talks

Indel-basedRealignment

Improvingtheoriginalalignmentsofthereadsbasedonmul8plesequence

(re-)alignment

Analysis-Ready Variants

111Raw Reads

Raw Variants IndelsSNPs

Analysis-ReadyReads

Indel Realignment

Base Recalibration

SNPs & Indels

Variants

IndelsSNPs

VariantAnnotation

Variant Evaluation

look good?

use in projecttroubleshoot

111Analysis-ReadyReads

Genotype Likelihoods

Joint Genotyping

Analysis-Ready

No

n-G

AT

K

Mark Duplicates& Sort (Picard)

Var. Calling HC in ERC mode

separately per variant type

Variant Recalibration

Map to Reference

BWA mem GenotypeRefinement

Data Pre-processing Variant Discovery>> >> Callset Refinement

YouarehereintheGATKBestPrac8cesworkflowforgermlinevariantdiscovery

InDels=inser8on/dele8on

AGCTAGGGTC AGCTAGGGTC

AGCTAGGGTC

TTC

AGCGGTC

Refseq

Sampleseq

Inser&on Dele&on

Theproblemwewanttofix

Severalconsecu3ve“SNPs”onlyfoundonreadsendingonthe

rightofthehomopolymer

Severalconsecu3ve“SNPs”onlyfoundonreadsendingonthe

le;ofthehomopolymer 7bp“T”

homopolymerrun

Addinga1-bpinser3onbringssanityto

theen3realignment

AlignmentbyBWA

A;errealignment

Whydoesthishappen?

þ  Localrealignmentaroundindels->mostparsimoniousalignment

þ  Improvesaccuracyofseveraldownstreamprocessingsteps

Ref T A C C C A T T T T T T T C T A A A A G C T BWA C C A T T T T T T C T A A A A A C T IR C C A – T T T T T T C T A A A A A C T

CATGCA CCA TGCA G

ref

del

ins

•  Mapperscannot“see”indelsnearendsofreads•  Becausemismatchesare“cheaper”thanagapinthis

context

Missmatch=-1Opengap=-3

Howdoweiden8fywhererealignmentisneeded?

•  Knownsites(e.g.dbSNP,1000Genomes)

•  Indelsseeninoriginalalignments(inCIGARs)

•  Siteswhereevidencesuggestsahiddenindel

-Entropycalcula8oniden8fies“messyareas”

1.Findthebestalternateconsensussequencethat,togetherwiththereference,bestfitsthereadsinapile(maximumof1indel)

3.Ifbestalternateconsensusissufficientlybe`erthantheoriginalalignments(usingLODscorethreshold)->acceptproposedrealignment

2.Scoreforalternateconsensus=totalsumofqualityscoresofmismatchingbases

Howdoestherealignmentalgorithmwork?

AAGAGTAGRef:

AAG---AGTAG

AAGAGTAG

Readpileconsistentwitha3bpinser8on

ReadpileconsistentwiththereferencesequenceRealigning

determineswhichisbe`er

ThreeadjacentSNPs

IndelRealignmentsteps/tools

•  Iden8fywhatregionsneedtoberealigned➔ RealignerTargetCreator

•  Performtheactualrealignment

➔ IndelRealigner

RealignerTargetCreator

•  Pre-processingsteptofindintervalsthatmayneedrealignment

•  InputBAMfilenotnecessaryifprocessingonlyatknownindels

•  Usingalistofknownindelswillbothspeedupprocessingandimproveaccuracy,butisnotrequired

Input BAM Target Intervals

Realigned BAM

RealignerTargetCreator

IndelRealigner

Known Sites

java –jar GenomeAnalysisTK.jar \ –T RealignerTargetCreator \ –R human.fasta \ –I original.bam \ –known indels.vcf \ –o realigner.intervals

IndelRealigner

•  A`emptsrealignmentatRealignerTargetCreatortargetintervals

•  Mustusesameinputfile(s)usedinRealignerTargetCreatorstep

•  Processingop8ons-  Onlyatknownindels:muchfaster,

accuratefor~90-95%ofindels-  AtindelsseenintheoriginalBAM

alignments:therecommendedmode

-  UsingfullSmith-Watermanrealignment:mostaccurate,butheavycomputa8onalcostandnotreallynecessarywiththenewtechs

Input BAM Target Intervals

Realigned BAM

IndelRealigner

Known Sites

java –jar GenomeAnalysisTK.jar \ –T IndelRealigner \ –R human.fasta \ –I original.bam \ –known indels.vcf \ –targetIntervals realigner.intervals \ –o realigned.bam

DePristo, M., Banks, E., Poplin, R. et. al, A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Gen.

ThisiswhatarealignedBAMlookslike

Before AierOlddata

(lowerquality)

Newdata(higherquality)

CanIseetheeffectsofrealignment?

•  IndelRealignerchangestheCIGARstringofrealignedreadsbutmaintainstheoriginalCIGAR(withOCtag)

->Cangrepforrealignedregionsandviewingenomebrowser(IGV)

20GAVAAXX100126:1:67:10041:180738 99 20 10011431 70 87M1D14M= 10011720 390

TTAAATGTGTTTATCTATTGTTCTACTATTCAGTTACCTGATTATAAAATCAAAGATTATTTCATGAAACTCAGTACCCCTTCAGGGAAAAAAAAAAAAAT

HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGGGGGGGG X0:i:1 X1:i:0 MC:Z:101M OC:Z:101M PG:Z:MarkDuplicates RG:Z:20GAV.1XG:i:0 AM:i:37

NM:i:1SM:i:37 XM:i:1 XO:i:0

BQ:Z:@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@cccddc``a`^\[Y MQ:i:60 XT:A:

Isrealignments8llnecessarywithlatestsoiware?

•  Variantcallerswithreassemblystep(HaplotypeCaller,MuTect2,Platypus)donotrequireindelrealignment

•  BUTpoten8alimprovementforBaseQualityScoreRecalibra8onwhenrunonrealignedBAMfiles(ar8factualSNPsarereplacedwithrealindels).

•  Alsos8llusefulforlegacytools–  UnifiedGenotyper–  MuTect1

Analysis-Ready Variants

111Raw Reads

Raw Variants IndelsSNPs

Analysis-ReadyReads

Indel Realignment

Base Recalibration

SNPs & Indels

Variants

IndelsSNPs

VariantAnnotation

Variant Evaluation

look good?

use in projecttroubleshoot

111Analysis-ReadyReads

Genotype Likelihoods

Joint Genotyping

Analysis-Ready

No

n-G

AT

K

Mark Duplicates& Sort (Picard)

Var. Calling HC in ERC mode

separately per variant type

Variant Recalibration

Map to Reference

BWA mem GenotypeRefinement

Data Pre-processing Variant Discovery>> >> Callset Refinement

YouarehereintheGATKBestPrac8cesworkflowforgermlinevariantdiscovery

talks

Furtherreading

h`p://www.broadins8tute.org/gatk/guide/best-prac8ces

h`p://www.broadins8tute.org/gatk/guide/ar8cle?id=38

h`ps://www.broadins8tute.org/gatk/gatkdocs/org_broadins8tute_gatk_tools_walkers_indels_IndelRealigner.php

h`ps://www.broadins8tute.org/gatk/gatkdocs/

org_broadins8tute_gatk_tools_walkers_indels_RealignerTargetCreator.php