Y-adapters better for low diversity library

nrobic · 10-08-2015, 10:32 AM

I have had a problem with base calling due to a low diversity library on Miseq. I used P5 and P7 adapters with two amplification steps. The library is a PCR product. Would Y adapters be a better option, or am I not on the right track.

microgirl123 · 10-08-2015, 11:54 AM

Your adapters probably aren't the problem, it's just the fact that it's a low diversity library. What % phiX did you spike in and what was your cluster density?

luc · 10-08-2015, 03:55 PM

Y-adapters could indeed be better (by 50%; because there will be no P5toP5 and no P7toP7 ligations) but they are unlikely to be the problem, in my eyes.
We do not know anything about your current adapters and objectives though.

nucacidhunter · 10-08-2015, 11:27 PM

Quote:

Originally Posted by nrobic

I have had a problem with base calling due to a low diversity library on Miseq. I used P5 and P7 adapters with two amplification steps. The library is a PCR product. Would Y adapters be a better option, or am I not on the right track.

With your approach, using adapters is not possible. There are some options to increase diversity of amplicons:
1- Sequencing pool of different amplicons
2- Spiking in a high diversity library
3- increasing amplicons library diversity by adding 0-3 diversity nucleotides to 5' end of target specific sequences of primers (3' to adapter overhang)

fanli · 10-09-2015, 10:43 AM

Also loading a lower concentration (ie lower cluster density) will help

nrobic · 10-13-2015, 04:36 AM

My first experiment with Y-adapters shows the base caller problem to be minimal as evaluated by the read 1-2 base intensity graph in Illumina SAV. The variant caller also shows a great reduction in false variants. I still need to load my control for comparison and repeat this finding again - Thanks

nrobic · 10-13-2015, 04:44 AM

I went as high as 20% with minimal improvement

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10-08-2015, 10:32 AM	#1
nrobic Junior Member Location: MA Join Date: Mar 2015 Posts: 4	Y-adapters better for low diversity library I have had a problem with base calling due to a low diversity library on Miseq. I used P5 and P7 adapters with two amplification steps. The library is a PCR product. Would Y adapters be a better option, or am I not on the right track.

10-08-2015, 03:55 PM	#3
luc Senior Member Location: US Join Date: Dec 2010 Posts: 453	Y-adapters could indeed be better (by 50%; because there will be no P5toP5 and no P7toP7 ligations) but they are unlikely to be the problem, in my eyes. We do not know anything about your current adapters and objectives though. Last edited by luc; 10-08-2015 at 06:09 PM.

10-13-2015, 04:44 AM	#7
nrobic Junior Member Location: MA Join Date: Mar 2015 Posts: 4	PhiX I went as high as 20% with minimal improvement

10-08-2015, 11:54 AM	#2
microgirl123 Senior Member Location: New England Join Date: Jun 2012 Posts: 200	Your adapters probably aren't the problem, it's just the fact that it's a low diversity library. What % phiX did you spike in and what was your cluster density?

10-09-2015, 10:43 AM	#5
fanli Senior Member Location: California Join Date: Jul 2014 Posts: 198	Also loading a lower concentration (ie lower cluster density) will help

10-13-2015, 04:36 AM	#6
nrobic Junior Member Location: MA Join Date: Mar 2015 Posts: 4	My first experiment with Y-adapters shows the base caller problem to be minimal as evaluated by the read 1-2 base intensity graph in Illumina SAV. The variant caller also shows a great reduction in false variants. I still need to load my control for comparison and repeat this finding again - Thanks