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  • BWA - Samtools problem

    Hello everyone

    There are some topics concerning BWA and Samtools, but i still haven't found the solution to my problem...

    Ok, here is what i do

    1. I run BWA aln and I get a .sai file
    2. I run BWA samse and I get a .sam file

    So far so good.

    But now i am having troubles using the 'pileup function'

    As far as i understand i should use ./samtools -f ref.fna testing.sam

    but it is giving me errors.

    I am probably just using a wrong command or maybe i should convert the .sam file to a .bam file? I have tried this using:
    samtools view -bt ref.fna -o out.bam testing.sam:sam_header_read2: Assertion `fp' failed.
    samtools view -b ref.fna -o out.bam testing.sam:segmentation error

    Anyone has the exact commando for me to got from BWA to 'pileup results' in samtools?

    thx
    Last edited by joa_ds; 08-05-2009, 01:05 AM.

  • #2
    Originally posted by joa_ds View Post
    Hello everyone

    There are some topics concerning BWA and Samtools, but i still haven't found the solution to my problem...

    Ok, here is what i do

    1. I run BWA aln and I get a .sai file
    2. I run BWA samse and I get a .sam file

    So far so good.

    But now i am having troubles using the 'pileup function'

    As far as i understand i should use ./samtools -f ref.fna testing.sam

    but it is giving me errors.

    I am probably just using a wrong command or maybe i should convert the .sam file to a .bam file? I have tried this using:
    samtools view -bt ref.fna -o out.bam testing.sam:sam_header_read2: Assertion `fp' failed.
    samtools view -b ref.fna -o out.bam testing.sam:segmentation error

    Anyone has the exact commando for me to got from BWA to 'pileup results' in samtools?

    thx
    If you have a "sam" file, then you must use the "-S" flag (for the input is SAM) with "samtools view". "samtools view" expects a ".bam" file, so you could also convert it to a BAM file.

    Comment


    • #3
      Try these two commands:

      >samtools pileup -cf ref.fa testing.bam
      or
      >samtools pileup -cf ref.fa -t ref.fa.fai testing.sam

      They are actually the same, but you would have to get bam file first which will require the indexed reference sequence (use faidx option to get it).

      Comment


      • #4
        For samtools view ..., I don't see any sense you need to use it, since I normally use it to convert bam file back to sam file.

        Comment


        • #5
          I have a quick question. How can I use 'samtools view' for converting bam file to sam file? I tried some times but I failed to do it...

          Comment


          • #6
            Originally posted by yasu View Post
            I have a quick question. How can I use 'samtools view' for converting bam file to sam file? I tried some times but I failed to do it...
            You can, but include the header ("-h").

            Comment


            • #7
              hi there again...

              The reasons I am trying BWA (still hasn't worked... ) is because I am analyzing illumina data for counting variant frequencies for a pool of several patients.

              So far so good, BowTie gives 15%, Maq gives 40% and Sanger validation gives 0% for a certain variant. We have around 1000 variants, so this is kinda annoying.

              I am doing a very simple thing: I align the reads using BowTie and i just parse the output file and count the variant frequencies. Some other people used MAQ and in the lab they verified using Sanger.

              Before I spend another 10h trying to figure out why this BWA thing ain't working, is it worth trying to determine variant frequencies in a pool? And if so, what is the best strategy to do so.

              And on the BWA/samtools thingie. I tryed using the -S flag but i keep getting "Segmentation fault"

              I must be doing something totally wrong here...


              Step 0:
              /data/illumina/bwa-0.4.9/bwa index -a is Final

              Step 1:
              /data/illumina/bwa-0.4.9/bwa aln -t 12 Final Risk_no_haplos.txt > Risk_no_haplos.sai

              Step 2:
              /data/illumina/bwa-0.4.9/bwa samse Final Risk_no_haplos.sai Risk_no_haplos.txt > Risk_no_haplos.sam

              Step 3:
              ../../samtools-0.1.5c_x86_64-linux/samtools view -S -h Risk_no_haplos.sam \
              is giving
              missing header? Abort!

              ../../samtools-0.1.5c_x86_64-linux/samtools pileup -S -cf Final Risk_no_haplos.sam
              is giving
              [samopen] no @SQ lines in the header.
              [sam_read1] missing header? Abort!


              aargh

              EDIT: Woohoo, finally figured it out... Apparently i had to use faidx before i could use pileup straight away... The question remains if using BWA is a wise thing for the presented problem...
              Last edited by joa_ds; 08-10-2009, 07:10 AM. Reason: Woops

              Comment


              • #8
                Originally posted by yasu View Post
                I have a quick question. How can I use 'samtools view' for converting bam file to sam file? I tried some times but I failed to do it...
                That is way simple, try this linux command

                samtools view test.bam > test.sam

                Comment


                • #9
                  Originally posted by joa_ds View Post
                  hi there again...


                  Step 3:
                  ../../samtools-0.1.5c_x86_64-linux/samtools view -S -h Risk_no_haplos.sam \
                  is giving
                  missing header? Abort!

                  ../../samtools-0.1.5c_x86_64-linux/samtools pileup -S -cf Final Risk_no_haplos.sam
                  is giving
                  [samopen] no @SQ lines in the header.
                  [sam_read1] missing header? Abort!


                  aargh

                  EDIT: Woohoo, finally figured it out... Apparently i had to use faidx before i could use pileup straight away... The question remains if using BWA is a wise thing for the presented problem...
                  Apparently, you shouldn't use samtools view sam file, that is for bam file. To pileup, you would either make bam file or get indexed reference sequence (.fai).
                  Last edited by totalnew; 08-10-2009, 08:27 AM.

                  Comment


                  • #10
                    When I attempt to align mated paired-end sequence reads and output the file
                    in SAM format, I receive a segmentation fault. If I try the same thing
                    without the -S/--sam option, it works fine. Here is what I am getting:

                    EEB-WITT5:Bowtie wittkopp-lab$ ./bowtie -q -k 1 --sam --best
                    --solexa1.3-quals dmel-all-CDS-r5.21 -1
                    ./mel_sim_data/Hybrids/s_2_1_sequence.txt -2
                    ./mel_sim_data/Hybrids/s_2_2_sequence.txt > s_2_sequence.sam

                    Segmentation fault

                    Any help in this matter would be greatly appreciated! Again, I would like
                    this output to be in SAM format. I tried converting the bowtie output to
                    SAM but the bowtie2sam.pl script from SAMtools doesn't do that for me.

                    Comment


                    • #11
                      I tried to use sort command :
                      samtools sort aln.sam aln.sam.sorted
                      i also got
                      Segmentation fault
                      is there anybody had this probelm before?

                      Comment


                      • #12
                        Originally posted by xuer View Post
                        I tried to use sort command :
                        samtools sort aln.sam aln.sam.sorted
                        i also got
                        Segmentation fault
                        is there anybody had this probelm before?

                        You should sort bam file, try this

                        samtools sort -n aln.bam sorted

                        then mv the sorted.bam file to whatever name you want.

                        Comment


                        • #13
                          Install SAM tool

                          Are there anyone know how to install SAM tool? I read in the SAM document and it didn't say anything about that. Thank you.

                          Comment


                          • #14
                            Originally posted by Victory View Post
                            Are there anyone know how to install SAM tool? I read in the SAM document and it didn't say anything about that. Thank you.
                            Download it from http://samtools.sourceforge.net/. Copy the binaries to your typical install directory (which depends on your system).

                            Comment


                            • #15
                              Thank you. I use SUN cluster computer. I have already copy whole folder of SAM version 0.1.6 to my working directory. However, it still doesn't work. I'm not sure which one is the install directory.

                              Comment

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