Different number of cycles for HiSeq PE sequencing

Hyeshik Chang · 07-02-2013, 06:34 PM

Hello,

I am planning to run a HiSeq 2500 rapid run with a different number of cycles for reads 1 and 2 (e.g. 250 x 50). I know that this layout produces a lot worse quality than 150 x 150, however what I need more in my current project is longer and more reads in spite of poor quality reads (I'm happy with phred score 20).

Does anyone have an experience of this kind of runs?

Thank you

GenoMax · 07-03-2013, 10:43 AM

It is feasible to set up asymmetric runs. You are aware of the limitations with read qualities. At 250 cycles on read 1 you are going well beyond the officially supported spec for HiSeq 2500. Make sure you have enough reagents available.

HESmith · 07-03-2013, 01:13 PM

Another option is to size-select your libraries to have an insert size of 270bp. Then 2x150bp generates overlapping reads that can be joined together, with the added benefit of error correction in the overlapping (lower-quality) ends.

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07-02-2013, 06:34 PM	#1
Hyeshik Chang Junior Member Location: Seoul Join Date: Apr 2009 Posts: 6	Different number of cycles for HiSeq PE sequencing Hello, I am planning to run a HiSeq 2500 rapid run with a different number of cycles for reads 1 and 2 (e.g. 250 x 50). I know that this layout produces a lot worse quality than 150 x 150, however what I need more in my current project is longer and more reads in spite of poor quality reads (I'm happy with phred score 20). Does anyone have an experience of this kind of runs? Thank you

07-03-2013, 10:43 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,088	It is feasible to set up asymmetric runs. You are aware of the limitations with read qualities. At 250 cycles on read 1 you are going well beyond the officially supported spec for HiSeq 2500. Make sure you have enough reagents available. Last edited by GenoMax; 07-03-2013 at 10:46 AM.

07-03-2013, 01:13 PM	#3
HESmith Senior Member Location: Bethesda MD Join Date: Oct 2009 Posts: 509	Another option is to size-select your libraries to have an insert size of 270bp. Then 2x150bp generates overlapping reads that can be joined together, with the added benefit of error correction in the overlapping (lower-quality) ends.