RE: Geeting from Ray L

[RayLangley]

Just wanted to say Hi. I have been working with Next-gen sequencing since 2008 as a post doc at NCGR in Santa Fe. I am currently and ARS at LRRI and an ARSe in life ;-). I have numerous pubs with analysis, bioinformatics and hopefully a couple of nice clinical sepsis data using metabolomics, proteomics and transcriptomic analysis. I am a very mid co-author on the STM carrier-screening diagnostic performed for Batten's Foundation. Most of the lab work was done by Darrell Dinwiddie. I developed a batch processing protocol at NCGR for maize samples, and I alpha tested the early TruSeq kit last year with Jim Huntely at NCGR that was presented at AGBT (yeah - those results sucked, but we learned a lot and the new TruSeq kits seem great). I have some experience with FFPE and the truseq lib prep. I am working on fibrosis and next gen models along with other systems analyses. I am very interested in DSN, FFPE, and transcriptomic analysis. Submitted my first K99 this year and hoping for the best.

Happy to discuss any of what I learned as well as gather advice.

Thanks, Ray

[upendra_35]

Hey Ray i have a question regarding TruSeq kit. Did you see any biases with TruSeq kit compared to old mRNA 8 sample preparation kit?

[RayLangley]

I have not seen any differences. The new kit is a lot easier to use than the old kits.

Since we are not using gel selection any more my average peak is around 300 BP. You get a lagging tail that I saw up to 1000 bases. Idealised libs are usually between 200 and 400 BP.

With poly A selection you are going to get some 3' bias. If that is a concern data from Shujun suggests that the DSN protocol gives much more even distribution across the transcript. However, that protocol is a bit tricky to utilise and Illumina won't guarantee the protocol with TruSeq. I haven't seen any issues - but I am still waiting for the sequence results.

I haven't heard about sequence bias with the MP adaptors that was an issue with the older kits. But I am not the best person to ask in regards to that.

Hope that helps,

Ray