When performing target enrichment of large panels of genes using a PCR based method such as Fluidigm or Raindance, primers of varying lengths are used to amplify targets. I am looking for a tool to remove the known primer sequences. I know Illumina has incorporated such a tool in the miseq reporter for truseq custom amplicons, but I have not been able to find a similar tool to incorporate into the standard CASAVA (or other) pipelines. I contacted Illumina about this, but, of course, they were no help. I have also attempted to use cutadapt, but it is not working. And, since the primer sequences are of varying length, I cannot simply remove X number of bases at the beginning of the read.
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In my pipeline for MiSeq amplicon analysis I align the reads keeping the primers on, and then just ignore the primer areas in downstream analysis. -
In some cases, there might be overlapping primers and amplicons where simply ignoring the areas wouldnt be a good idea. In those cases, trimming is still important as the variant allele frequencies might be inaccurate, but seems there are no ready to use tools out there!Originally posted by frozenlyse View Post--
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NGS has revolutionized clinical microbiology by offering new ways to identify and surveil pathogens without prior knowledge of the target. One popular approach is 16S rRNA and ITS amplicon sequencing, which targets specific microbial markers. But studies are increasingly incorporating shotgun metagenomics, a technique that sequences all DNA from a sample without targeting specific microbial markers. This approach allows more detailed taxonomic classification,...10-30-2026, 06:46 AM
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