Identifying recessive gene candidates with GEMINI

Transcription

1 Identifying recessive gene candidates with GEMINI Aaron Quinlan University of Utah! quinlanlab.org Please refer to the following Github Gist to find each command for this session. Commands should be copy/pasted from this Gist 1

2 Compound heterozygote detective work with GEMINI 2

3 Compound het refresher 3

4 Example compound heterozygote Dad Mom G a G G Kid C C t C a G C t T 4

5 Phasing genotypes 5 Jessica Chong

6 The result of phasing by transmission 6 Jessica Chong

7 Phasing a VCF file by transmission with GATK Jessica Chong 7 Jessica Chong

8 Phasing a VCF file by transmission with GATK Jessica Chong G/G 7 Jessica Chong

9 Phasing a VCF file by transmission with GATK Jessica Chong G/G 7 Jessica Chong

10 Phasing a VCF file by transmission with GATK Jessica Chong G/G 7 Jessica Chong

11 Phasing a VCF file by transmission with GATK Jessica Chong G/G? 7 Jessica Chong

12 Phasing a VCF file by transmission with GEMINI 8

13 Phasing a VCF file by transmission with GEMINI G/G 8

14 Phasing a VCF file by transmission with GEMINI G/G 8

15 Phasing a VCF file by transmission with GEMINI G/G 8

16 Phasing a VCF file by transmission with GEMINI G/G? 8

17 Phasing by transmission C/C 9 * Convention for phased genotype is maternal allele first

18 Phasing by transmission C/C G A C T Both sites phasable: high confidence as deleterious alleles on different chromosomes 9 * Convention for phased genotype is maternal allele first

19 Phasing by transmission C/C G/A C/C G A C T Both sites phasable: high confidence as deleterious alleles on different chromosomes 9 * Convention for phased genotype is maternal allele first

20 Phasing by transmission C/C G/A C/C G A C T G A T C Both sites phasable: high confidence as deleterious alleles on different chromosomes Both sites phasable: yet exclude as deleterious alleles on same chromosomes 9 * Convention for phased genotype is maternal allele first

21 Phasing by transmission C/C G/A C/C G A C T G A T C Both sites phasable: high confidence as deleterious alleles on different chromosomes Both sites phasable: yet exclude as deleterious alleles on same chromosomes G/G 9 * Convention for phased genotype is maternal allele first

22 Phasing by transmission C/C G/A C/C G A C T G A T C Both sites phasable: high confidence as deleterious alleles on different chromosomes Both sites phasable: yet exclude as deleterious alleles on same chromosomes G/G G A? Only one site is phasable: lower confidence but cannot necessarily exclude. 9 * Convention for phased genotype is maternal allele first

23 Phasing by transmission C/C G/A C/C G A C T G A T C Both sites phasable: high confidence as deleterious alleles on different chromosomes Both sites phasable: yet exclude as deleterious alleles on same chromosomes G/G G A? Only one site is phasable: lower confidence but cannot necessarily exclude. 9 * Convention for phased genotype is maternal allele first

24 Phasing by transmission C/C G/A C/C G A C T G A T C Both sites phasable: high confidence as deleterious alleles on different chromosomes Both sites phasable: yet exclude as deleterious alleles on same chromosomes G/G G A??? Only one site is phasable: lower confidence but cannot necessarily exclude. Neither site is phasable: lower confidence but cannot necessarily exclude (recombination?). 9 * Convention for phased genotype is maternal allele first

25 Compound het test case 10 Jessica Chong

26 The comp_hets tool in GEMINI Requires a PED file #family_id sample_id paternal_id maternal_id sex phenotype family family family

27 Create a GEMINI database from a VCF Notes: 1. The VCF has been normalized and decomposed with VT 2. The VCF has been annotated with VEP. $ curl tutorials/trio.trim.vep.vcf.gz > trio.trim.vep.vcf.gz $ curl tutorials/recessive.ped > recessive.ped $ gemini load - - cores 4\ - v trio.trim.vep.vcf.gz \ - t VEP \ - - skip- gene- tables \! - p recessive.ped \ trio.trim.vep.recessive.db Note: copy and paste the full command from the Github Gist to avoid errors

28 Running the comp_hets tool. gemini comp_hets trio.trim.vep.recessive.db Note: copy and paste the full command from the Github Gist 13

29 Again, we can limit the attributes returned w/ the --columns option. gemini comp_hets --columns "chrom, start, end, gene, impact, cadd_raw" trio.trim.vep.recessive.db Note: copy and paste the full command from the Github Gist chrom start end gene impact cadd_raw variant_id family_id family_members family_genotypes samples family_count comp_het_id priority chr AAK1 UTR_3_prime family1 1805;unaffected,1847;unaffected,4805;affected G/C,G/C,G/C _1638_ chr AAK1 UTR_5_prime family1 1805;unaffected,1847;unaffected,4805;affected G/G,G/A,G A _1638_ chr AAK1 UTR_3_prime family1 1805;unaffected,1847;unaffected,4805;affected A/C,A/C,A/C _1637_ chr AAK1 UTR_3_prime family1 1805;unaffected,1847;unaffected,4805;affected G/C,G/C,G/C _1637_ chr AAK1 UTR_3_prime family1 1805;unaffected,1847;unaffected,4805;affected T/T,T/T,T/C _1636_ chr AAK1 UTR_5_prime family1 1805;unaffected,1847;unaffected,4805;affected G/G,G/A,G A _1636_ chr AAK1 UTR_3_prime family1 1805;unaffected,1847;unaffected,4805;affected G/C,G/C,G/C _1638_ chr AAK1 UTR_5_prime None 1645 family1 1805;unaffected,1847;unaffected,4805;affected AT/A,AT/A,AT/A _1638_ chr AAK1 UTR_3_prime family1 1805;unaffected,1847;unaffected,4805;affected T/T,T/T,T/C _1636_

30 Start with highest priority compound heterozygote candidates C/C 15

31 Start with highest priority compound heterozygote candidates C/C G A C T Both sites phasable: high confidence as deleterious alleles on different chromosomes 15

32 Restrict to highest priority (i.e, priority==1) candidates $ gemini comp_hets \ --columns "chrom, start, end, gene, impact, cadd_raw" \ trio.trim.vep.recessive.db \ awk '$14==1' \ head chrom start end gene impact cadd_raw variant_id family_id family_members family_genotypes samples family_count comp_het_id priority chr ACAN non_syn_coding family1 1805;unaffected,1847;unaffected,4805;affected C/A,C/C,A C _9519_ chr ACAN non_syn_coding family1 1805;unaffected,1847;unaffected,4805;affected,,A G _9519_ chr ACAN non_syn_coding family1 1805;unaffected,1847;unaffected,4805;affected C/A,C/C,A C _9519_ chr ACAN splice_region family1 1805;unaffected,1847;unaffected,4805;affected G/G,G/A,G A _9519_ chr ACOXL non_syn_coding family1 1805;unaffected,1847;unaffected,4805;affected C/C,,C T _3247_ chr ACOXL non_syn_coding family1 1805;unaffected,1847;unaffected,4805;affected,C/C,T C _3247_ chr AKAP1 non_syn_coding family1 1805;unaffected,1847;unaffected,4805;affected,C/C,T C _13305_ chr AKAP1 synonymous_coding family1 1805;unaffected,1847;unaffected,4805;affected G/G,G/A,G A _13305_ chr ALK non_syn_coding family1 1805;unaffected,1847;unaffected,4805;affected T/T,T/G,T G _839_841 1 chr ALK non_syn_coding family1 1805;unaffected,1847;unaffected,4805;affected A/T,,T A _839_841 1 $ gemini comp_hets \ --columns "chrom, start, end, gene, impact, cadd_raw" \ trio.trim.vep.recessive.db \ awk '$14==1' \ wc -l 612 lines Note: copy and paste the full command from the Github Gist Each compund heterozygote is a set of two lines, so we have 306 (612 / 2) compound heterozygote candidates 16

33 So many candidates. Time to start --filtering! $ gemini comp_hets \ --columns "chrom, start, end, gene, impact, cadd_raw" \ --filter "impact_severity!= 'LOW'" \ trio.trim.vep.recessive.db \ awk '$14==1' \ wc -l Note: copy and paste the full command from the Github Gist 260 lines (130 comp_hets) 17

34 Use ESP and ExAC to focus on rare variants $ gemini comp_hets \ --columns "chrom, start, end, gene, impact, cadd_raw" \ --filter "impact_severity!= 'LOW' \ and ((aaf_esp_ea <= 0.01 or aaf_esp_ea is NULL) \ and (aaf_exac_all <= 0.01 or aaf_exac_all is NULL)) \ trio.trim.vep.recessive.db \ awk '$14==1' \ wc -l Note: copy and paste the full command from the Github Gist 8 lines, 4 comp_hets 18

35 Use ESP and ExAC to focus on rare variants $ gemini comp_hets \ --columns "chrom, start, end, gene, impact, cadd_raw" \ --filter "impact_severity!= 'LOW' \ and ((aaf_esp_ea <= 0.01 or aaf_esp_ea is NULL) \ and (aaf_exac_all <= 0.01 or aaf_exac_all is NULL)) \ trio.trim.vep.recessive.db \ awk '$14==1' Note: copy and paste the full command from the Github Gist chr GAA non_syn_coding family1 1805;unaffected,1847;unaffected4805;affected T/T,T/C,T C _14401_ chr GAA non_syn_coding family1 1805;unaffected,1847;unaffected4805;affected G/A,G/G,A G _14401_ chr HS6ST1 non_syn_coding family1 1805;unaffected,1847;unaffected4805;affected C/A,C/C,A C _3657_ chr HS6ST1 non_syn_coding family1 1805;unaffected,1847;unaffected4805;affected G/G,G/C,G C _3657_ chr PRR5- ARHGAP8 non_syn_coding family1 1805;unaffected,1847;unaffected4805;affected G/G,G/T,G T _16838_ chr PRR5- ARHGAP8 non_syn_coding family1 1805;unaffected,1847;unaffected4805;affected,C/C,T C _16838_ chr THSD4 non_syn_coding family1 1805;unaffected,1847;unaffected4805;affected C/C,,C T _8777_ chr THSD4 non_syn_coding family1 1805;unaffected,1847;unaffected4805;affected G/A,G/G,A G _8777_

36 Load the following files into IGV (Load from URL) and inspect your candidates BAM alignment files:! tutorials/1805.workshop.bam tutorials/1847.workshop.bam tutorials/4805.workshop.bam VCF variant file:! tutorials/trio.trim.vep.vcf.gz! 20

37 Finding recessive genes with GEMINI assuming consanguinuity 21

38 The autosomal_recessive tool. 22

39 The autosomal_recessive tool. Default behavior: 23

40 The autosomal_recessive tool. The - - min- kindreds option: This specifies the number of families required to have a variant in the same gene in order for it to be reported. For example, we may only be interested in candidates where at least 2 families have a variant in that gene. 24

41 The autosomal_recessive tool. - - filter for variants with potential functional consequence: 25

42 The autosomal_recessive tool: other options The gt- pl- max option: In order to eliminate less confident genotypes, it is possible to enforce a maximum PL value for each sample. On this scale, lower values indicate more confidence that the called genotype is correct. 10 is a reasonable value: What is the PL? What is a Phred scaled genotype likelihood? 26

43 The autosomal_recessive tool: other options What is a Phred scaled genotype likelihood? Example calculation based on the GATK HaplotypeCaller 27

44 Runs of homozygosity Method 1: intersecting with previously known regions 28

45 Intersect with observed homozygosity region(s) (Example commands) 1. Tabix a BED file with the observed homozygosity regions bgzip homoz_region.bed tabix -p bed homoz_region.bed.gz 2. Use the annotate tool to flag variants that overlap these regions. gemini annotate -f homoz_region.bed.gz \ c homoz_region \ -t boolean \ AR.db 3. Filter variants for those that overlap these regions. gemini autosomal_recessive AR.db --columns "chrom, start, end, ref, alt, filter, qual, gene, impact, aaf_esp_ea, aaf_1kg_eur - filter "filter is NULL and aaf_esp_ea < 0.1 and (impact_severity = 'HIGH' or impact_severity = 'MED') and region ==1 29

46 Runs of homozygosity Method 2: search for runs of homozygosity 30

47 Intersect with observed homozygosity region(s) Run the roh tool to search for candidate runs of homozygosity. gemini roh AR.db 31

48 Intersect with observed homozygosity region(s) Run the roh tool to search for candidate runs of homozygosity. gemini roh AR.db sort -k7nr chrom start end sample num_of_snps density_per_kb run_length_in_bp! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S ! chr S

49 Caveats when screening for runs of homozygosity 1. Difficult with exome data. Density of markers. 2. Shorter runs of homozygosity happen often by chance. 3. Density of homozygotes is important. 33