Confusion about single end vs paired end read output on Illumina HiSeq

acidcoated · 03-19-2015, 09:44 AM

Hello,

I think I'm confused about read output for Illumina's HiSeq. I think I'm not understanding why it is possible to get 150M single end reads in a run, but you can get 300M pairs (is this double the data)? So what is the maximum number of TOTAL reads you can get (either on rapid-run or high-output).
I'm trying to understand multiplexing better. Because multiplexing is based on how many reads you desire, you determine how many libraries you can load in a single lane based on the max output (~140-150M reads) of a lane.
So say I want 20M single end reads, I can load 6 libraries safely with some buffer room and reach 20M reads per library. But if you want paired end reads, how does this work say if I want 20M pairs (40M total) per library; can I still load 6 libraries and reach that output? Is this purely based on maximum data that can be produced or on the chemistry of the SBS reagents in single end vs paired end mode?

Any resources are greatly appreciated. Thanks!

GenoMax · 03-19-2015, 09:56 AM

Unique clusters is what you need to consider.

If you sequence only one end then you get 150M reads but if you sequence the other then you get 300M reads. Each unique cluster can generate n x 2 number of reads. Illumina tends to quote number of paired end reads in their specifications (so that corresponds to half the number of unique clusters being sequenced).

In example above you don't change the loading concentration but just sequence the other end of the fragment to get a pair of reads for each cluster.

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03-19-2015, 09:44 AM	#1
acidcoated Junior Member Location: Chicago, IL Join Date: Jun 2014 Posts: 3	Confusion about single end vs paired end read output on Illumina HiSeq Hello, I think I'm confused about read output for Illumina's HiSeq. I think I'm not understanding why it is possible to get 150M single end reads in a run, but you can get 300M pairs (is this double the data)? So what is the maximum number of TOTAL reads you can get (either on rapid-run or high-output). I'm trying to understand multiplexing better. Because multiplexing is based on how many reads you desire, you determine how many libraries you can load in a single lane based on the max output (~140-150M reads) of a lane. So say I want 20M single end reads, I can load 6 libraries safely with some buffer room and reach 20M reads per library. But if you want paired end reads, how does this work say if I want 20M pairs (40M total) per library; can I still load 6 libraries and reach that output? Is this purely based on maximum data that can be produced or on the chemistry of the SBS reagents in single end vs paired end mode? Any resources are greatly appreciated. Thanks!

03-19-2015, 09:56 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	Unique clusters is what you need to consider. If you sequence only one end then you get 150M reads but if you sequence the other then you get 300M reads. Each unique cluster can generate n x 2 number of reads. Illumina tends to quote number of paired end reads in their specifications (so that corresponds to half the number of unique clusters being sequenced). In example above you don't change the loading concentration but just sequence the other end of the fragment to get a pair of reads for each cluster.