Amplicon Sequencing Data Analysis

Question

[t_stranger]

I am looking for a tutorial and journal of amplicon sequencing data analysis. Is there any specific free website to teach beginner the pipeline of data analysis and what should we report in our result. Any journal recommended?

Answer 1

[WouterDeCoster]

I would suggest you to follow the GATK best practices. Amplicon sequencing mainly follows the guidelines for exome sequencing/panel sequencing, with two important exceptions:

• You shouldn't mark duplicates because all your reads are duplicates
• You should softmask your primer sequences: you can't get valid variant calls in those

[t_stranger]

Thanks for your suggestion. May I know why we should not mark the duplicates before variant call? Because I read some resource they included it in the pipeline.

[WouterDeCoster]

Since you are doing amplicon sequencing all reads mapping to the same location will mimic duplicates so you will only keep one read per amplicon per direction.

[lamteva.vera]

Can one use BAMClipper instead of softmasking primers?

[WouterDeCoster]

Assuming you mean bamclipper which removes primers using soft-clipping, the answer would be yes, although I haven't tested the software I assume it works as described in their README.