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  • BSmooth alignment problem

    I was aligning short reads obtained from Illumina but always got an error as following:

    ###############################################
    perl bswc_bowtie2_align.pl --out=./outReadslevel/ --sam=./outputSAM/chr ./h.sapiens.bsIndex/chr ./h.sapiens.ref/chr*.fa ./SRA/sra_data.fastq
    ###############################################
    Error: <mate1s/reads> not specified

    The alignment pipeline is followed by:
    Alignment of bisulfite sequence reads and tabulation of read-level methylation measurements - BenLangmead/bsmooth-align

    Alignment of bisulfite sequence reads and tabulation of read-level methylation measurements - BenLangmead/bsmooth-align

    # sra_data.fastq is built successfully in the first step..
    Does anyone know how to solve this problem?
    Thank you in advance!!

  • #2
    Originally posted by genefan View Post
    I was aligning short reads obtained from Illumina but always got an error as following:

    ###############################################
    perl bswc_bowtie2_align.pl --out=./outReadslevel/ --sam=./outputSAM/chr ./h.sapiens.bsIndex/chr ./h.sapiens.ref/chr*.fa ./SRA/sra_data.fastq
    ###############################################
    Error: <mate1s/reads> not specified

    The alignment pipeline is followed by:
    Alignment of bisulfite sequence reads and tabulation of read-level methylation measurements - BenLangmead/bsmooth-align

    Alignment of bisulfite sequence reads and tabulation of read-level methylation measurements - BenLangmead/bsmooth-align

    # sra_data.fastq is built successfully in the first step..
    Does anyone know how to solve this problem?
    Thank you in advance!!
    While I've never actually used bsmooth, I can tell you that the syntax isn't quite what you would expect. You might try:

    Code:
    perl bswc_bowtie2_align.pl --out=outReadslevel/ --sam=outputSAM/chr -- h.sapiens.bsIndex/chr -- h.sapiens.ref/chr*.fa -- -- SRA/sra_data.fastq --
    The "--" is important for the parsing. I think you need the "-- --" part near the end as well as it may otherwise assume that the fastq file is actually an option for bowtie2.
    Last edited by dpryan; 11-18-2013, 04:38 AM.

    Comment


    • #3
      Originally posted by dpryan View Post
      While I've never actually used bsmooth, I can tell you that the syntax isn't quite what you would expect. You might try:

      Code:
      perl bswc_bowtie2_align.pl --out=outReadslevel/ --sam=outputSAM/chr -- h.sapiens.bsIndex/chr -- h.sapiens.ref/chr*.fa -- -- SRA/sra_data.fastq --
      The "--" is important for the parsing. I think you need the "-- --" part near the end as well as it may otherwise assume that the fastq file is actually an option for bowtie2.
      Thanks a lot, the problem is solved! The "-- --" part near the end is mattered..

      Comment


      • #4
        Glad that fixed things. I've seen enough code from Ben Langmead to know that his stuff is rather particular about input syntax.

        Comment


        • #5
          Originally posted by dpryan View Post
          Glad that fixed things. I've seen enough code from Ben Langmead to know that his stuff is rather particular about input syntax.
          Yes, this is quite tricky..

          Comment


          • #6
            Originally posted by dpryan View Post
            Glad that fixed things. I've seen enough code from Ben Langmead to know that his stuff is rather particular about input syntax.
            One more question:
            bowtie2_bs_align.pl [options*] -- <index> -- <refs> -- <bowtie2-args> -- [<mate2s>]

            <index>: Name prefix for index files. E.g. if index is in /tmp/idx.*.watson.bt2 and /tmp/idx.*.crick.bt2, specify "/tmp/idx"..

            chr1.crick.1.bt2, chr1.crick.2.bt2,chr1.crick.3.bt2, chr1.crick.4.bt2,
            chr1.watson.3.bt2 ,chr1.watson.4.bt2, chr1.watson.rev.1.bt2, chr1.watson.rev.2.bt2
            chr1.crick.rev.1.bt2 ,chr1.crick.rev.2.bt2 ,chr1.watson.1.bt2 ,chr1.watson.2.bt2
            chr2.... and all the other chromosomes..

            How should I define the index correctly?
            if /tmp/chr is used, it gives "Index with basename does not exist"... when I use /tmp/chr*.bt2, the program works fine, but is this the right solution?
            The output is given: chrY.ev.tsv
            Is there any way to combine the bt2 files?
            Many thanks!!

            Comment


            • #7
              How exactly did you create the index files? You should have only a set of index files each strand, not one for each chromosome for each strand.

              Comment


              • #8
                Originally posted by dpryan View Post
                How exactly did you create the index files? You should have only a set of index files each strand, not one for each chromosome for each strand.
                I used the following command:
                perl bswc_bowtie2_index.pl --name=chr --bowtie2-build=/.../bowtie2-build ./ref/*.fa
                Alignment of bisulfite sequence reads and tabulation of read-level methylation measurements - BenLangmead/bsmooth-align

                there are 24 fasta files (chr1.fa, chr2.fa....) in the ref folder..
                This will then output the above index files.

                Comment


                • #9
                  You can "cat" the *.fa files into a single multi-fasta file and then use that single file as your "reference" for bowtie2-build. That will create one set of index files for the entire genome.

                  Comment


                  • #10
                    Originally posted by GenoMax View Post
                    You can "cat" the *.fa files into a single multi-fasta file and then use that single file as your "reference" for bowtie2-build. That will create one set of index files for the entire genome.
                    Thank you for the suggestion! It should work well in this way..

                    Comment

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