Modified seq Illumina Primers for ddRAD

JQ.ACGT · 04-13-2018, 05:35 AM

Hello, my ddRAD libraries (mean 550 pb) contain the first 5 identical bases at their ends, belonging to the partial restriction site. They will be paired-end sequenced in a MiSeq. I would like to modify the Illumina sequencing primers by adding those 5 bases at the 3 prime end of each primer. Thus, a higher diversity is expected in the first sequenced bases and also an adequate cluster generation. Does anyone have experience with this approach? and the use or not, and its PhiX concentration. Thank you. Best regards.

luc · 04-13-2018, 07:01 PM

It could work - however you will not be able to sue the PhiX libraries as spike in since you will can't use the Illumina sequencing primers together with your extended versions.

JQ.ACGT · 04-15-2018, 03:10 AM

Thank you. It is right. So the question is what can work best. 1) Use modified primers to bypass the restriction site or 2) use a high concentration of PhiX (how much?).

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04-13-2018, 05:35 AM	#1
JQ.ACGT Junior Member Location: Spain Join Date: Mar 2018 Posts: 2	Modified seq Illumina Primers for ddRAD Hello, my ddRAD libraries (mean 550 pb) contain the first 5 identical bases at their ends, belonging to the partial restriction site. They will be paired-end sequenced in a MiSeq. I would like to modify the Illumina sequencing primers by adding those 5 bases at the 3 prime end of each primer. Thus, a higher diversity is expected in the first sequenced bases and also an adequate cluster generation. Does anyone have experience with this approach? and the use or not, and its PhiX concentration. Thank you. Best regards.

04-13-2018, 07:01 PM	#2
luc Senior Member Location: US Join Date: Dec 2010 Posts: 360	It could work - however you will not be able to sue the PhiX libraries as spike in since you will can't use the Illumina sequencing primers together with your extended versions.

04-15-2018, 03:10 AM	#3
JQ.ACGT Junior Member Location: Spain Join Date: Mar 2018 Posts: 2	Thank you. It is right. So the question is what can work best. 1) Use modified primers to bypass the restriction site or 2) use a high concentration of PhiX (how much?).