Identifying de novo mutations with GEMINI

1.

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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4.


5.

Common options for disease model tools. 4

6.

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

7.

High impact variants Brian O’Roak 6

8.

De novo mutations 7

9.

How many de novo mutations should we expect? 8

10.

De novo mutations (rough expectations) 9

11.

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

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11

13.

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

16.

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

17.

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

18.

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

19.

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

20.

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

22.

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

24.

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

26.

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

Identifying de novo mutations with GEMINI

Identifying de novo mutations with GEMINI

Aaron Quinlan

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Transcript

Identifying de novo mutations with GEMINI Please refer to the

Automated tools for disease inheritance models 2

Automated tools for disease inheritance models 2

Automated tools for disease inheritance models 3

Common options for disease model tools. 4

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

High impact variants Brian O’Roak 6

De novo mutations 7

How many de novo mutations should we expect? 8

De novo mutations (rough expectations) 9

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

11

Why are there so many artifacts? • Prior probabilities -

Detective work with GEMINI 13

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

Create a GEMINI database from a VCF Notes: 1. The

Normalization and decomposition are required preprocessing steps Variant decomposition http://genome.sph.umich.edu/wiki/Vt#Decompose

Running the de_novo tool $ gemini de_novo trio.trim.vep.denovo.db Note: copy

Information overload There are currently 115 columns in the variants

Limit the attributes returned w/ the -‐-‐columns option. $ gemini

Limit the attributes returned w/ the -‐-‐columns option. http://gemini.readthedocs.org/en/latest/content/tools.html#common-args-common-arguments $

Better, but there are still so many (likely false) candidates.

Causes of erroneous genotype predictions: lack of depth 22

Let’s enforce a minimum sequence depth for each subject: -‐d

Causes of erroneous genotype predictions: low quality variants 24

Require that the mutation passes GATK QC with -‐-‐filter $

Require that the mutation is likely to have functional consequence

Require that the mutation is likely to have functional consequence

Require that the mutation is not likely to be a

Require that the mutation is not likely to be a

6 candidates. Which is causal? Requires manual inspection… chrom

Load the following ﬁles into IGV (Load from URL) and