16Mar

bowtie2 aligment problems

Posted by: admin Categories: Genetics
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gravatar for amitpande74

2 hours ago by

Hi,

I have reads from DNA sequencing,5 pools. The reads look like this:

 @NS500455:80:HG7TNBGXB:1:11101:17723:1055 1:N:0:ATCACG
ACTTANGTGTATGTAAACTTCCGACTTCAACTGTATAGGGATCCNAGCTCCAATTCGCCCTATAGTGAGTCGTAT
+
/AAAA#EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE#EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE

It's illumina primer followed by the construct of a transposon which I had to filter. So, I used cutadapt:

for file in /FASTQ/*
do
  cutadapt -g ACTTAAGTGTATGTAAACTTCCGACTTCAACTG --discard-untrimmed --minimum-length=35 "$file" -o "`basename -s .fastq &
done

Then I tried aligning this with Bowtie2 using the command :

./bowtie2-align-s -x rat/rn4 -U A1_S1_R1_001.fastq --sensitive-local -S A_align.sam

Bowtie2 fails to align majority if them :

2648325 reads; of these:
  2648325 (100.00%) were unpaired; of these:
    2242006 (84.66%) aligned 0 times
    309307 (11.68%) aligned exactly 1 time
    97012 (3.66%) aligned >1 times
15.34% overall alignment rate

I tool the aligned and the unaligned sequences and used BLAT to figure out any contamination and to see if the sequences are from the rat genome. All positive.
I don't have any clue as to why this is happening.
Can someone help please.
regards to all.



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