Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Invalid format for mpileup use with VarScan

    Dear all,

    I'm trying to call variants on RNA-seq data.
    I aligned the paired-end reads with Bowtie/Tophat and then generated a mpileup file with Samtools version 0.1.16 using the following command:
    samtools mpileup -q 1 -Q 13 -f human_g1k_v37.fasta test.sorted.bam > test.sorted.mpileup

    This mpileup file looks ok. Then I try to run VarScan version 2.3.2, with the following code:
    java -jar VarScan.v2.3.2.jar mpileup2snp /test.sorted.mpileup --min-var-freq 0.08 --p-value 0.01 > /test.sorted.mpileup.varscan

    Only SNPs will be reported
    Min coverage: 8
    Min reads2: 2
    Min var freq: 0.08
    Min avg qual: 15
    P-value thresh: 0.01
    Reading input from /test.sorted.mpileup

    Initially, everything seems fine, but then VarScan throws me this error:

    Error: Invalid format for pileup at line 419813325
    22 31372123 A 1018 ...,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,......,.....,,,,,,,,,,,,,,............,,,,,,,,,,,,,,.....................,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.......................,,,,,,,,,,.........t,,..........................................,,,,,,,,,,,,,,,,,,,,,,,,,,,..........,,,,,,,,,,,,,,,,,,,,,,..,,,,,,,,,.,,,,.................,,...........................,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,..,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,....,,,,,,,,,,,...,,..,......................................................,,,........,,,,.....................................,,,,,,,............,,................................................,.........,.....,..............,.,,,,,........,.,,,,,,,..,,,,,,.,,,,,,,,.,,..,,,,,,,,,,,,,,,,,,,,,,,,,,,,.......,,,,,,,,,,,,,,,,...........,,,,.,,,,,,,,,,,,,,,,,,,,,,,,,......,,,,,,,..,,,,..,,,,,,,,,,,,,,,,,.............,,,,,.,,,,,,,,.....,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.GHJHHIJH@JIEECJI39<IG:9JI<DCDCD?JJJJIFJFGJ1JJIIHC)3DAHCC9G)?JJEEJHJBJ@:JIIHIJJGFAIIJIH@IDI@H?IIIJJIGEJJGJH1IHGJEJC?@EIIIJHJ@JIJJJFEJJHIIJFJEGJJFGHJGJHJIIIJHCGJJ?GJHJ?F<ACJIGEEFJBD9DACCC?FJHJJCIJEJJIGJJJIJJG@EJAJCJHEFJID?DCDC?#CDCCDC?DGJCDGIEFEHFBJH?GJIJJGGIC?<JD?IG@JAIFIIDIJHCD<CAIIGJJJJDJ;JEJIIIJGIEJEDHJ;GJJJIJGJIGG>@IJJIJCJJJGGJJC?HAHGFHADGCGHH?3<?DDH<<DGCFFDFDF?FDF#C???D@D#FFDDCCD?(3CDC<CCCCCC<D@+#DBDC#DDDA?C#9C?D@=CD@=9

    The generated varscan-file up till that line seems fine. However, I don't really know how to work around this corrupt line. Would it be easiest if I just removed that line? And how can I do this on the mpileup file? Or are there other options?

    Many thanks,
    Lien

  • #2
    It's strange to suddenly encounter an invalid-format line in SAMtools mpileup output. If I take it *exactly* as you pasted it, there's no delimiter (tab) between the last base call in column 5 and the first base quality value ("G"). However, if I put a tab between those, VarScan read the line just fine.

    In terms of immediate action, you could simply remove the line (99.9% of reads show no variant anyway) with grep or vi or another command-line tool. You might also want to send me lines 419813320-419813330 of your pileup file and I'll take a look.

    Comment


    • #3
      Hi Dan,

      I also don't know how this strange line is formed. I performed the exact same commands on similar files, and they seem to work fine.
      I just removed this line, so hopefully everything will work out now. I just wasn't sure if I could just delete this file without consequence.

      Thanks for your help,
      Lien

      Comment


      • #4
        I have a similar error and am curious if the reason for this is found? Im running it in a pipeline and it would be easier if I could do something as a remedial step without having to check for the error messages.

        Thank you,
        Teja

        Code:
        Error: Invalid format for pileup at line 71
        1       10277   C       0

        Comment


        • #5
          Vyellapa, can you send me the first 75 lines of your pileup file? Send it to dkoboldt (at) genome [dot] wustl [dot] edu

          Comment


          • #6
            Hello all,

            We have just released VarScan v2.3.5 which should correct the invalid mpileup warning:

            Comment


            • #7
              v2.3.5 similar format error

              I am also trying to call snps and indels using VarScan. I have the latest release (v2.3.5) installed so I can output to vcf format. I am using the following command and getting the invalid format error:

              -bash-3.2$ java -jar VarScan.v2.3.5.jar mpileup2cns /research/sample.mpileup -min-coverage 8 --min-reads2 2 --min-var-freq 0.01 --min-avg-qual 15 --p-value 0.01 --strand-filter 0 --output-vcf 1 --variants 0 > /research/sample.vcf
              Only variants will be reported
              Min coverage: 8
              Min reads2: 2
              Min var freq: 0.01
              Min avg qual: 15
              P-value thresh: 0.01
              Reading input from /research/sample.mpileup
              Error: Invalid format for pileup at line 1
              ï¿ï¿½BC��<�BCFYc1c2c3c4c5c6c7c8c9c10c11c12c13c14c15c16c17c18c19c20c21c22cXcYcMt5/research/Wdemos_work/sample_reordered_sorted.bam%##samtoolsVersion=0.1.17 (r973:277)

              Can anyone please lend me a hand to figure out why it isn't working please? Also, I do not understand why the sorted. bam file in another directory is being referred to in the error message.

              This is how I generated my pileup file:
              samtools mpileup -q 1 -C50 -DSuf /ref/human_v37.fa /research/sample_reordered_sorted.bam > /research/sample.pileup

              thanks
              Last edited by wdemos; 03-18-2014, 05:57 AM.

              Comment


              • #8
                the -u option was causing an issue. I was running it in a wrapper and not technically piping it in to VarScan

                Comment


                • #9
                  Thanks for letting me know!

                  Comment


                  • #10
                    short solution

                    Try to use this command before you call variants:

                    sed -n '/\t0\t/!p' file.mpileup > file.mpileup2

                    use the file.mpileup2 to call variants.

                    Comment


                    • #11
                      Originally posted by vyellapa View Post
                      I have a similar error and am curious if the reason for this is found? Im running it in a pipeline and it would be easier if I could do something as a remedial step without having to check for the error messages.

                      Thank you,
                      Teja

                      Code:
                      Error: Invalid format for pileup at line 71
                      1       10277   C       0
                      Hi, Teja:

                      I wonder whether you solved the problem with mpileup, if so, would you mind giving me some suggestions on that? Thanks.

                      Best,
                      Liye

                      Comment


                      • #12
                        Looks like Dan Kabolt fixed it the next release of Varscan. If youre still having an error with the new release too, im not sure what else could help.

                        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
                        10 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, Yesterday, 06:07 PM
                        0 responses
                        9 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, 03-22-2024, 10:03 AM
                        0 responses
                        49 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, 03-21-2024, 07:32 AM
                        0 responses
                        67 views
                        0 likes
                        Last Post seqadmin  
                        Working...
                        X