paired-end RNA-Seq of short transcripts

fbarreto · 11-23-2011, 11:19 AM

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!

pmiguel · 11-23-2011, 11:39 AM

Nearly all RNAseq protocols fragment either the RNA or cDNA during library preparation. So long transcripts should show up.

--
Phillip

fbarreto · 11-23-2011, 11:52 AM

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.

pmiguel · 11-23-2011, 12:43 PM

Sorry, misread you. You are correct, if you ask for 180bp+ inserts, then any transcript smaller than that will be lost.

--
Phillip

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11-23-2011, 11:19 AM	#1
fbarreto Member Location: San Diego Join Date: Jan 2010 Posts: 15	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!

11-23-2011, 11:39 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Nearly all RNAseq protocols fragment either the RNA or cDNA during library preparation. So long transcripts should show up. -- Phillip

11-23-2011, 11:52 AM	#3
fbarreto Member Location: San Diego Join Date: Jan 2010 Posts: 15	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.

11-23-2011, 12:43 PM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Sorry, misread you. You are correct, if you ask for 180bp+ inserts, then any transcript smaller than that will be lost. -- Phillip