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Old 05-07-2018, 02:09 AM   #1
SDH
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Location: Western Australia

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Default Extract 'amplicon' based on custom primers

Argh – first time post as in depth searching here normally answers all my questions… Anyway, I am undertaking targeted amplicon sequencing using a modification of the Illumina 16S workflow. Rather than utilising 16S primers however I have a number of custom primers targeting specific genes. They are run as a multiplex and the amplicons are barcoded with dual indexes post-PCR. Therefore after merging and demultiplexing (based on illumina indexes) I am left with a number of reads pertaining to the original sample. I however then need to separate further based on the custom primers to determine the number of reads pertaining to each individual PCR from within the multiplex (and ideally the primers removed) ie I am left with the amplicon sequence. To compound the issue the amplicon sizes vary.

This can be achieved in principle using Geneious via the trim primers function where reads can be annotated based on the presence of a custom oligo and then extracted. This however is not practical for a high-throughput workflow. With my limited knowledge I have also written a script to handle this however it is really pushing the limits of bash.

Is there a program that that can handle this data? It may well be obvious but I am at a loss. I have unsuccessfully trialled Mothur and BBmap’s ./msa.sh function in combination with ./cutprimers.sh however this is not stringent enough (I want exact f/r primer matches only). I believe the extract_barcodes.py function in QIIME1 may work however I cannot get QIIME1 to install properly with the new miniconda version. QIIME2 at this stage does not appear to have this function.

Any help is most appreciated.
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Old 05-07-2018, 02:58 AM   #2
GenoMax
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I believe that Geneious may actually be using `bbduk.sh` from BBMap internally. Have you tried that program?
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Old 05-07-2018, 08:38 AM   #3
SDH
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Thanks GenoMax - I reinvestigated bbduk and have been able to get it working. I'm not sure why I wrote it off in the first place...I hope it wasn't because I was using 'out=' instead of 'outm='...I should never have doubted the bbmap suite.

Cheers!
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Old 05-07-2018, 08:57 AM   #4
GenoMax
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Good to know that bbduk worked. When you want to collect something that matches outm= is the way to go.
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