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Old 04-09-2017, 10:37 AM   #1
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Location: St. Louis, MO

Join Date: Apr 2017
Posts: 1
Default Overcoming PCR Primer Bias with MiSeq?

Hi, I am a grad student new to MiSeq and HTS in general. I'm trying to figure out which sequencing kit/method to use for my project, and I'm getting some mixed responses. I have spoken to 5-6 people on the matter (some of whom work at Illumina), and both TruSeq Custom Amplicon sequencing and Nextera XT have been suggested - I'm just trying to figure out the best way to go about this, since my budget is limited.

Background: I am trying to detect co-infections of malaria strains in host DNA samples. There are 5 lineages/strains that I am looking for, all of which we have unique sequence fragments for from PCR/Sanger methods.

Issue: The primers and PCR/Sanger sequencing protocol we have are great for amplifying and identifying the dominant lineage present, but the issue is that the primers are biased - they will amplify some lineages over others for various reasons. We want to study co-infections of multiple lineages in a single sample, but our PCR/Sanger methods cannot determine co-infection due to that bias.

The sequences we have are all from the malaria cyt-b gene (mtDNA), fragments are about 500-550 bp long, and the sequences are fairly conserved between lineages - they average about 10 nucleotides different from each other. As such, I can't develop lineage-specific primers for PCR, as the sequences are too similar.

Goal: To use a high-throughput technique to target that same region (so that we can use our lab database to identify the lineages). I had thought to use a
generic malaria probe (one that will bind with any malaria present, but not with any host DNA), but I need to ensure somehow that the same primer bias described before won't negatively affect results. I would think that would be remedied by read depth, but I admit that I am new to HTS-related techniques so I'm not sure.

I had been considering TruSeq Custom Amplicon sequencing, but because my target area is so short (~600 bp), that method would probably not be cost effective. Can anyone give any advice on this? Thanks.
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