions).  This data allows researchers to close contigs that wouldn’t normally be easy to close.  In addition since most PacBio libraries are created without amplification they still have the methylation information included on the DNA – which can be recovered without expensive, labor intensive steps.  Of course this is mainly cost effective for smaller genomes (bacteria and some fungi) as the coverage required for higher eukaryotes is somewhat restrictive.

If you are planning on doing a PacBio run here are a few things to keep in mind.  First, the best input material is high quality intact DNA.  It doesn’t make a lot of sense to use highly fragmented DNA if you are trying to get long reads.  Second, it takes a lot of DNA to make a library.  Libraries can be made with as little as 1ug but you will have lower success.  Standard amounts are 5ug-10ug of DNA.  Third, samples are sequenced on SMRT cells.  Each SMRT cell generates about 50k reads, with an average read length of 10kb.  There will be smaller fragments, but there will also be much longer fragments (80kb is possible).  Fourth, for smaller bacterial genomes you can generally close the genome with a single SMRT cell.  For eukaryotic genomes you will need 20-200 SMRT cells to close a genome.  Many people will generate hybrid genomes, using PacBio data to build a 5-10x scaffold and underlying that with 50-80x Ilumina data.

It is also possible to do amplicon sequencing on the PacBio using fusion primers.  This is great if you are looking at a long amplicon (1kb-10kb) and want to get it in a single read – however because the through put of the PacBio is lower than other machines it is more expensive per read.

One last thing – in the past PacBio has had a bad reputation for generating low quality reads.  Although this is still the case if you look at individual reads– the consensus reads that are able to be bioinformatically processed can in many cases have much higher Q-scores than any other chemistry.  So if you are interested in long reads but are worried about the quality of PacBio (or if you just want more information on setting up an order) feel free to contact us for more information and a more technical explanation.  As always the best way to get a hold of us is through info@rtlgenomics.com

Let’s get to sequencing!