Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • How to extract a regional consensus sequence of reads from BAM file

    Deal all,

    How do I get a regional consensus sequence, say ch01: 77295611-77295961, from a BAM file by Samtools?

    I extracted bam file for this region: ch01: 77295611-77295961 (region.bam) from all_aln.bam and used the recommanded command:

    samtools mpileup -uf ref.fa region.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq

    However, the cns.fq seems contain sequence for the whole reference (took longer time), not just the region ch01: 77295611-77295961.

    How to fix this?

    Thanks.

    Jeff

  • #2
    anyone knows or do I need to write a script to parse it out?

    Comment


    • #3
      Looking at the command line options for mpileup, why didn't you use -r there to specify a region?

      Usage: samtools mpileup [options] in1.bam [in2.bam [...]]

      Input options:

      -6 assume the quality is in the Illumina-1.3+ encoding
      -A count anomalous read pairs
      -B disable BAQ computation
      -b FILE list of input BAM files [null]
      -C INT parameter for adjusting mapQ; 0 to disable [0]
      -d INT max per-BAM depth to avoid excessive memory usage [250]
      -E extended BAQ for higher sensitivity but lower specificity
      -f FILE faidx indexed reference sequence file [null]
      -G FILE exclude read groups listed in FILE [null]
      -l FILE list of positions (chr pos) or regions (BED) [null]
      -M INT cap mapping quality at INT [60]
      -r STR region in which pileup is generated [null]
      -R ignore RG tags
      -q INT skip alignments with mapQ smaller than INT [0]
      -Q INT skip bases with baseQ/BAQ smaller than INT [13]

      Comment


      • #4
        Thanks. Tried:

        samtools mpileup -uf ref.fa region.bam -r ch01: 77295611-77295961 | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq

        the consensus is indeed calculated for region 77295611-77295961 on ch01, however, the cns.fq contains sequence sprend over all ch01, in the form of NNNNN-consensus-NNNNN format. (Need to parse out by writing a script).

        Jeff

        Comment


        • #5
          I believe that your problem is the space between the "ch01:" and "77295611-77295961". Perhaps try it without the space.

          Comment


          • #6
            which command is used to get the complete consensus from the bam file?

            Comment


            • #7
              Hi all,

              I tried this and ran into the same problem. When I broke up the pipe, it looked like bcftools was giving the expected output, but vcfutils then tried to recreate the entire chromosome.

              My solution was to make a small modification to vcfutils so that it does not add a "gap" at the beginning of the sequence.

              Line 500 now reads:
              if (($t[1] - $last_pos > 1) && ($last_pos > 0)) {

              That seems to do it...

              Comment


              • #8
                to adamr:
                Thanks a lot it works!

                Comment


                • #9
                  Ive been trying to work with this pipe in order to generate short sequences.

                  Code:
                  samtools mpileup -uf ref.fa region.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq
                  Ive even included adamr's changes to vcfutils.pl. The above code works but only for a single region. If I try to give a bed file there are issues. The sequences are not named based on the BED format column.

                  Sample Bed:
                  Code:
                  1	1158620	1158631	"SDF4.1:1158631"
                  1	1650793	1650804	"CDK11A,CDK11B;CDK11B.1:1650797"
                  Also if just does not work for the multiple regions. I will get something like

                  Code:
                  @1
                  CATTTTGCnnnnnnnnnnnnnnnnnnnnnnnnnnnn....
                  !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!....
                  ~~~~~~~~~~~~~~
                  At the very least I was hoping for a multiple sequence fq file. Does anyone have any insight?

                  Comment

                  Latest Articles

                  Collapse

                  • seqadmin
                    Strategies for Sequencing Challenging Samples
                    by seqadmin


                    Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                    03-22-2024, 06:39 AM
                  • seqadmin
                    Techniques and Challenges in Conservation Genomics
                    by seqadmin



                    The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                    Avian Conservation
                    Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                    03-08-2024, 10:41 AM

                  ad_right_rmr

                  Collapse

                  News

                  Collapse

                  Topics Statistics Last Post
                  Started by seqadmin, Yesterday, 06:37 PM
                  0 responses
                  11 views
                  0 likes
                  Last Post seqadmin  
                  Started by seqadmin, Yesterday, 06:07 PM
                  0 responses
                  10 views
                  0 likes
                  Last Post seqadmin  
                  Started by seqadmin, 03-22-2024, 10:03 AM
                  0 responses
                  51 views
                  0 likes
                  Last Post seqadmin  
                  Started by seqadmin, 03-21-2024, 07:32 AM
                  0 responses
                  68 views
                  0 likes
                  Last Post seqadmin  
                  Working...
                  X