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SOLiD sequencing protocol and how to obtain quality of SOLiD reads?

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  • SOLiD sequencing protocol and how to obtain quality of SOLiD reads?

    I have just started working on NGS few weeks back and I was trying to get the pipeline for SOLiD right.

    From what I understand the basic pipeline goes something like this (please do correct me if I'm wrong):

    > get xsq files -> convert them to *.csfasta or *.qual files (using xsq tools) -> check for quality control (by converting *.csfasta and *.qual files to *.fastq files) -> mapping (obtain *.sam files) -> convert them to *.bam (and sort it) -> check for duplicates -> detect variants -> annotate variants

    I have the files in *.csfasta and *.qual format with me now. While trying to assess the quality of the reads, I realized that not many tools use *.csfasta and *.qual files as input. I read some place else that, we can convert these files to *.fastq format and use 'fastx tools' to check for quality of the reads. One such tool was 'bfast' and it also maps the reads to reference genome.

    Using solid2fastq in bfast when I did the conversion, I think the output I received was wrong. I can tell because the output_file.fastq file looked something like this:

    @1_28_69
    T..................................................
    +
    !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
    @1_28_95
    T..................................................
    +
    !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!


    I used the following command to generate the output:

    scripts user$ solid2fastq -o output_file sample.csfasta sample.qual

    I'm not sure where I'm making the mistake.

    And also I wanted to know if converting *.csfasta files to *.fastq files is the right thing to do? Wouldn't you lose the quality? Is there any tool which takes in the native *.csfasta files and computes quality of the reads and also mapping instead?

  • #2
    you want to map with either the xsq or csfasta file. I would avoid converting to fastq for anything other than a quality check.

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    • #3
      Conversion is clearly wrong. If my memory serves, ! is zero quality in Sanger-type fastq, and dots are missing colors (which are either 0 or -1 quality depending on version of the machine). Are csfasta/qual files OK?
      Anyway, as already said somewhere in this forum, google up SEAStAR package from Armbrust lab. Currently it can do quality trimming and prepare data for assembly, but in couple of weeks they promised to add loads of stuff, mostly assembly and metagenomics, but still useful for any solid applications.

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      • #4
        I rechecked the fasta files again. The first 100 reads looked like this but later on they seemed to be fine.

        Someone else mentioned to me that, it might be due to that fact that the reference file has NNNNNNNNNNNNNNN in the first few lines.

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        • #5
          hey hi..I have also started working de novo assembly on .qual format. I am new into this thing. I dont even have idea about protocol. I have tried to search but ended of nothing. I saw ur post so can u please tell me the refined protocol of doing assembly on .qual files.

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