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Old 07-15-2012, 09:25 PM   #1
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Location: China

Join Date: Jun 2012
Posts: 2
Default Calling for help in using samtools mpileup: all Ns in reference bases

I'm mapping 36bp single ended reads to C.elegans cDNA reference by bwa, trying to make variant calling after getting mpileup files. The problem is the samtools mpileup doesn't work well with my reference sequence and show all Ns in the third column.
Like this:
AC8.4 3 N 1 ^!G I
AC8.4 4 N 1 A I
AC8.4 5 N 1 A I
AC8.4 6 N 1 T I
AC8.4 7 N 1 G I
AC8.4 8 N 1 C I
Do you know what's wrong?
P.S. my reference consists of 55783 chromosomes (transcripts), each one with about 20~10k bp length.
My commanding lines are like this:
bwa -n 1 -t 7 -l 10000 -o 0 XXX.fa XXX.fastq > XXX.sai
bwa samse -n 10 XXX.fa XXX.sai XXXfastq > XXX.sam
samtools view -S -b XXX.sam > XXX.bam
samtools sort XXX.bam XXX_sorted
samtools mpileup -f XXX.fa XXX_sorted.bam > result.mpileup

Plz help me, thank you very much!
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Old 07-16-2012, 06:10 AM   #2
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Location: China

Join Date: Jun 2012
Posts: 2

Solved by myself already!
Somebody told me that samtool mpileup doesn't work with references with "|" or "." symbol in their chromosome names. So I tried first by simplifying chromosome (transcript) names to refrain "|" and ".". However it didn't work.
Later I found empty lines and Ns in some of sequence of my transcripts. After I removed all the bad transcripts, samtools worked quite well!
Hope anybody has similar problems will get help!
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bwa, reference base, samtools mpileup

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