GATK SNP calling complete workflow

[thedamian]

Hi All,
I wolud like to consult my GATK workflow for a pair end Illumina data.
Generally I'm calling SNPs using following steps:

bwa aln -t 4 hg19.fa seq1.fastq > 1.sai
bwa aln -t 4 hg19.fa seq2.fastq > 2.sai
bwa sampe -r "@RG\tID:exomeID\tLB:exomeLB\tSM:exomeSM\tPL:illumina\tPU:exomePU" hg19.fa 1.sai 2.sai seq1.fastq seq2.fastq > original.sam

java -Xmx5g -jar FixMateInformation.jar I=original.sam O=fixed.sam SO=coordinate VALIDATION_STRINGENCY=LENIENT
java -Xmx5g -jar SortSam.jar I=fixed.sam SO=coordinate O=first.bam VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=true
java -Xmx5g -jar MarkDuplicates.jar I=first.bam O=marked.bam METRICS_FILE=metricsFile CREATE_INDEX=true VALIDATION_STRINGENCY=LENIENT REMOVE_DUPLICATES=true

java -Xmx5g -jar GenomeAnalysisTK.jar -nt 4 -T RealignerTargetCreator -R hg19.fa -o intervalsList -I marked.bam -known Mills_and_1000G_gold_standard.indels.hg19.vcf
java -Xmx5g -jar GenomeAnalysisTK.jar -nt 4 -T IndelRealigner -R hg19.fa -I marked.bam -targetIntervals intervalsList -known Mills_and_1000G_gold_standard.indels.hg19.vcf -o realigned.bam
java -Xmx5g -jar GenomeAnalysisTK.jar -nt 4 -T CountCovariates -l INFO -R hg19.fa -I realigned.bam -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov DinucCovariate -recalFile recalFile -knownSites dbsnp_135.hg19.vcf
java -Xmx5g -jar GenomeAnalysisTK.jar -nt 4 -T TableRecalibration -R hg19.fa -I realigned.bam -o recalibrated.bam -recalFile recalFile
java -Xmx5g -jar GenomeAnalysisTK.jar -nt 4 -T UnifiedGenotyper -R hg19.fa -I recalibrated.bam -o resultSNPs.vcf -D dbsnp_135.hg19.vcf -metrics UniGenMetrics -stand_call_conf 50.0 -stand_emit_conf 10.0 -dcov 1000 -A DepthOfCoverage -A AlleleBalance -L exomes.bed

The SNP and indels databases I've downloaded from ftp://[email protected] (bunlde -> 1.5 -> hg19)

The exome intervals I've gained using UCSC Table Browser http://genome.ucsc.edu/cgi-bin/hgTables?command=start

I'm not sure if I'm doing it in a correct way. Is my way compact enough? What about yours workflows? Are your steps look simillar? Should I use more GATK subprograms to obtain more accurate results?

Thanks in advance for suggestions

[Rocketknight]

The Mills and 1000G gold standard indels are a very stringently curated list of indels. For the purposes of indel realignment in GATK you probably want one or more of the broader sets from the GATK bundle instead, though which ones I'm not completely sure.

Overall though, your workflow looks fine. Bear in mind though, that it will only call SNPs and not indels (but since you're calling your output file resultSNPs I assume you know that already).

[thedamian]

Is the order of funtions important?
Does this:

FixMateInformation
SortSam
MarkDuplicates
RealignerTargetCreator
IndelRealigner

mean the same as this:

SortSam
FixMateInformation
RealignerTargetCreator
IndelRealigner 
MarkDuplicates

[thedamian]

Rocketknight indeed I didn't want indels, but now I need it. Do you know which option should I use to obtain a final file with SNPs and Indels?

[Rocketknight]

Yep, the only line you need to change is UnifiedGenotyper. Add the option -glm BOTH and the output VCF file will have SNPs and indels in it. Note that if you want to do Variant Quality Score Recalibration you'll have to do some things differently if you're recalibrating indels too.

[ymc]

Some indel positions are off in dbsnp135_b37.vcf. How do u deal with them??

[vd4mindia]

"The exome intervals I've gained using UCSC Table Browser http://genome.ucsc.edu/cgi-bin/hgTables?command=start"
Can you tell me how to get the exome intervals as you have referred

[thedamian]

"The exome intervals I've gained using UCSC Table Browser http://genome.ucsc.edu/cgi-bin/hgTables?command=start"
Can you tell me how to get the exome intervals as you have referred

set these paramaters: http://pokazywarka.pl/1ve6r7/ and then click "get output". Adjust your parameters and then click "get BED".

[vd4mindia]

Hi I am facing some problems with the Local realignment step around the indels, I am using the marked bam file after the PCR duplicate marking step but I am getting the following error
##### ERROR MESSAGE: Invalid command line: Cannot process the provided BAM file(s) because they were not indexed. The GATK does offer limited processing of unindexed BAMs in --unsafe mode, but this GATK feature is currently unsupported.

I donot see such errors in the forums. Is it that after marknig the PCR duplicates the bam files needs to be sorted and indexed again? or just indexing and running the target realigner step will be sufficient? Am using the GATK version 2.3 am sure its not a serious problem but am not being able to troubleshoot it. I am trying to get a bit stringent in this local indel realignment so for more specificity am actually using the DBSNP137.vcf , 1000G indel vcf and the Mills and 1000 G gold standard vcf. Please guide me about this. I am using the GATK for the first time and trying to develop a pipeline. Below is the command line am using for this step

java -Xmx14g -jar /data/PGP/gmelloni/GenomeAnalysisTK-2.3-4-g57ea19f/GenomeAnalysisTK.jar -R /scratch/GT/vdas/test_exome/exome/hg19.fa -I SRR062634_marked.sorted.bam -T RealignerTargetCreator -known /scratch/GT/vdas/test_exome/exome/databases/dbsnp_137.hg19.vcf -known /scratch/GT/vdas/test_exome/exome/databases/1000G_phase1.indels.hg19.vcf -known /scratch/GT/vdas/test_exome/exome/databases/Mills_and_1000G_gold_standard.indels.hg19.vcf -o SRR062634_marked.sorted.bam.inervals -S LENIENT

[thedamian]

@vd4mindia: I don't know, maybe try to index it. You shoud have in your directory files SRR062634_marked.sorted.bam and SRR062634_marked.sorted.bai.
If this won't help I'm not able to solve this issue.

[choishingwan]

I thought fixMate is no longer required by the GATK pipeline? And if you want to perform the fixMate then sortSam, you can run FixMate with the SO options in SortSam, then your output will be automatically sorted in one go. It is also a good idea to always include Create_Index=TRUE in the pipeline to make sure you have the index for every steps just to be safe

[vd4mindia]

@thedamian,

Yes I fixed it , I indexed it again and then made the call and the local realignment step is running now. But i need some inputs for the base quality recalibration step. In the above thread this step is written as below
java -Xmx5g -jar GenomeAnalysisTK.jar -nt 4 -T CountCovariates -l INFO -R hg19.fa -I realigned.bam -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov DinucCovariate -recalFile recalFile -knownSites dbsnp_135.hg19.vcf

Since during my Realigner Target Creator step I used dbSNP_137.vcf for known sites I should be using the same as well for the Base quality step right?

java -Xmx14g -jar /data/PGP/gmelloni/GenomeAnalysisTK-2.3-4-g57ea19f/GenomeAnalysisTK.jar -R /scratch/GT/vdas/test_exome/exome/hg19.fa -I SRR062634_marked.sorted.bam -T RealignerTargetCreator -known /scratch/GT/vdas/test_exome/exome/databases/dbsnp_137.hg19.vcf -known /scratch/GT/vdas/test_exome/exome/databases/1000G_phase1.indels.hg19.vcf -known /scratch/GT/vdas/test_exome/exome/databases/Mills_and_1000G_gold_standard.indels.hg19.vcf -o SRR062634_marked.sorted.bam.inervals -S LENIENT

As you can see the known sites /scratch/GT/vdas/test_exome/exome/databases/dbsnp_137.hg19.vcf

So my script for this step should be

For the Base calibrator( i see some place its written count covariates, which should I do) any suggestion but am following base calibrator below is the command

java -Xmx14g -jar /data/PGP/gmelloni/GenomeAnalysisTK-2.3-4-g57ea19f/GenomeAnalysisTK.jar -nt 8 -T BaseRecalibrator -R /scratch/GT/vdas/test_exome/exome/hg19.fa -knownSites /scratch/GT/vdas/test_exome/exome/databases/dbsnp_137.hg19.vcf -I SRR062634.realigned.bam -o SRR062634.realigned.recal.csv -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov ContextCovariate -S LENIENT

for table recalibration

java -Xmx14g -jar /data/PGP/gmelloni/GenomeAnalysisTK-2.3-4-g57ea19f/GenomeAnalysisTK.jar -T TableRecalibration -R /scratch/GT/vdas/test_exome/exome/hg19.fa -I SRR062634.realigned.bam -o SRR062634.realigned.bam.recal.bam -BQSR SRR062634.realigned.recal.csv -S LENIENT

Please let me know if this looks correct or not?

[vd4mindia]

Yes @choishingwan I checked and the fixmate step is retired and am not using it but I have another query in the above line please let me know and share your views

[choishingwan]

@vd4mindia: From your code, I guess you are running GATK version before 2.6 right? You are right about using the dbsnp137. From the look of it it seems correct. In case if you want to use the latest gatk, you can refer to the following site: http://gatkforums.broadinstitute.org...se-2-0-retired

Personally, I use the following code if it helps:

samtools sort <sample>.bwa.bam <sample>.bwa.sort

#Index
samtools index <sample>.bwa.sort.bam

#mark duplicate
java -Xmx5g -jar MarkDuplicates.jar INPUT=<sample>.bwa.sort.bam OUTPUT=<sample>.bwa.sort.deduped.bam METRICS_FILE=<sample>.duplicates REMOVE_DUPLICATES=TRUE VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=TRUE


#Realignment based on known insert sites (Using Java 1.7 from now on as required by GATK)
java -Xmx5g -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R Reference.fa -I <sample>.bwa.sort.deduped.arg.bam -known 1000G_phase1.indels.hg19.vcf -known Mills_and_1000G_gold_standard.indels.hg19.vcf -o <sample>.realign.intervals -S LENIENT

java -Xmx5g -jar GenomeAnalysisTK.jar -T IndelRealigner -R Reference.fa -I <sample>.bwa.sort.deduped.arg.bam -targetIntervals <sample>.realign.intervals -known 1000G_phase1.indels.hg19.vcf -known Mills_and_1000G_gold_standard.indels.hg19.vcf -o <sample>.bwa.sort.deduped.arg.realigned.bam -S LENIENT

java -Xmx5g -jar GenomeAnalysisTK.jar -T BaseRecalibrator -R Reference.fa -l INFO -I <sample>.bwa.sort.deduped.arg.realigned.bam -knownSites 1000G_phase1.indels.hg19.vcf -knownSites Mills_and_1000G_gold_standard.indels.hg19.vcf -knownSites dbsnp_137.hg19.vcf -o <sample>.recalibration_report.grp -S LENIENT

java -Xmx5g -jar GenomeAnalysisTK.jar -T PrintReads -R Reference.fa -l INFO -I <sample>.bwa.sort.deduped.arg.realigned.bam -BQSR <sample>.recalibration_report.grp -o <sample>.bwa.sort.deduped.arg.realigned.recalibrated.bam -S LENIENT

This is how we do the analysis here in our lab, not necessarily the correct way. We did use the latest GATK though, so some of the code might differ from yours.

[vd4mindia]

@thedamain,

I need some information about the exomes.bed file which you are using. I see that most of the pipelines say that they generate it from the UCSC genome browser to restrict the output to exonic sequences, is there any specific cut off that must be used to generate this bed file for the hg19 ref gene or a simple selection will do? or do i have to specify some extra bp addition for both end to catch the splice sites? Please let me know. On what criteria is the bed file selected or it is just a representation bed file of the whole ref genome restricted only to the exonic regions? Please let me know