Cycle Transferring Question (uneven ends instead of 2x150)

Danner · 08-07-2020, 03:48 AM

Hey All,
I have increased the length of index runs and decreased the length of the main reads. There is an interfance to say how long you want each read to go for. So in principle instead of 2x150 you could do one of the ends with 300 reads as there is enough reagents. However I assume the quality goes down.

I am using a miniSeq or HiSeq. I normally would use kits that are 2x150. However on one of my ends the primer had to be further away then I would like. Could I get Read1 to go 200 cycles and read2 to go 100 cycles? I know that I can program it in, but what problems may I encounter? Would the read quality fall and I end up with NNNN for the last 50 cycles? I know that this happens if you try and go 400 cycles on one end with MiSeq.

Thanks!

GenoMax · 08-07-2020, 05:44 AM

You should be able to do asymmetric runs like that. But wait for @luc or one of the experimental people to confirm for certain.

As long as there is sequenceable material left there should be no NNN calls. Quality will suffer like you expect. My assumption is you got NNN calls in your prior run because you read through the adapter on other end and kept on going.

itstrieu · 08-07-2020, 06:47 AM

While not asymmetric, we did a 2x300 run on a 500 cycle kit by accident and read 1 looked good but read 2 quality dropped after 230 cycles due to reagents starting to run out.

I don't see any issue with asymmetric runs other than Q30 scores dropping the longer the read length. Especially if it is >300 cycle on the PE side.

HESmith · 08-07-2020, 06:59 AM

Yes, you can perform asymmetric runs - it's routine for single-cell RNA-Seq. Quality will decline with read length, and (in our experience) drops to unacceptable levels when you exceed the recommended maximum (e.g., 150bp on HiSeq 4000). Better to design a custom primer that's closer to region you wish to sequence (assuming that's an option).

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08-07-2020, 03:48 AM	#1
Danner Junior Member Location: Berlin, Germany Join Date: Aug 2020 Posts: 1	Cycle Transferring Question (uneven ends instead of 2x150) Hey All, I have increased the length of index runs and decreased the length of the main reads. There is an interfance to say how long you want each read to go for. So in principle instead of 2x150 you could do one of the ends with 300 reads as there is enough reagents. However I assume the quality goes down. I am using a miniSeq or HiSeq. I normally would use kits that are 2x150. However on one of my ends the primer had to be further away then I would like. Could I get Read1 to go 200 cycles and read2 to go 100 cycles? I know that I can program it in, but what problems may I encounter? Would the read quality fall and I end up with NNNN for the last 50 cycles? I know that this happens if you try and go 400 cycles on one end with MiSeq. Thanks!

08-07-2020, 05:44 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	You should be able to do asymmetric runs like that. But wait for @luc or one of the experimental people to confirm for certain. As long as there is sequenceable material left there should be no NNN calls. Quality will suffer like you expect. My assumption is you got NNN calls in your prior run because you read through the adapter on other end and kept on going.

08-07-2020, 06:47 AM	#3
itstrieu Member Location: Atlanta Join Date: Nov 2018 Posts: 27	While not asymmetric, we did a 2x300 run on a 500 cycle kit by accident and read 1 looked good but read 2 quality dropped after 230 cycles due to reagents starting to run out. I don't see any issue with asymmetric runs other than Q30 scores dropping the longer the read length. Especially if it is >300 cycle on the PE side.

08-07-2020, 06:59 AM	#4
HESmith Senior Member Location: Bethesda MD Join Date: Oct 2009 Posts: 509	Yes, you can perform asymmetric runs - it's routine for single-cell RNA-Seq. Quality will decline with read length, and (in our experience) drops to unacceptable levels when you exceed the recommended maximum (e.g., 150bp on HiSeq 4000). Better to design a custom primer that's closer to region you wish to sequence (assuming that's an option).