Identifying de novo mutations with GEMINI

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Identifying de novo mutations with GEMINI Please refer to the following Github Gist to ﬁnd each command for this session. Commands should be copy/pasted from this Gist Aaron Quinlan University of Utah ! ! ! ! ! quinlanlab.org 1 https://gist.github.com/arq5x/9e1928638397ba45da2e#ﬁle-denovo-sh

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Automated tools for disease inheritance models 2

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Automated tools for disease inheritance models 2

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Automated tools for disease inheritance models 3

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Common options for disease model tools. 4

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Why search for de novo mutations? Brian O’Roak 5

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High impact variants Brian O’Roak 6

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De novo mutations 7

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How many de novo mutations should we expect? 8

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De novo mutations (rough expectations) 9

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In practice, it’s not so simple. Brian O’Roak 10

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Why are there so many artifacts? • Prior probabilities - the more interesting something is, the less likely it is to be real ! • If something can go wrong, it will. • Incorrect genotype assignment • Low coverage in one or more of the individuals in the family (especially the parents…why?) • Mismapping • Misalignment • Paralogy • Systematic artifacts • Somatic events 12

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Detective work with GEMINI 13

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The de_novo tool in GEMINI http://gemini.readthedocs.org/en/latest/content/tools.html#de-novo-identifying-potential-de-novo-mutations 14

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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 https://s3.amazonaws.com/gemini-‐tutorials/trio.trim.vep.vcf.gz > trio.trim.vep.vcf.gz $ curl https://s3.amazonaws.com/gemini-‐tutorials/denovo.ped > denovo.ped $ gemini load -‐-‐cores 4 \ -‐v trio.trim.vep.vcf.gz \ -‐t VEP \ -‐-‐skip-‐gene-‐tables -‐-‐skip-‐cadd -‐-‐skip-‐gerp-‐bp \ -‐p de_novo.ped \ ! trio.trim.vep.denovo.db Note: copy and paste the full command from the Github Gist to avoid errors ~8 minutes http://gemini.readthedocs.org/en/latest/content/preprocessing.html#step-1-split-left-align-and-trim-variants 15

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Normalization and decomposition are required preprocessing steps Variant decomposition http://genome.sph.umich.edu/wiki/Vt#Decompose Variant normalization http://genome.sph.umich.edu/wiki/File:Normalization_mnp.png http://gemini.readthedocs.org/en/latest/ content/preprocessing.html#preprocessing- and-loading-a-vcf-ﬁle-into-gemini Details can be found in the GEMINI documentation  16

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Running the de_novo tool $ gemini de_novo trio.trim.vep.denovo.db Note: copy and paste the full command from the Github Gist 17

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Information overload There are currently 115 columns in the variants table. Perhaps a bit of overkill for a typical analysis http://gemini.readthedocs.org/en/latest/content/database_schema.html#the-variants-table 18

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Limit the attributes returned w/ the -‐-‐columns option. $ gemini de_novo \ -‐-‐columns "chrom, start, end, ref, alt, \ filter, qual, gene, impact" \ trio.trim.vep.denovo.db Note: copy and paste the full command from the Github Gist 19

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Limit the attributes returned w/ the -‐-‐columns option. http://gemini.readthedocs.org/en/latest/content/tools.html#common-args-common-arguments $ gemini de_novo \ -‐-‐columns "chrom, start, end, ref, alt, \ filter, qual, gene, impact" \ trio.trim.vep.denovo.db Note: copy and paste the full command from the Github Gist 20

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Better, but there are still so many (likely false) candidates. $ gemini de_novo \ -‐-‐columns "chrom, start, end, ref, alt, \ filter, qual, gene, impact" \ trio.trim.vep.denovo.db | wc -‐l Note: copy and paste the full command from the Github Gist 771 candidates! 21

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Causes of erroneous genotype predictions: lack of depth 22

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Let’s enforce a minimum sequence depth for each subject: -‐d $ gemini de_novo \ -‐-‐columns "chrom, start, end, ref, alt, \ filter, qual, gene, impact" \ -‐d 15 \ trio.trim.vep.denovo.db | wc -‐l Note: copy and paste the full command from the Github Gist 676 candidates 23

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Causes of erroneous genotype predictions: low quality variants 24

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Require that the mutation passes GATK QC with -‐-‐filter $ gemini de_novo \ -‐-‐columns "chrom, start, end, ref, alt, \ filter, qual, gene, impact" \ -‐d 15 \ -‐-‐filter "filter is NULL" \ trio.trim.vep.denovo.db | wc -‐l Note: copy and paste the full command from the Github Gist 55 candidates 25

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Require that the mutation is likely to have functional consequence 26

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Require that the mutation is likely to have functional consequence $ gemini de_novo \ -‐-‐columns "chrom, start, end, ref, alt, \ filter, qual, gene, impact" \ -‐d 15 \ -‐-‐filter "filter is NULL and impact_severity != ‘LOW’” \ trio.trim.vep.denovo.db | wc -‐l Note: copy and paste the full command from the Github Gist 13 candidates 27

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Require that the mutation is not likely to be a known polymorphism 28

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Require that the mutation is not likely to be a known polymorphism Note: copy and paste the full command from the Github Gist $ gemini de_novo \ -‐-‐columns "chrom, start, end, ref, alt, \ filter, qual, gene, impact" \ -‐d 15 \ -‐-‐filter "filter is NULL \ and is_coding = 1 and impact_severity != ‘LOW’ \ and (aaf_1kg_eur <= 0.005 or aaf_1kg_eur is NULL) \ and (aaf_esp_ea <= 0.005 or aaf_esp_ea is NULL)" \ trio.trim.vep.denovo.db | wc -‐l 6 candidates! 29

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6 candidates. Which is causal? Requires manual inspection… chrom start end ref alt filter qual gene impact variant_id family_id family_members family_genotypes samples family_count chr2 96525735 96525736 T C None 1929.31 ANKRD36C non_syn_coding 2537 family1 1805,1847,4805 T/T,T/T,T/C 4805 1 chr2 96525749 96525750 T A None 1513.36 ANKRD36C non_syn_coding 2538 family1 1805,1847,4805 T/T,T/T,T/A 4805 1 chr2 96525754 96525755 A T None 1699.28 ANKRD36C non_syn_coding 2539 family1 1805,1847,4805 A/A,A/A,A/T 4805 1 chr15 41229630 41229631 T G None 2116.49 DLL4 non_syn_coding 7892 family1 1805,1847,4805 T/T,T/T,T/G 4805 1 chr17 55183812 55183813 A G None 2155.84 AKAP1 non_syn_coding 13311 family1 1805,1847,4805 A/A,A/A,A/G 4805 1 chr22 43027436 43027437 C T None 1320.03 CYB5R3 non_syn_coding 16718 family1 1805,1847,4805 C/C,C/C,C/T 4805 1 Phenotype: blue skin disease $ gemini de_novo \ -‐-‐columns "chrom, start, end, ref, alt, \ filter, qual, gene, impact" \ -‐d 15 \ -‐-‐filter "filter is NULL \ and is_coding = 1 and impact_severity != ‘LOW’ \ and (aaf_1kg_eur <= 0.005 or aaf_1kg_eur is NULL) \ and (aaf_esp_ea <= 0.005 or aaf_esp_ea is NULL)" \ trio.trim.vep.denovo.db Which gene can we rule out at a glance? 30

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Load the following files into IGV (Load from URL) and inspect your candidates BAM alignment files: ! https://s3.amazonaws.com/gemini-‐tutorials/1805.workshop.bam https://s3.amazonaws.com/gemini-‐tutorials/1847.workshop.bam https://s3.amazonaws.com/gemini-‐tutorials/4805.workshop.bam VCF variant file: ! https://s3.amazonaws.com/gemini-‐tutorials/trio.trim.vep.vcf.gz ! 31