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Old 09-10-2015, 07:13 AM   #1
danova
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Location: France

Join Date: Sep 2010
Posts: 27
Default Bowtie2 - align two PCR primers help

Hi,
Not sure its the correct strategy by im trying to achieve the following:

1- I have two primers (see below) that map to 16s amplicons and they generate an insert of 1200 to 1400 nt long.

The two primers are described below.
>forward_primer
CCTACGGGNGGCWGCAG
>reverse_primer
GGATTAGATACCCBDGTAGTC

When i try to use bowtie2 to align the two primers to a reference database of different 16S sequences they are supposed to match i only get 600 hits (expected 400000 hits), meaning this primer set will align perfectly to 400000 different sequences of the database.

bowtie2 -f -t -a --no-dovetail --no-contain --no-overlap --no-mixed --fr --minins 800 --maxins 1500 -x 16S_database -1 forward_primer -2 reverse_primer


I have checked the orientation of the primers , -fr is the correct orientation.

It looks too me that bowtie2 handles degenerated primers differently then what i expected. Does anybody know how to improve the alignement of the degenerated primers (-N 1 option does not really improve). ANy idea ???
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