Practical exercises Day 2. Variant Calling

Transcription

1 Practical exercises Day 2 Variant Calling

2 Samtools mpileup Variant calling with samtools mpileup + bcftools Variant calling with HaplotypeCaller (GATK Best Practices) Genotype GVCFs Hard Filtering Variant normalization Variant annotation (1st step)

3 Samtools mpileup Variant calling with samtools mpileup + bcftools Variant calling with HaplotypeCaller (GATK Best Practices) Genotype GVCFs Hard Filtering Variant normalization Variant annotation (1st step)

4 SAMTOOLS MPILEUP Perform Mpileup on realigned, duplicate marked sorted Bam for patient 4805 Find Samtools Mpileup tool

5 SAMTOOLS MPILEUP Perform Mpileup on realigned, duplicate marked sorted Bam for patient 4805 Find Samtools Mpileup RD.MD.RG.SORT.4805.bam

6 SAMTOOLS MPILEUP Perform Mpileup on realigned, duplicate marked sorted Bam for patient 4805 Visualize Mpileup

7 Samtools mpileup Variant calling with samtools mpileup + bcftools Variant calling with HaplotypeCaller (GATK Best Practices) Genotype GVCFs Hard Filtering Variant normalization Variant annotation (1st step)

8 SAMTOOLS MPILEUP Perform Mpileup on realigned, duplicate marked sorted Bam for patient 4805 Now, Output vcf from Mpileup

9 SAMTOOLS MPILEUP Perform Mpileup on realigned, duplicate marked sorted Bam for patient 4805 Visualize vcf output from Mpileup QUESTION : HOW MANY VARIANTS IN THE VCF?

10 SAMTOOLS MPILEUP + BCFTOOLS CALL Copy vcf from dataset_formation Drag and drop to your current history

11 SAMTOOLS MPILEUP + BCFTOOLS CALL Visualize vcf output from Mpileup + bcftools call QUESTION : HOW MANY VARIANTS ARE CALLED?

12 Samtools mpileup Variant calling with samtools mpileup + bcftools Variant calling with HaplotypeCaller (GATK Best Practices) Genotype GVCFs Hard Filtering Variant normalization Variant annotation (1st step)

13 Select GATK tools

14 Select HaplotypeCaller

15 Select intervals (for speed-up) 1 2

16 Select intervals (to speed-up process)

17 Select bam file and GVCF mode 1 2

18 Select ploidy

19 Check log status

20 Check GVCF content

21 Samtools mpileup Variant calling with samtools mpileup + bcftools Variant calling with HaplotypeCaller (GATK Best Practices) Genotype GVCFs Hard Filtering Variant normalization Variant annotation (1st step)

22 Select GATK tools

23 Select GenotypeGVCFs

24 Select input files Hold Ctrl key

25 Check VCF content (Header section)

26 Check VCF content (INFO field)

27 Check VCF content (Genotypes) Sample Genotypes and Information

28 GATK Calling Variant workflow

29 Samtools mpileup Variant calling with samtools mpileup + bcftools Variant calling with HaplotypeCaller (GATK Best Practices) Genotype GVCFs Hard Filtering Variant normalization Variant annotation (1st step)

30 Split SNPs and Indels : SelectVariants

31 Select dataset and enable advanced options 1 2

32 Select SNPs

33 Edit attributes

34 Put an informative name

35 SNPs and Indels extracted

36 GATK VariantFiltration

37 Select SNP dataset, unset mask and insert filter 1 3 2

38 Filter expression and name Boolean expression : OR ( ), AND (&&), NOT (!), etc. Operators : LESS (<), LESS OR EQUAL (<=), EQUAL (==), DIFFERENT (!=), PLUS (+), etc. Float numbers warning : use 2.0 instead of 2

39 Filter SNPs according to GATK recommandations

40 Check SNPs VCF content

41 Filter Indels according to GATK recommandations

42 Combine SNPs and Indels

43 Select SNPs and Indels datasets Hold Ctrl key

44 Check combined VCF content

45 GATK Hard Filtering workflow

46 Samtools mpileup Variant calling with samtools mpileup + bcftools Variant calling with HaplotypeCaller (GATK Best Practices) Genotype GVCFs Hard Filtering Variant normalization Variant annotation (1st step)

47 VARIANT NORMALIZATION STEP 1 Split multi-allelic variants into multiple records before normalizing Use VCFbreakCreateMulti

48 VARIANT NORMALIZATION VT Normalize

49 VARIANT NORMALIZATION Normalized variants output TO DO : FIND VARIANTS WHICH HAVE THEIR POSITIONS CHANGED

50 VARIANT COMPARISON Want to compare variants called for sample 1847 by GATK HC/Genotype GVCF and Mpileup/bcftools call? First, use GATK SelectVariants to extract variants called in 1847 only from GATK GenotypeGVCFs.vcf!

51 VARIANT COMPARISON Want to compare variants called for sample 1847 by GATK HC/Genotype GVCF and Mpileup/bcftools call? Comparison can be done by VCF-VCFintersect How many variants are called by both variant callers?

52 Samtools mpileup Variant calling with samtools mpileup + bcftools Variant calling with HaplotypeCaller (GATK Best Practices) Genotype GVCFs Hard Filtering Variant normalization Variant annotation (1st step)

53 VARIANT ANNOTATION LAUNCH VeP WHICH ASSEMBLY ARE YOU WORKING ON?

54 VARIANT ANNOTATION GO TO GRCh37 Ensembl AND LAUNCH VeP