These days, we’re all about multi-tasking—talking on our cell phones while placing our cappuccino order at Starbucks and tweeting about it all on our tablets. In that same vein, researchers report an optimized protocol for multi-tasking (well, technically “multiplexing”) reduced representation bisulfite sequencing (RRBS) for ramped-up DNA methylation data.
RRBS is great, churning out methylation info at a reduced cost compared to whole-genome methylation sequencing. But with the increased capacity on Illumina sequencing platforms (now up to ~200 million reads per lane) comes the ability to seriously multiplex RRBS sequencing for even more cost-effectiveness. And in this cash-strapped economy, who wouldn’t want that?
But RRBS poses some rather unique challenges to multiplexing, so this team from New Zealand picked apart the TruSeq Sample Preparation protocol and optimized it so that the whole process goes smoothly and efficiently. Here’s some of what they did:
- They improved strategies for RRBS library preparation, including a move away from two rounds of bisulfite conversion with Qiagen’s Epitect system to one round (but longer incubation) with Zymo’s bisulfite kits.
- They also optimized the PCR amplification step in library consruction which wasn’t performing optimally on bisulfite converted template. They used Agilent’s PfuTurbo Cx DNA polymerase instead
- They developed a bioinformatics pipeline that increased the output and improved data quality.
Now that they’ve improved multiplexed RRBS, the team warns that the next challenge is for molecular biologists to keep up with all the volumes of data they can now generate!
Put down that coffee and focus on the details at the Journal of Biomedicine and Biotechnology, September 2012.