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Paired-end data, solexa, Gerald

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  • Paired-end data, solexa, Gerald

    Dear group,
    I have data from an experimen where 4 biological replicates were sequenced (exon-capture).
    The experiment is paired-end method.
    Gerald output the sequences for 4 lanes as S-1-sequence.txt,---, S-4-sequence.txt.

    I will be using BWA and samtools:

    1. Solexa sequece -> sanger FASTQ
    2. Sanger FASTQ + BWA -> .sai
    3. .sai -> .sam
    4. sam -> bam
    5. sort bam -> snp/indel calls.

    Since i have to use sampe, there are few questions:

    1. sampe requires, s1.fq, s2.fq, s1.sai, s2.sai - how do I get these two s1.fq and s2.fq.

    2. What are insert sizes option in sampe.

    3. I have only one sequence file per lane. How does paired end data looks .

    4. Do i have to seperate one sequence file from one lane to s1.fq and s2. fq

    Please help me.

    thank you.

  • #2
    Originally posted by adrian View Post
    1. sampe requires, s1.fq, s2.fq, s1.sai, s2.sai - how do I get these two s1.fq and s2.fq.
    You should have s_N_1_sequence.txt and s_N_2_sequence.txt for each lane N.

    Originally posted by adrian View Post
    2. What are insert sizes option in sampe.
    Code:
    $ bwa sampe
    
    Usage:   bwa sampe [options] <prefix> <in1.sai> <in2.sai> <in1.fq> <in2.fq>
    
    Options: -a INT   maximum insert size [500]
             -o INT   maximum occurrences for one end [100000]
             -n INT   maximum hits to output for paired reads [3]
             -N INT   maximum hits to output for discordant pairs [10]
             -c FLOAT prior of chimeric rate [1.0e-05]
             -P       preload index into memory (for base-space reads only)
             -s       disable Smith-Waterman for the unmapped mate
    
    Notes: 1. For SOLiD read, <in1.fq> corresponds R3 reads and <in2.fq> to F3.
           2. For reads shorter than 30bp, applying a smaller -o is recommended to
              to get a sensible speed at the cost of pairing accuracy.
    I guess you are looking for the '-a' option

    Originally posted by adrian View Post
    3. I have only one sequence file per lane. How does paired end data looks .
    Uh? The _qseq.txt files should also have a _1 or _2 notation... you should be able to find out which are the first and the second read in PE

    Originally posted by adrian View Post
    4. Do i have to seperate one sequence file from one lane to s1.fq and s2. fq
    If you are using the sampe module, yes.

    d

    Comment


    • #3
      Yes, I was referring to -a option.

      What is insert size. Why insert size plays role here.

      Does insert size mean length of the read?

      thanks for your reply.

      Comment


      • #4
        Originally posted by adrian View Post
        Yes, I was referring to -a option.

        What is insert size. Why insert size plays role here.

        Does insert size mean length of the read?

        thanks for your reply.
        The insert size is the average length of the fragment in your library (tipically 200-300 bp, but you'd better ask lab people). It is important because you expect your pairs being 200-300 bp apart. If one pair is beyond that distance it may be due to a big insertion in your chromosome. Also, paired end sequencing allows you to find out deletions, insertions, traslocations, inversions and so on...

        d

        Comment


        • #5
          As dawe said, you should use the _sequence.txt files as fastq (fq) files. The problem is, however, that Solexa fastq files use another encoding for the quality strings than bwa expects. If you omit the conversion your result may be inaccurate.

          http://en.wikipedia.org/wiki/FASTQ_format

          Comment

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