Cnvnator Error

[SalendraSingh]

We have installed Root and tested that it works. The SpeedSeq sv also works without the -d option. We have added source /mnt/pan/Data4/speedseq/root-v5-34/bin/thisroot.sh to the end of the speedseq.config. When we run the speedseq example (run_speedseq.sh) with the -d option to get CNV, we receive the following message: --Example script: ../bin/speedseq sv
-o example
-B example.bam
-S example.splitters.bam
-D example.discordants.bam
-R data/human_g1k_v37_20_42220611-42542245.fasta
-d ***_Below is the message on the terminal_ Calculating read depth Traceback (most recent call last): File "/mnt/pan/Data4/local/vxv89/sxs1528/speedseq/speedseq/bin/cnvnator_wrapper.py", line 350, in chroms_list = get_chroms_list(args.bam) File "/mnt/pan/Data4/local/vxv89/sxs1528/speedseq/speedseq/bin/cnvnator_wrapper.py", line 153, in get_chroms_list proc = subprocess.Popen(['samtools', 'view', '-H', bam_fn], stdout = subprocess.PIPE) File "/home/sxs1528/anaconda/lib/python2.7/subprocess.py", line 710, in init errread, errwrite) File "/home/sxs1528/anaconda/lib/python2.7/subprocess.py", line 1335, in _execute_child raise child_exception OSError: [Errno 2] No such file or directory

---

Please give us suggestions as what to do next.

Thank you

[cc2qe]

might be a duplicate of #40 . Is samtools in your PATH?

[SalendraSingh]

Hi Colby,

Thanks for the fast reply, Yes the PATH was one issue, but now i am getting a lot of other error messages while running the same example. the following is the log of it. Your help will be really grateful

################################################################# [sxs1528@hpcviz example]$ ../bin/speedseq align \

-o example2 \
-M 3 \
-p \
-R "@RG\tID:SampleName\tLB:SampleName\tSM:SampleName\tPL:ILLUMINA" \
data/human_g1k_v37_20_42220611-42542245.fasta \
data/NA12878.20slice.30X.fastq.gz

Sourcing executables from ../bin/speedseq.config ... Aligning... samblaster: Version 0.1.22 samblaster: Inputting from stdin samblaster: Outputting to stdout samblaster: Opening example2.VlJLfHVa3Em3/disc_pipe for write. samblaster: Opening example2.VlJLfHVa3Em3/spl_pipe for write. [M::main_mem] read 95830 sequences (9678830 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (21, 46044, 29, 11) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat](25, 50, 75) percentile: (85, 213, 382) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 976) [M::mem_pestat] mean and std.dev: (217.79, 181.51) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1273) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat](25, 50, 75) percentile: (275, 318, 363) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (99, 539) [M::mem_pestat] mean and std.dev: (318.39, 70.18) [M::mem_pestat] low and high boundaries for proper pairs: (11, 627) [M::mem_pestat] analyzing insert size distribution for orientation RF... [M::mem_pestat](25, 50, 75) percentile: (139, 169, 239) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 439) [M::mem_pestat] mean and std.dev: (179.10, 63.43) [M::mem_pestat] low and high boundaries for proper pairs: (1, 539) [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat](25, 50, 75) percentile: (297, 318, 1164) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 2898) [M::mem_pestat] mean and std.dev: (659.64, 531.94) [M::mem_pestat] low and high boundaries for proper pairs: (1, 3765) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 95830 reads in 3.650 CPU sec, 3.649 real sec samblaster: Loaded 1 header sequence entries. [main] Version: 0.7.10-r789 [main] CMD: /mnt/pan/Data4/local/vxv89/sxs1528/speedseq/speedseq//bin/bwa mem -t 1 -p -R @RG\tID:SampleName\tLB:SampleName\tSM:SampleName\tPL:ILLUMINA data/human_g1k_v37_20_42220611-42542245.fasta data/NA12878.20slice.30X.fastq.gzsamblaster: Output 110 discordant read pairs to example2.VlJLfHVa3Em3/disc_pipe

[main] Real time: 4.431 sec; CPU: 4.218 sec samblaster: Output 82 split reads to example2.VlJLfHVa3Em3/spl_pipe samblaster: Marked 225 of 47915 (0.47%) read ids as duplicates using 2336k memory in 0.164S CPU seconds and 5S wall time. Done ##################################################################################### [sxs1528@hpcviz example]$ ../bin/speedseq var \

-o example2 \
data/human_g1k_v37_20_42220611-42542245.fasta \
example2.bam

Sourcing executables from ../bin/speedseq.config ... Calling variants... Done

######################################################################################

../bin/speedseq sv -d -o example2 -B example2.bam -S example2.splitters.bam -D example2.discordants.bam -R data/human_g1k_v37_20_42220611-42542245.fasta > output.txt

==============Error================================== Sourcing executables from ../bin/speedseq.config ...

Checking for required python modules (/home/sxs1528/anaconda/bin/python2.7)...

Running LUMPY express Sourcing executables from ../bin/speedseq.config ...

Checking for required python modules (/home/sxs1528/anaconda/bin/python2.7)...

create temporary directory

Calculating insert distributions... Library read groups: SampleName Library read length: 101 done 0 Running LUMPY...

/mnt/pan/Data4/local/vxv89/sxs1528/speedseq/speedseq//bin/lumpy
-t example2.cglDWOgxA3L7/temp_lumpyexpress/example2.sv.vcf
-msw 4
-tt 0

-pe bam_file:example2.discordants.bam,histo_file:example2.cglDWOgxA3L7/temp_lumpyexpress/example2.sv.vcf.sample1.lib1.x4.histo,mean:319.045536728,stdev:73.6969198516,read_length:101,min_non_overlap:101,discordant_z:5,back_distance:10,weight:1,id:SampleName,min_mapping_threshold:20,read_group:SampleName
-sr bam_file:example2.splitters.bam,back_distance:10,min_mapping_threshold:20,weight:1,id:SampleName,min_clip:20
> example2.cglDWOgxA3L7/example2.sv.vcf LUMPY Express done Calculating read depth 95226 alignments were processed corresponding to chrom 20_slice Filling and saving tree for '20_slice' ... A total of 95226 aligned reads have been processed. Writing histograms ... Allocating memory ... Done. Calculating histograms with bin size of 100 for '20_slice' ... Processing data from the following chromosomes: ['20_slice'] ===== Running tree on input data ===== Running histograms on input data for input bin size

Removed 16 outliers with isize >= 769 20_slice 1000000

Error computing histograms (input bin size). Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::Fill: Invalid signature - do nothing Error in TH2I::LabelsDeflate: Invalid axis option \ufffd\ufffd\ufffd*?

*** Break *** segmentation violation

There was a crash.

This is the entire stack trace of all threads:

#0 0x0000003b564ac6ce in waitpid () from /lib64/libc.so.6 #1 0x0000003b5643e609 in do_system () from /lib64/libc.so.6 #2 0x00007f3d9eb4bf98 in TUnixSystem::StackTrace() () from /mnt/pan/Data4/local/exome_processing/speedseq/root-v5-34/lib/libCore.so #3 0x00007f3d9eb4ae13 in TUnixSystem::DispatchSignals(ESignals) () from /mnt/pan/Data4/local/exome_processing/speedseq/root-v5-34/lib/libCore.so #4 #5 0x00007f3d9d103459 in TH1::SetContent(double const_) () from /mnt/pan/Data4/local/exome_processing/speedseq/root-v5-34/lib/libHist.so #6 0x000000000041fb06 in HisMaker::produceHistograms(std::basic_string<char, std::char_traits, std::allocator >_, int, std::basic_string<char, std::char_traits, std::allocator >*, int, bool) ()

#7 0x000000000040825b in main ()

The lines below might hint at the cause of the crash. If they do not help you then please submit a bug report at http://root.cern.ch/bugs. Please post the ENTIRE stack trace from above as an attachment in addition to anything else

that might help us fixing this issue.

#5 0x00007f3d9d103459 in TH1::SetContent(double const_) () from /mnt/pan/Data4/local/exome_processing/speedseq/root-v5-34/lib/libHist.so #6 0x000000000041fb06 in HisMaker::produceHistograms(std::basic_string<char, std::char_traits, std::allocator >_, int, std::basic_string<char, std::char_traits, std::allocator >*, int, bool) ()

#7 0x000000000040825b in main ()

#################################################################

Thank you,

Salendra

Salendra Singh Bioinformatics Analyst Case Comprehensive Cancer Center Case Western Reserve University 2103 Cornell Rd, WRB 3144 Cleveland, OH-44106 Email : [email protected] [email protected] PH : +1 (608)421-3696

On Fri, Aug 28, 2015 at 11:35 AM, Colby Chiang [email protected] wrote:

might be a duplicate of #40 https://github.com/hall-lab/speedseq/issues/40 . Is samtools in your PATH?

— Reply to this email directly or view it on GitHub https://github.com/hall-lab/speedseq/issues/45#issuecomment-135808286.

[cc2qe]

I think this is because you're running CNVnator on the example file, which has nonsense contigs ("20_slice"). CNVnator expects human contigs from GRCh37 (without "chr" prefixes). I think it will work if you run it on an actual human dataset.

[SalendraSingh]

Thanks Colby for the help,even I thought so, let me run a real sample. I will bother you again, if it fails. I really appreciate your help

Best regards,

Salendra Singh

Salendra Singh Bioinformatics Analyst Case Comprehensive Cancer Center Case Western Reserve University 2103 Cornell Rd, WRB 3144 Cleveland, OH-44106 Email : [email protected] [email protected] PH : +1 (608)421-3696

On Fri, Aug 28, 2015 at 12:37 PM, Colby Chiang [email protected] wrote:

I think this is because you're running CNVnator on the example file, which has nonsense contigs ("20_slice"). CNVnator expects human contigs from GRCh37 (without "chr" prefixes). I think it will work if you run it on an actual human dataset.

— Reply to this email directly or view it on GitHub https://github.com/hall-lab/speedseq/issues/45#issuecomment-135827389.