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  • How working with sam format and blast?

    Hi,

    I'm trying to work with the BWA, but I'm having a problem.
    After doing the alignment with BWA, I need to pass the generated file to fasta format, so I can make a blast.
    But when I do this, apparently the fasta file generated comter seems only the reads. Lose alignment. Anyone know what's happening?
    I use te samtools to pass the file in sam format for bam format, and after I use the blast2fastx to convert the alignment in bam format to fasta format.
    I don't know if have some influence, but I'm work with the paired-end alignment.

  • #2
    Converting from SAM or BAM to fasta will result in information loss, unless the converter happens to store all of the alignment information in the description line (and then blast keeps it).

    After using bwa your reads are aligned, why are you then blasting things? If you describe what you're really trying to do we might be able to tell you a more efficient way.

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    • #3
      I did the alignment using BWA, with a reference genome and my reads. Then I had to make a blast alignment I generated to verify that the generated alignment contained some sequence of chloroplast DNA.

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      • #4
        Originally posted by Guigra View Post
        I did the alignment using BWA, with a reference genome and my reads. Then I had to make a blast alignment I generated to verify that the generated alignment contained some sequence of chloroplast DNA.
        How about doing it the other way around? Filter your reads using BLAST against a chloroplast DNA database, and then use the remaining reads with BWA to map against the genome

        Unless you want to identify chloroplast insertions in the nuclear genome?

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        • #5
          Actually I'm trying to check if material extraction for sequencing was done correctly without leftover remnants of chloroplast DNA.
          Your tip solved my problem, thank you! You know how to make this filter in the BWA or bowtie, or some other program? If you know tell me how can I do?

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          • #6
            Originally posted by Guigra View Post
            Actually I'm trying to check if material extraction for sequencing was done correctly without leftover remnants of chloroplast DNA.
            Your tip solved my problem, thank you! You know how to make this filter in the BWA or bowtie, or some other program? If you know tell me how can I do?
            Well you could align reads to the chloroplast genome of your choice, then extract unmapped reads (something like samtools view -F 4....you'll have to double check the flags) and use that to input into BWA for your actual alignment.

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            • #7
              Originally posted by jimmybee View Post
              Well you could align reads to the chloroplast genome of your choice, then extract unmapped reads (something like samtools view -F 4....you'll have to double check the flags) and use that to input into BWA for your actual alignment.
              Thanks for the help! I will use your tip.

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              • #8
                Or just included the chloroplast genome in the reference with the nuclear genome and do the entire alignment in one go.

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