I recently have amplicons sequenced (Illumina PE250) to investigate microbial community. I want to know what quality detection, and what kind of trims of raw reads by what programs should be performed before joining paired-end raw reads? I think low-quality parts, potential barcode or primer sequences existed in raw reads should be excluded in advance. Is this right?
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Yongjie Zhang
Here is a good example to start: http://www.mothur.org/wiki/MiSeq_SOP
Very useful link. Thanks. I found the join_paired_ends.py in Qiime is not very ideal to my data. Mothur might be better.
You could use SeqPrep for this. see https://github.com/jstjohn/SeqPrep or my fork https://github.com/dakl/SeqPrep which adds two parameters for more stringent handling of mismatches (set to N, essentially).
You should be able to find everything you need for removing adapters, splitting the sample based on barcodes and joining the pairs with a combination of the tools in the fastx_toolkit and ea-utils.
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I finally merged reads pairs using usearch -fastq_mergepairs with -fastq_allowmergestagger enabled, but before merging, I cut primer/adapters using CutAdapt and trimed low-quality regions from ends of reads using Trimmomatric, and after merging, I cut potential primer/adapter again by CutAdapt and filtered reads with >0.5 expected errors by usearch -fastq_filter. Then I can do downstream analyses.