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Old 03-19-2017, 08:35 AM   #1
seb42
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Default Degenerate sequencing primers

Hello,

We are using custom sequencing primers containing multiple degenerate bases for MiSeq sequencing of the V4 SSU region. We have included these degenerate bases to hit more taxa during the PCR steps. Since the degenerate bases are in the V4 primer region they are pretty close to the start of the sequenced region so might have a disproportional affect on sequencing.

We have been getting poor quality scores for multiple libraries (40-60% reads pass) and only getting between 3-5 million reads back. We were wondering if anyone else has had any trouble with this and if so how they overcame the problem.

We were thinking that due to the mix of primers the poor results could be due to low sequencing primer density. Has anyone seen results similar to ours due to low primer densities or mixing of multiple sequencing primers?

Thank you for any advice you can give.
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Old 03-19-2017, 01:04 PM   #2
barrmur
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Quote:
Originally Posted by seb42 View Post
Hello,

We are using custom sequencing primers containing multiple degenerate bases for MiSeq sequencing of the V4 SSU region. We have included these degenerate bases to hit more taxa during the PCR steps. Since the degenerate bases are in the V4 primer region they are pretty close to the start of the sequenced region so might have a disproportional affect on sequencing.

We have been getting poor quality scores for multiple libraries (40-60% reads pass) and only getting between 3-5 million reads back. We were wondering if anyone else has had any trouble with this and if so how they overcame the problem.

We were thinking that due to the mix of primers the poor results could be due to low sequencing primer density. Has anyone seen results similar to ours due to low primer densities or mixing of multiple sequencing primers?

Thank you for any advice you can give.
You should make sure the sequencing run is been carried out with about 20% PhiX in order to increase the base diversity during the run. Sounds like the low diversity is leading to a number of the clusters being removed during filtering.
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Old 03-19-2017, 11:41 PM   #3
nucacidhunter
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You have not mentioned what protocol you have used to prepare the libraries. But poor quality and low outputs are characteristics of libraries prepared based on Earth Microbiome Project protocols even though they include primer pads to increase Tm.

In my experience two step PCR following Illumina's protocol gives better results and all library fragments are sequenced using standard Illumina primers that eliminates sequencing bias caused by degenerate sequencing primers.

Edit: 20-30% PhiX spike in increases quality but output still would be lower than two step PCR method.

Last edited by nucacidhunter; 03-19-2017 at 11:44 PM.
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Old 03-20-2017, 04:55 AM   #4
seb42
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Thank you for your answers. We do have a phiX spike in our runs and we are using a modified version of the Earth Microbiome Project. Our primers are a bit different than the ones mentioned in that protocol however.
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Old 03-20-2017, 04:03 PM   #5
nucacidhunter
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This is the limitation of using fusion primers. Using gel purified primers for both PCR steps and sequencing will improve performance by minimizing miss-synthesized oligos containing indels and mismatches.
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Old 03-21-2017, 07:30 AM   #6
thermophile
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check the tm of your primers. The reverse emp primer is too cool (62-65, should be min 65). If your degeneracies are even cooler you will likely not get good annealing. I've had decent luck with locking nucleotide sequencing primers from http://www.exiqon.com/lna-technology to raise the annealing temp of the custom sequencing primers.
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Old 03-21-2017, 09:12 PM   #7
nucacidhunter
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I think in addition to Tm, sequencing primers has to be full length because any truncation from 3 end will affect phasing/prephasing and therefore quality and PF%.
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