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Old 11-28-2012, 12:37 PM   #1
onconaut
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Location: South Central US

Join Date: Aug 2012
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Default ds cDNA fragmentation with Covaris

Hello Seqers,
I am preparing libraries for a 2x100nt sequencing run for mRNA-seq on a HiSeq2000. We are using the TruSeq mRNA protocol. Illumina suggests chemical fragmentation, but offers usltrasonication with Covaris as an alternative. We have tried their suggested settings and "titrated" them and we have tried settings suggested by Covaris. When we analyze those libraries on a bioanalyzer the Covaris fragmentation gives me a nicer peak and a tighter distribution. The peaks range from 280bp to 320bp (corresponding to fragment inserts of approx. 160 and 200bp respectively).

I am trying to generate larger fragments of about 250-300bp (final library size 370 - 420 bp) in order to avoid/minimize the overlap between the paired end reads, but I am somehow stuck and seem to not be able to increase my fragment size peak. Are any of you preparing libraries for 2x100bp reads?
If so what fragment size are you aiming at and how do you fragment?
Does anyone have settings for the Covaris S2 or S220 that she/he would be willing to share?
Thanks, O.
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Old 11-28-2012, 01:11 PM   #2
kwaraska
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Location: Boston,MA

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Default

Covaris has a paper on their website on how to get certain fragment sizes. I printed it out or I would send an e-copy, but if you go to www.covarisinc.com they will be able to guide you.
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