removing primer sequences from overlapping amplicons

eeyun · 06-02-2013, 11:52 AM

Hi all -

I'm wondering what methods you'd recommend to remove primer sequences from amplicon sequencing data where multiple amplicons for the same specimen in the same run will overlap. Simply removing the primers using one of the trimming tools seems like it will appropriately remove the primers for the amplicon they are producing, but also any neighboring sequence in an adjacent amplicon which overlaps the sequence.

One strategy I had thought might work is to align the reads to each amplcon sequence separately, and process them like that, but I was hoping there might be an easier way. I'm also concerned that since the amplicons overlap that reads will inappropriately align to the incorrect amplicon, depending on the order they're processed.

Please forgive me if there is an obvious and well known solution to this, I did search for quite a while.

Thanks in advance!

JackieBadger · 06-02-2013, 02:16 PM

why don't you first de-multiplex your sequences based on priming sequence (so then you will not have over lapping amplicons in the files) then just trim of the primers separately ?
I dont fully understand your situation so forgive me if I'm misinterpreting.

vstamborski · 06-19-2013, 07:41 AM

I have a similar (or perhaps the same) problem. I created amplicons pools, then added adapters and barcoded them. I've already separated out samples by index, and removed everything "extra". What remains is my amplicons of interest flanked by the amplification primers. There are hundreds of primers, and in some cases they are offset by 5-30bp. I want to get rid of them.

I tried using NextGENe to trim the primer sequnces, and in most cases it worked; however, NextGENe removes sequences up to 3x the primer length into the amplicon (to remove dimers, trimers, etc) and there is no option to disable this. The result is that instead of trimming a 25bp primer, in some cases it trims the next primer too (instead?) resulting in 50bp being trimmed off the end. I'd like to trim only the actual primer sequence, not additional ones further 3'. Does anyone know of a way to do this?
Thank you,
Valerie

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Removing overlapping genes from annotation for RNAseq read count	DRAT	Bioinformatics	2	04-11-2014 03:53 AM
Looking to find number overlapping sequences between fastq files	jme	Bioinformatics	2	01-17-2012 09:16 AM
Create one sequence based on overlapping primer sequences in amplicon	ketan_bnf	Bioinformatics	2	09-15-2011 12:33 AM
PubMed: Removing Noise From Pyrosequenced Amplicons.	Newsbot!	Literature Watch	0	02-01-2011 02:00 AM
Removing an unknown primer sequence from reads.	mchotalia	Bioinformatics	2	09-12-2009 10:57 PM

06-02-2013, 11:52 AM	#1
eeyun Junior Member Location: 90049 Join Date: Jan 2013 Posts: 9	removing primer sequences from overlapping amplicons Hi all - I'm wondering what methods you'd recommend to remove primer sequences from amplicon sequencing data where multiple amplicons for the same specimen in the same run will overlap. Simply removing the primers using one of the trimming tools seems like it will appropriately remove the primers for the amplicon they are producing, but also any neighboring sequence in an adjacent amplicon which overlaps the sequence. One strategy I had thought might work is to align the reads to each amplcon sequence separately, and process them like that, but I was hoping there might be an easier way. I'm also concerned that since the amplicons overlap that reads will inappropriately align to the incorrect amplicon, depending on the order they're processed. Please forgive me if there is an obvious and well known solution to this, I did search for quite a while. Thanks in advance!

06-02-2013, 02:16 PM	#2
JackieBadger Senior Member Location: Halifax, Nova Scotia Join Date: Mar 2009 Posts: 381	why don't you first de-multiplex your sequences based on priming sequence (so then you will not have over lapping amplicons in the files) then just trim of the primers separately ? I dont fully understand your situation so forgive me if I'm misinterpreting.

06-19-2013, 07:41 AM	#3
vstamborski Junior Member Location: WI Join Date: Nov 2012 Posts: 4	I have a similar (or perhaps the same) problem. I created amplicons pools, then added adapters and barcoded them. I've already separated out samples by index, and removed everything "extra". What remains is my amplicons of interest flanked by the amplification primers. There are hundreds of primers, and in some cases they are offset by 5-30bp. I want to get rid of them. I tried using NextGENe to trim the primer sequnces, and in most cases it worked; however, NextGENe removes sequences up to 3x the primer length into the amplicon (to remove dimers, trimers, etc) and there is no option to disable this. The result is that instead of trimming a 25bp primer, in some cases it trims the next primer too (instead?) resulting in 50bp being trimmed off the end. I'd like to trim only the actual primer sequence, not additional ones further 3'. Does anyone know of a way to do this? Thank you, Valerie