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Old 11-23-2011, 11:19 AM   #1
fbarreto
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Default paired-end RNA-Seq of short transcripts

Hi,

We're planning to start getting ours hands in some of this next-gen stuff, and I have a basic question regarding the paired-end (PE) method for sequencing and mapping in an RNA-Seq experiment.

If my library is constructed for 100bp PE, with ~180-200bp insert, does that mean that only transcripts that are >380 bp are getting sequenced? In other words, if a transcript is only 100 bp long, does it get excluded from library construction because it doesn't fall into the size range?

Pardon my ignorance if this should be obvious. I'm assuming the smart people designing these methods have accounted for this issue, but I don't understand how quite yet.

Thanks for any insight!
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Old 11-23-2011, 11:39 AM   #2
pmiguel
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Nearly all RNAseq protocols fragment either the RNA or cDNA during library preparation. So long transcripts should show up.

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Old 11-23-2011, 11:52 AM   #3
fbarreto
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I understand how long transcripts show up. I'm curious as to how very short transcripts get sequenced through this method (if they do), since some of my genes of interest have short transcripts and I don't want them excluded.
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Old 11-23-2011, 12:43 PM   #4
pmiguel
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Sorry, misread you. You are correct, if you ask for 180bp+ inserts, then any transcript smaller than that will be lost.

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