Problem generating consensus calls

[barraza....@gmail.com]

Hi, mi name is Leticia and I´m having troubles with the consensus calls.
I´m analysing sequences from exomes using Seqmule pipeline, and I always have the same error message:

[ => SeqMule Execution Status: step 21 FAILED at mié nov 30 19:23:05 CST 2016, Extract consensus calls]
ERROR: command failed
/home/angie/SeqMule/bin/secondary/../../bin/secondary/worker /home/angie/Plex3B/Illumina14B_2/seqmule.11302016.14920.logs 21 "/home/angie/SeqMule/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -ref /home/angie/SeqMule/bin/secondary/../../database/human_g1k_v37.fasta -jmem 1750m -jexe java -t 2 -c-vcf Illumina14_result/Illumina14_bwamem.merge.realn.recal.0_gatk_hc.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.recal.0_gatk_hc.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.0_samtools.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.0_varscan.extract.vcf -p Illumina14_result/Illumina14.extract "
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
After fixing the problem, please execute 'cd /home/angie/Plex3B/Illumina14B_2' and 'seqmule run Illumina14.script' to resume analysis.
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

I have already tried many things but it always fails at the same step.
Please, help me to fix it.


Best Regards.

[Yunfei Guo]

Hi there,

Could you please send me the log in /home/angie/SeqMule/bin/secondary/../../bin/secondary/worker /home/angie/Plex3B/Illumina14B_2/seqmule.11302016.14920.logs/21 ?

Also what was your command? Thanks.

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[Leticia Barraza]

Of course:
The JOBID and the PID.3720 doesn´t say anything. The MSG says Consensus Calls and the Status just say error.
This was my comand:

seqmule pipeline -a M14B_forwardpairedT.fq.gz -b M14B_reversepairedT.fq.gz -capture /home/angie/SeqMule/database/hg19illumina/nexterarapidcapture_exome_targetedregions.bed -m -e -advanced /home/angie/SeqMule/misc/predefined_config/bwa_gatk_HaplotypeCaller_samtools_varscan.config -t 4 -prefix Illumina14

Thank you.

[Yunfei Guo]

Okay, so could you go to the directory where you ran sequel and try the following command and send me the output?

/home/angie/SeqMule/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -ref /home/angie/SeqMule/bin/secondary/../../database/human_g1k_v37.fasta  -jmem 1750m  -jexe java  -t 2 -c-vcf Illumina14_result/Illumina14_bwamem.merge.realn.recal.0_gatk_hc.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.recal.0_gatk_hc.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.0_samtools.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.0_varscan.extract.vcf -p Illumina14_result/Illumina14.extract 

Thanks.

[Leticia Barraza]

Thank you.

Done, this was the output:

##### ERROR ------------------------------------------------------------------------------------------
##### ERROR A GATK RUNTIME ERROR has occurred (version 2.3-9-gdcdccbb):
##### ERROR
##### ERROR Please visit the wiki to see if this is a known problem
##### ERROR If not, please post the error, with stack trace, to the GATK forum
##### ERROR Visit our website and forum for extensive documentation and answers to
##### ERROR commonly asked questions http://www.broadinstitute.org/gatk
##### ERROR
##### ERROR MESSAGE: Code exception (see stack trace for error itself)
##### ERROR ------------------------------------------------------------------------------------------
ERROR: Failed to split VCF by sample
NOTICE: Cleaning...

[Yunfei Guo]

Thanks for your patience. I might know what’s going on.

To confirm my guess, in the same directory, could you run?

perl -ane 'if(/^#CHROM/){print $#F,"\n"}' Illumina14_result/Illumina14_bwamem.merge.realn.recal.0_gatk_hc.extract.vcf Illumina14_result/Illumina14_bwamem.merge.recal.0_gatk_hc.extract.vcf Illumina14_result/Illumina14_bwamem.merge.0_samtools.extract.vcf Illumina14_result/Illumina14_bwamem.merge.0_varscan.extract.vcf

for i in Illumina14_result/*.bam; do echo $i; samtools view -H $i | grep -v '@SQ’;done

Thanks.

[Leticia Barraza]

Thank you.

I´m pretty confused, I run it and this was the output:

angie@angie-bioinfo:~/Plex3B/Illumina14B_2$ perl -ane 'if(/^#CHROM/){print $#F,"\n"}' Illumina14_result/Illumina14_bwamem.merge.realn.recal.0_gatk_hc.extract.vcf Illumina14_result/Illumina14_bwamem.merge.recal.0_gatk_hc.extract.vcf Illumina14_result/Illumina14_bwamem.merge.0_samtools.extract.vcf Illumina14_result/Illumina14_bwamem.merge.0_varscan.extract.vcf
9
9
9
9
angie@angie-bioinfo:~/Plex3B/Illumina14B_2$ for i in Illumina14_result/*.bam; do echo $i; samtools view -H $i | grep -v '@SQ’;done

[Yunfei Guo]

Hmmm,

I wanted to know if the number of samples is consistent across variant callers. It looks fine to me. But why didn’t you have any BAM file in the result folder? Anyway, let’s try this:

/home/angie/SeqMule/bin/seqmule update -git
#in the directory where you ran seqmule

/home/angie/SeqMule/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -ref /home/angie/SeqMule/bin/secondary/../../database/human_g1k_v37.fasta  -jmem 1750m  -jexe java  -t 2 -c-vcf Illumina14_result/Illumina14_bwamem.merge.realn.recal.0_gatk_hc.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.recal.0_gatk_hc.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.0_samtools.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.0_varscan.extract.vcf -p Illumina14_result/Illumina14.extract

Thanks.

[Leticia Barraza]

Thank you.

I updated it, and tried to run the last command you send me: 

/home/angie/SeqMule/bin/

secondary/../../bin/seqmule stats -tmpdir /tmp -ref /home/angie/SeqMule/bin/secondary/../../database/human_g1k_v37.fasta  -jmem 1750m  -jexe java  -t 2 -c-vcf Illumina14_result/Illumina14_bwamem.merge.realn.recal.0_gatk_hc.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.recal.0_gatk_hc.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.0_samtools.extract.vcf,Illumina14_result/Illumina14_bwamem.merge.0_varscan.extract.vcf -p Illumina14_result/Illumina14.extract

And this was the output:

##### ERROR ------------------------------------------------------------------------------------------
##### ERROR A GATK RUNTIME ERROR has occurred (version 2.3-9-gdcdccbb):
##### ERROR
##### ERROR Please visit the wiki to see if this is a known problem
##### ERROR If not, please post the error, with stack trace, to the GATK forum
##### ERROR Visit our website and forum for extensive documentation and answers to
##### ERROR commonly asked questions http://www.broadinstitute.org/gatk
##### ERROR
##### ERROR MESSAGE: Code exception (see stack trace for error itself)
##### ERROR ------------------------------------------------------------------------------------------
ERROR: Failed to split VCF by sample
NOTICE: Cleaning...

Regards.

[Yunfei Guo]

Could you send me the INFO level logs as well? Thanks.

[Leticia Barraza]

OK.

1
QC assesment on input. STATUS.finished
2
QC assesment on input. STATUS.finished
3
Generate QC stat. STATUS.finished
4
bwamem alignment. STATUS.finished
5
Remove duplicates in Illumina14_result/Illumina14.0.0_bwamem.sort.bam. STATUS.finished
6
Filter BAM file by mapping quality. STATUS.finished
7
Merge BAM by bwamem and Illumina14. STATUS.finished
8
gatkfull realn. STATUS.finished
9
gatkfull recal. STATUS.finished
10
gatkfull recal. STATUS.finished
11
gatk_hc variant calling. STATUS.finished
12
gatk_hc variant filtering. STATUS.finished
13
gatk_hc variant calling. STATUS.finished
14
gatk_hc variant filtering. STATUS.finished
15
samtools variant calling. STATUS.finished
16
varscan variant calling. STATUS.finished
17
Extract variants in custom regions. STATUS.finished
18
Extract variants in custom regions. STATUS.finished
19
Extract variants in custom regions. STATUS.finished
20
Extract variants in custom regions. STATUS.finished
21
Extract consensus calls. STATUS.error
22
Generate Venn digram. STATUS.finished
23
Generate alignment and coverage status. STATUS.finished
24
Generate variant stat. STATUS.waiting
25
Remove intermediate files. STATUS.finished
26
Generating html report. STATUS.waiting

Did I missed something?

Thank you.
Regards.

[Yunfei Guo]

What I am asking for is something like

INFO ...
INFO ...
INFO ...
INFO …

Generated by GATK after you run the command “/home/angie/SeqMule/bin/secondary/../../bin/seqmule stats …”.

Thanks.

[Yunfei Guo]

Don’t worry. I should have explained it better.

Do you mind if I take a look at the first 100 lines of your VCF files? If not, please run the following command and send me the vcf.gz file. Thanks!

for i in Illumina14_result/Illumina14_bwamem.merge.realn.recal.0_gatk_hc.extract.vcf Illumina14_result/Illumina14_bwamem.merge.recal.0_gatk_hc.extract.vcf Illumina14_result/Illumina14_bwamem.merge.0_samtools.extract.vcf Illumina14_result/Illumina14_bwamem.merge.0_varscan.extract.vcf; do perl -ne 'BEGIN{print "<<",$ARGV[0],">>\n"}if(/^#/){print}else{print if $c++<100}' $i;done | gzip -9 > vcf.gz


> On Dec 2, 2016, at 2:40 PM, Leticia Barraza <barraza....@gmail.com> wrote:
>
> I´m sorry, do you mean these two?
> Illumina14_bwamem.merge.realn.recal.0_gatk_hc_var_stat.txt
> Illumina14_bwamem.merge.recal.0_gatk_hc_var_stat.txt
>
> Sorry if I do not understand many things.
>
> Regards.

[Leticia Barraza]

I´m sorry, do you mean these two?

Illumina14_bwamem.merge.realn.recal.0_gatk_hc_var_stat.txt

Illumina14_bwamem.merge.recal.0_gatk_hc_var_stat.txt.

Sorry if I don´t understand many things. If the answer is yes, then I´ll upload the info they have.

Thank you

Regards. 

[Yunfei Guo]

Not these files. If you can, please run the command I sent to you and send me the vcf.gz file. Thanks.

[Leticia Barraza]

Thank you. I ran the command, I adjunt the file.

Regards,

[Yunfei Guo]

Hi

After examining your files, it seems there is a NullPointerException error from gatklite. 

I planned to add GATK full version as an alternative to gatklite. This may take some time, however. Please stay tuned. Thanks.

Nonetheless, you can still use these VCFs for analysis. It’s just that there are no consensus calling results.

<vcf.vcf>

[Leticia Barraza]

Ok, thank you so much, I´ll stay tuned.

Regards.