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  • 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!

  • #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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