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  • bowtie2 multiple single and paired-end files

    Hi everyone,

    I a newbie in Bowtie2, so I have a question about bowtie2 commands for single and paired-end reads.

    I have three paired .fastq files, and two single .fastq files.
    When I execute the paired files, I use:
    bowtie2 -x index -1 file1.fastq -2 file2.fastq -3 file3.fastq ...
    When I execute the paired files, I use:
    bowtie2 -x index -U file4.fastq file5.fastq ...

    The paired files have different number of reads and lines.
    These commands are still running, so I wonder did I use them in a right way?
    If not, I would appreciate if you could guide me how to use multiple single and paired-end reads.

    Thank you very much,
    Natasha

  • #2
    bowtie2 multiple single and paired-end files

    Hi Natasha,


    I think when you have more than 1 file of each type you should separate them with commas, not spaces, and the -3 flag means something related to the index for paired end reads.

    so something like this:

    paired reads:
    -1 filea_read1.fastq,fileb_read1.fastq -2 filea_read2.fastq,fileb_read2.fastq

    and for unpaired reads:
    -U filec.fastq,filed.fastq,filee.fastq

    see the Bowtie2 and bowtie manuals.

    Hope this helps,
    Maria

    Comment


    • #3
      Hi,

      Maria, thank you so much for your reply.
      You are right that files should be separated by comma.
      But, I am still confused about the paired reads.

      The three paired Illumina files I have are:
      3 ILLUMINA (Illumina Genome Analyzer IIx) runs: 9.5M spots, 2.7G bases

      So, my question now is:
      should I run these files separately (3 bowtie2 commands, only -1 mate):
      bowtie2 -x index -1 file1.fastq ...
      bowtie2 -x index -1 file2.fastq ...
      bowtie2 -x index -1 file3.fastq ...

      or should I run them in a same command (1 bowtie2 command, only -1 mate, separated by commas):
      bowtie2 -x index -1 file1.fastq,file2.fastq,file3.fastq ...

      Thank you very much,
      Natasha

      Comment


      • #4
        bowtie2 multiple single and paired-end files

        Hi Natasha,

        If you have 3 files for paired-end reads from Illumina GA, one of the files is probably an index read, so you would have to demultplex the reads in the other 2 files based on the index, before attempting to align the reads, if this hasn't been done already. Check with whoever ran the samples, and have a look at the Illumina website

        Illumina sequencing and array technologies fuel advancements in life science research, translational and consumer genomics, and molecular diagnostics.


        for manuals related to the version of the Illumina technology used to generate your reads.

        How many samples are in the 3 paired-end files?

        As for running paired-end reads with bowtie, you use the flag -1 before the name of the file containing the first reads of the pair, and the flag -2 before the name of the file containing the second reads of the pair.

        bowtie2 (other parameters) -1 read1.fastq -2 read2.fastq

        see the bowtie manual


        best wishes,
        Maria

        Comment


        • #5
          Maria, thank you very much for your help.
          After few tries, I think I have the right scripts now.

          Regards,
          Natasha

          Comment


          • #6
            Illumina put mate 1 and mate 2 in a same file, and when using SraToolkit to convert .sra files to .fastq files, for the paired end files the following command should be executed:
            fastq-dump --split-3 file.sra.
            That is how the mate 1 and 2 will be created.
            This is the answer for my question

            Thank you

            Comment


            • #7
              Mapping paired-end and Mate-pair reads together

              Hi,


              I was wondering, How to use Bowtie2 to map paired-end and mate-pair reads simultaneously.

              I saw in manual that there are --fr option for PE reads and --rf option for MP reads. But it did not mentioned about using them together.

              So if I specify,

              Code:
              bowtie2 -x CLC -a --no-mixed --fr -1 frags.A.fastq -2 frags.B.fastq --rf -1 jumps.A.fastq -2 jumps.B.fastq -S CLC_mapping.sam
              would that work?

              Thanks
              Sagar

              Comment


              • #8
                I'm pretty sure that won't work. Your best option, depending on what your goals are, might be to map the two libraries separately and then merge the resulting SAM/BAM files.

                Comment

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