Two reverse primers

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manoj kumar

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Apr 5, 2016, 11:19:52 AM4/5/16
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Hi,

I used two reverse primers with one base pair change to do my sequence analysis (Primer 1 = ADNGCCATCATYTCNCC, Primer 2 = ANDGCCATCATYTCNCC). While trimming with split library command I can only include one reverse primer in my mapping file. So I decide to add the command of reverse primer mismatch "--reverse_primer_mismatches 1" to complement one base pair change in my reverse primer. I just wanted to know what I did was correct. 

Kindly advise.

Cheers,
Manoj

jonsan

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Apr 5, 2016, 11:58:12 AM4/5/16
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Hi Manoj,

That sounds to me like it should work; however, it might have some complicated unintended consequences, as sequences amplified with the listed primer will tolerate one bp mismatch anywhere else in the primer, while those amplified with the alternative primer will not; so, on balance, you might observe a bias in the quality between sequences.

An alternative that might avoid that problem would be to combine those two primers using additional degeneracy where they differ. That would look like:

Primer 1 = ADNGCCATCATYTCNCC
Primer 2 = ANDGCCATCATYTCNCC
Primer X = ANNGCCATCATYTCNCC

Because the IUPAC nucleotide code 'N' can match any base, including two Ns will permit sequences amplified with both primers to match even if you specify 0 primer mismatches. 

Hope this helps!
-jon

manoj kumar

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Apr 5, 2016, 1:00:17 PM4/5/16
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Hi Jon,

I hope that will also work and will give few errors. Thanks.

Cheers,
Manoj
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