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Old 08-19-2016, 01:12 PM   #7
giderk
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Location: Saint Louis, MO

Join Date: Apr 2015
Posts: 12
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I haven't run the MiSeq yet, but I will soon. My funding for re-runs in the event of failures is really limited, so I am trying to weigh my options.

In response to kerplunk412, I didn't think that extending primers into the P5 and P7 was an option because I have 16 different P5 indices and 24 different P7s. Wouldn't the sites be too variable to be good priming sites?

After speaking with Illumina support some more, they seem least confident in lowering the annealing temperature. It seems my best option is to extend the sequencing primers into my amplicon to increase the Tm, but there is a loss of coverage when I do this. I am now thinking that I will create a couple different sequencing primers and mix them. This won't recover all the diversity I was hoping to, but it will catch some taxonomic groups that I really didn't want to miss. I'm now wondering what the best way to combine sequencing primers is. One of my sequencing primers has a decent amount of degeneracy, the other does not. It makes sense to me that I should calculate the proportion of each sequencing primer variant (resulting from a degenerate primer) and combine my non-degenerate primer in equal proportion with all the variants. Does that make sense to anyone out there?

Thanks already for all the response to this thread.
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