BICFVariantAnalysisTools

Using the BioHPC Workflow Launching Tool Astrocyte

Prioritization of Variants

SNP

INDEL

SV

Allows groups to give easy-access to their analysis pipelines via the web

Astrocyte– BioHPCWorkflowPlatform

StandardizedWorkflows

SimpleWebForms

Onlinedocumentation&resultsvisualization*

WorkflowsrunonHPCclusterwithoutdeveloperoruserneedingclusterknowledge

Slidecontribution:DavidTrudgian@BioHPC

astrocyte.biohpc.swmed.edu

https://astrocyte.biohpc.swmed.edu/brand/bicf/browse/

Alignments

FASTQTrim Adapters

Low quality ends (Q< 25)Remove short reads (<35bp)

TrimFASTQ

Trim Galore

DedupBAM

BWAPicard

Realigned, Recalibrated

BAM

GATK Reaalignment

& Base Recalibration

TypesofVariation

Germline Somatic

GermlineWorkflows

GATK VCF

SAM VCF

SV VCF

Lumpy

SS VCF

Speed Seq

GATK Haplotype

CallerSamtools Mpileup

Platypus

Platypus VCF+ + + = Union

VCFHotspot

VCF

DedupBAM

Realigned, Recalibrated

BAM

KeyFiles• VCF file — SNPs/Indels for each sample

• SampleID.annot.vcf.gz• Coverage Histogram for each sample

• SampleID.coverage_histogram.png• Cumulative Distribution Plot for all samples

• coverage_cdf.png• QC for all samples

• sequence.stats.txt• Structural Variants (unfiltered)

• SampleID.sssv.sv.vcf.gz.annot.txt

RecommendedFilteringforGermlineTesting

• ExAC POPMAX AF (0.01-0.05) -• depends on rarity of the phenotype of the

proband• Depth >10• LOF or Misssense (Coding Changes)• Alt Read Ct > 3• Mutation Allele Frequency (MAF) > 0.15• If novel:

– Called by 2+ callers

AccuracyinGIABSample

Sample Fixed Adapters SNV-SN Indel-SN SNV/Indel PPV

NA12878_1_HFVC2BBXX Fresh 1 99.6% 100% 98.9%

NA12878_2_HFVC2BBXX Fresh 1 99.6% 100 98.7%

NA12878_1_HFYWMBBXX Fresh 1 99.7% 100 98.8%

NA12878_2_HFYWMBBXX Fresh 1 99.6% 100 98.5%

GM12878_Fresh_1adapter Fresh 1 99.6% 100 98.6%

GM12878_Fresh_4adapter Fresh 4 99.6% 100 99.0%

GM12878_FFPE_1adapter FFPE 1 99.5% 100 98.5%

GM12878_FFPE_4adapter FFPE 4 99.6% 100 98.4%

Normal Tumor

Tumors are Heterogeneous

SomaticWorkflows

Mutect VCF

VarScan VCF

SS VCF

Speed SeqMuTect2 VarScan Virmid

Virmid VCF+ + + = Union

VCFShimmer

VCF

Shimmer

+

Check Mate

QC Pairs Same

Subject

DedupBAM

Realigned, Recalibrated

BAM

KeyFiles

• VCF file — SNPs/Indels for each sample• TumorID_NormalID.annot.vcf.gz

• Match Check File• TumorID_NormalID_matched.txt

RecommendedFilteringforSomaticMutations

• ExAC POPMAX AF > 0.01• Depth < 20• LOF or Misssense• MAF (Normal) * 10.< MAF (Tumor)• In COSMIC > 5 Subject

– Tumor: Alt Read Ct < 3– Tumor: MAF < 0.01

• Others– Tumor: Alt Read CT < 8– Tumor: MAF < 0.05– Tumor: Called by 2+ callers

SimulatedDatasetstoEvaluateSensitivityandSpecificityofSomaticMutationCalling

• We generated 3 sets of 18 SNVs and 16 Indels • We inserted each set into 4 normal alignment

files (1 cell line (Depth of Coverage) and 3 Saliva samples (Depth of Coverage) using BamSurgeon

• We calculated the observed mutation allele frequency (MAF) using bamreadct

• We ran our somatic mutation workflow using the original bam (Normal) and the altered bam (Tumor)

BioinformaticsSomaticMutationSensitivity

Somatic Germline FP

SNV Obs MAF > 5%

100% novel and known hotspots

80.5% novel, 88.3% known

hotspotsGermline: 0; Somatic: 0

Indel MAF > 5% and Alt Read CT >

8

86.2% novel, 95.4% known

hotspots

86.3% novel, 87.5% known

hotspotsGermline: 0; Somatic: 0

Indel MAF > 10% and Alt Read CT >

893.2% novel, 100%

known hotspots100% novel and known hotspots

Germline: 0; Somatic: 0

Createanewproject

Adddatatoyourproject

Adddatatoyourproject

ForNGSexperiment,thisisrecommended.

MakeyourdesignfileGermlineWorkflowSampleID

This ID will be used to name all workflow produced files ie S0001 will produce S0001.bam

FullPathToFqR1

Name of the fastq file R1 (not the full path)

FullPathToFqR2

Name of the fastq file R2 (not the full path)

SampleID FullPathToFqR1 FullPathToFqR2

GM12877 GM12877_S124_R1_001.fastq.gz GM12877_S124_R2_001.fastq.gz

GM12878 GM12878_S124_R1_001.fastq.gz GM12878_S124_R2_001.fastq.gz

GM12879 GM12879_S124_R1_001.fastq.gz GM12879_S124_R2_001.fastq.gz

Tipsonmakingyourdesignfile• Use tab as delimiter

– Excel save as “Text (tab delimited)”• If no SubjectID, use same number/character for

all rows• SampleID and SampleName • If no FqR2, leave them empty• For all contents, no “-”• For all contents, no spaces• Columns names MUST be exactly the same as

documented

Selectyourdatafilesandsetupworkflowandsubmit

SELECTYOURFILES

Projectisrunning

Timelineofthewholerun

Workingwiththeoutput

ExportalloutputtoAstrocyteOutgoingDirectory

HowtoTransferdatatoRunSomaticWorkflow

• Mount BioHPC on your computer (see BioHPC Introduction slides)

• Login into Cluster

MakeyourdesignfileSomaticWorkflowTumorID

NormalID

The TumorID and NormalID are used for naming the files TumorID_NormalID.annot.vcf.gz

TumorBam

Name of the bam file for the Tumor sample

NormalBam

Name of the bam file for Normal sample

TumorID NormalID TumorBAM NormalBAM

Patient1_tumor Patient1_normal p1_tumor.bam p1_normal.bam

Patient2_tumor Patient2_normal p2_tumor.bam p2_normal.bam

Commonerrorsandsolutions

• Make sure the delimiter is tab• Make sure the column name are the same

as mentioned in documentation• Make sure the file names match

Common errors and solutions

• Not all files are uploaded

• It’s about the proxy setting

• Use auto-detect proxy

Downstreamvisualizationofvariantsfromauserperspective

LingCaiQBRC/CRI-GMDP

Visualizationtools

• IGV– Somaticmutationexamplefromacancersample

• gene.iobio– Germlinemutationexamplesfromgeneticdiseasepatientsamples

IGVuserguide:http://software.broadinstitute.org/software/igv/book/export/html/6

gene.iobio tutorials:http://gene.iobio.io/help_resources.html

UsingIGVonBioHPC – gettingstarted1. LaunchaWebGUI sessionfrom“WebVisualization”under“Cloud

Services”fromBioHPC portal2. Openterminalandtypeincommand

moduleloadIGV/2.3.90;igv.sh

3. Specifygenome(shouldmatchtothereferencegenomefromwhichthevariantswerecalled)

UsingIGVonBioHPC – loadingfilesandsearch1. File->LoadfromFile->Select

2. Search• Alocus(forexample,chr5:90,339,000-90,349,000)• Agenesymbolorotherfeatureidentifier(e.g.,DPYDorNM_10000000)• Amutation(EGFR:T790MorEGFR:2369C>T)

Havetheindexfilesinthesamefolder!

UsingIGVonBioHPC – customizingvisualization• Collapsetracks,displayalternativereadingframes

• Coloralignmentsbyreadstrand• Sortalignmentsbybase

UsingIGVonBioHPC– customizingvisualization

UsingIGVonBioHPC – gettingdetailedinformation• Variant,bamcoverage,read,nucleotidepositionondifferent

transcripts

Usinggene.iobio tovisualizevariantsforgeneticdiseases

Geneticdiseases• Inherited

• Autosomal• Sex-linked

• Denovo

http://www.nature.com/nrg/journal/v18/n10/full/nrg.2017.52.html

http://gene.iobio.io/

Usinggene.iobio – loadingfiles

Selectionofindexfilesisrequiredduringupload

Usinggene.iobio – viewingrankedvariantsandcallvariants

Usinggene.iobio – examiningvariants

Usinggene.iobio – transcriptspecificannotation

Usinggene.iobio – multi-geneanalysis(importgenelist)

Usinggene.iobio – multi-geneanalysis(Viewingresult)

Usinggene.iobio – multi-geneanalysis(Filteringresult)Loss/Gainoffunctionmutations• Splice• Stopgain/loss• Startgain/loss• CodingframeshiftsNon-synonymousMutations• AminoAcidChangesVariantsLikelytoChangeExpression• TranscriptionFactorBindingSites• miRNATargets

Usinggene.iobio – multi-geneanalysis(bookmarks)

DeterminingGeneticCausesofDiseaseinExomesisNotTrivial• ThecausalvariantisidentifiedinMendelianDisease(Inherited)fromexomesisabout

30%ofcases.

• Ageneticmutationcanexpressarangeofphenotypes(Penetrance)

• Notallfunctionalmutationsareincodingregions(ncRNAsorregulatoryregions)

• Sporaticgeneticdiseasesoftenhaveapolygeniccauses,sometimeswithacombinationofinheritedandsomatic(denovo)mutation

• Mutationscanbelocalizedtoaparticulartissuetypeorregionofthebody(Mosaicism)

https://omictools.com/interpretation5-category

GUItoolsforvariantfiltering(notfree)

Commandlinetoolsforvariantfiltering(free!)

https://gemini.readthedocs.io/en/latest/#

vcftools,bcftools:manipulateVCFpeddy:ped correspondencecheck,ancestrycheck,sexcheck.directly,quicklyonVCF