Recommendation 150nt single-end vs 2x75nt paired-end

atpoint · 02-28-2018, 09:00 AM

We are in the planning phase for an experiment that requires sequencing of a DNA pool of length about 140bp. It is crucial that the entire fragment is sequenced. Platform will be Nextseq500 or MiSeq, depending on what the Power Analysis will demand as sufficient read depth for what we want to do. Anyway, I would like to ask for your advice on the running mode. Should we perform 150bp single-end or 2x75bp paired-end sequencing? I ask because typically base quality decreases towards the end 3' end. Is it therefore advisable to run it paired and then merge the two reads into a consensus sequence?

Bukowski · 02-28-2018, 11:55 AM

Quote:

Originally Posted by atpoint

We are in the planning phase for an experiment that requires sequencing of a DNA pool of length about 140bp. It is crucial that the entire fragment is sequenced. Platform will be Nextseq500 or MiSeq, depending on what the Power Analysis will demand as sufficient read depth for what we want to do. Anyway, I would like to ask for your advice on the running mode. Should we perform 150bp single-end or 2x75bp paired-end sequencing? I ask because typically base quality decreases towards the end 3' end. Is it therefore advisable to run it paired and then merge the two reads into a consensus sequence?

I don't really know what your application is, but leaving yourself only 5 bp overlap for the reads at 2x75 doesn't sound like its the ideal strategy for flashing reads. How much fidelity do you need over the length? What is wrong with 2x150 anyway?

nucacidhunter · 02-28-2018, 02:00 PM

150 cycle kit have enough reagents for 175 cycles so if you are doing single index read or no index read then you can can increase paired end read length to paired end 83-84 to have enough sequence for flashing. Paired end data quality should be better then single end longer read.

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02-28-2018, 09:00 AM	#1
atpoint Junior Member Location: Muenster Join Date: Apr 2016 Posts: 5	Recommendation 150nt single-end vs 2x75nt paired-end We are in the planning phase for an experiment that requires sequencing of a DNA pool of length about 140bp. It is crucial that the entire fragment is sequenced. Platform will be Nextseq500 or MiSeq, depending on what the Power Analysis will demand as sufficient read depth for what we want to do. Anyway, I would like to ask for your advice on the running mode. Should we perform 150bp single-end or 2x75bp paired-end sequencing? I ask because typically base quality decreases towards the end 3' end. Is it therefore advisable to run it paired and then merge the two reads into a consensus sequence?

02-28-2018, 02:00 PM	#3
nucacidhunter Senior Member Location: Melbourne Join Date: Jan 2013 Posts: 1,138	150 cycle kit have enough reagents for 175 cycles so if you are doing single index read or no index read then you can can increase paired end read length to paired end 83-84 to have enough sequence for flashing. Paired end data quality should be better then single end longer read.