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  • why can't I get alignment with BWA?

    What's the general steps to align a set of reads to ref?
    Is the flowing right?
    1.build index -> bwa index -c -a is ecoli.fa
    2.convert csfasta and correspond qual file into fastq
    3.Find the SA coordinates of the input reads -> bwa aln -n 3 -t 4 -M 3 -c ecoli.fa t_ecoli.fastq >map.sai
    4.Generate alignments in the SAM format given single-end reads -> bwa samse -n 1 ecoli.fa map.sai t_ecoli.fastq >map.sam
    Do as the above, I get a sam file with no reads mapped. All the 25000 items have flag 4.
    What's more, I have 50000 reads in the input fastq file, why does it only process 25000?
    If I delete one read form the fastq file, BWA will process 24999 reads. Why?
    And what is the bwasw for?
    Thank you !
    Last edited by gigigou; 09-06-2012, 05:54 AM.

  • #2
    I think in step 3 you are missing the output character. It should be:

    bwa aln -n 3 -t 4 -M 3 -c ecoli.fa t_ecoli.fastq > map.sai

    See if that helps

    Comment


    • #3
      Originally posted by jimmybee View Post
      I think in step 3 you are missing the output character. It should be:

      bwa aln -n 3 -t 4 -M 3 -c ecoli.fa t_ecoli.fastq > map.sai

      See if that helps
      Oh, sorry that I missed the ">" in the post, but I did add it in the command line.
      So it can't be the reason.
      Actually I do get the align file, but in the file I find there is no mapped items. All the 25000 reads are reported unmapped.
      thank you all the same

      Comment


      • #4
        Originally posted by gigigou View Post
        What's the general steps to align a set of reads to ref?
        Is the flowing right?
        1.build index -> bwa index -c -a is ecoli.fa
        2.convert csfasta and correspond qual file into fastq
        3.Find the SA coordinates of the input reads -> bwa aln -n 3 -t 4 -M 3 -c ecoli.fa t_ecoli.fastq >map.sai
        4.Generate alignments in the SAM format given single-end reads -> bwa samse -n 1 ecoli.fa map.sai t_ecoli.fastq >map.sam
        Do as the above, I get a sam file with no reads mapped. All the 25000 items have flag 4.
        What's more, I have 50000 reads in the input fastq file, why does it only process 25000?
        If I delete one read form the fastq file, BWA will process 24999 reads. Why?
        And what is the bwasw for?
        Thank you !
        You are making a color space index, but converting your reads to fastq? Are you sure that's right?

        Comment


        • #5
          I agree with swbarnes, after creating the color-space index then give bwa the color-space input file and not a nucleotide file.

          Comment


          • #6
            Originally posted by swbarnes2 View Post
            You are making a color space index, but converting your reads to fastq? Are you sure that's right?
            The reads are converted into fastq format, but they are still in color space
            @SRR001354.lite.sra.1 461_28_1048
            T23331323333132332323133333332333232
            +
            %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

            I tried to input the original csfasta file, it processed 50000 reads, but still with no reads mapped.

            50000 + 0 in total (QC-passed reads + QC-failed reads)
            0 + 0 duplicates
            0 + 0 mapped (0.00%:-nan%)
            0 + 0 paired in sequencing
            0 + 0 read1
            0 + 0 read2
            0 + 0 properly paired (-nan%:-nan%)
            0 + 0 with itself and mate mapped
            0 + 0 singletons (-nan%:-nan%)
            0 + 0 with mate mapped to a different chr
            0 + 0 with mate mapped to a different chr (mapQ>=5)

            Comment


            • #7
              Originally posted by westerman View Post
              I agree with swbarnes, after creating the color-space index then give bwa the color-space input file and not a nucleotide file.
              In the fsatq file that converted from csfasta, the reads are still in color space, as above.

              Comment


              • #8
                Since we do not have access to your files it is hard to troubleshoot problems. But a couple of pieces of general advice in troubleshooting a problem:

                1) Do not use any non-standard options; e.g., try 'aln' and 'samse' without any parameters.

                2) Try a smaller input file. Especially one with a known-to-be-good sequence. In other words take a bit of your ecoli reference and make it into a color-space read.

                Good luck in solving this.

                Comment


                • #9
                  Provided you have done what others have already suggested before ...

                  (and this may be a stupid suggestion) but are you sure the data you have is from your samples (i.e. there was no mix-up at the place where got it sequenced).

                  Comment


                  • #10
                    Originally posted by westerman View Post
                    Since we do not have access to your files it is hard to troubleshoot problems. But a couple of pieces of general advice in troubleshooting a problem:

                    1) Do not use any non-standard options; e.g., try 'aln' and 'samse' without any parameters.

                    2) Try a smaller input file. Especially one with a known-to-be-good sequence. In other words take a bit of your ecoli reference and make it into a color-space read.

                    Good luck in solving this.
                    Thank you.
                    All the parameters are specified as the manual says.
                    I think the input file is small enough, only 50000 reads.
                    Thank you for your help.
                    I'll look into it.

                    Comment


                    • #11
                      Originally posted by GenoMax View Post
                      Provided you have done what others have already suggested before ...

                      (and this may be a stupid suggestion) but are you sure the data you have is from your samples (i.e. there was no mix-up at the place where got it sequenced).
                      I think the data is not the problem cause I have used other align tools to do the alignment, and they all give perfect results. So I think the problem is BWA, maybe I didn't specify the parameters properly, I'll try to solve it.
                      Thank you!

                      Comment


                      • #12
                        I have solved the problem.
                        In step 2, the file's name should be specified as the pl says.
                        But I think it is a little inconvenient, so I modified the pl to let it work more efficiently.

                        Comment

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