Demultiplexing and adapter/barcode trimming from PacBio amplicon reads #847
Description
Hi there!
I'm new to bioinformatic analyis of PacBio amplicon data and while I was searching for possible tools for demultiplexing/adapter/primer trimming I was suggested to try with cutadapt.
But since we are talking about three different actions I would ask for some guidance how to perform it properly. The way the HiFi reads are oriented is the following: Barcode1-Adapter-FW_Primer-Sequence-RW_Primer-Adapter'-Barcode1'. Adapters and barcodes on both sides are different and while barcodes depend on the sample, adapters only refer to FW_ and RW_Primers.
My question is the following: Would it be possible to perform demultiplexing step by cutadapt by including both adapter and barcode as part of a single "ADAPTER" in a single step or is this not a good practices for some reason? Or would it be better to first demultiplex the samples based on the barcode and then consecutively remove adapters and primers.
It's also important to note that as emphasized in #627 not all reads are oriented the same way so I should somehow account for that as well.
I already started with demultiplexing sequences using lima tool by PacBio but now I'm wondering whether it would be the best to perform all three steps using the same software.
If you could give me some suggestions what would be the best way to handle the data in my case I would really appreciate it.
Thank you very much in advance!