I have a question about differences in read count for Illumina MISeq data. I am attempting to sequence a highly polymorphic duplicated gene family (MHC) using amplicon sequencing. I have 5 total exons (amplicons) that are generated by first using PCR and then sent for amplicon sequencing. I am using the same PCR program (different primers, though) for each individual locus. Upon receiving my first round of sequences back, I am noticing that I have a substantially higher number of reads for certain exons when compared to others. Before sending for sequencing, PCR products were visualized using an agarose gel and all bands looked about equal in terms of brightness and were all the expected product size for each locus. In case it's of use, my amplicons are typically 200-400 bp in length.
Can anyone offer any troubleshooting advice? We originally used Taq polymerase and currently are looking to switch to a higher fidelity polymerase as our next step. Would appreciate any help!
Any normalization of the individual libraries before sequencing?
It may be best to post this question over at SeqAnswers.com.
That said, are the libraries being made after pooling the amplicons? You may want to make the libraries independently and then pool them. Smaller fragments always cluster better so if your amplicons are not all of same/similar size then you would need to account for this in your pooling/prep.