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Path-OS: The Curation of
Cancer Samples
Ken Doig – Bioinformatics Research Core
Peter MacCallum Cancer Centre
ken.doig@petermac.org
Agenda
•  Context
•  System overview
•  Amplicon Panels
•  Filtering
•  Futures
22 May 2014 HVP5 Path-OS 2
The Context
What we do
•  Peter MacCallum Cancer Centre
–  Molecular Pathology Department
•  Provide pathology services to the hospital and ext. labs.
•  Blood and tumour tissue samples
•  Targeted genetic sequencing using amplicon panels
•  Between 4-50 cancer specific genes
•  Looking for needles in haystacks
•  Very sensitive assays
...
...
AAAAGCAGGT TATATAGGCT AAATAGAACT AATCATTGTT TTAGACATAC TTATTGACTC TAAGAGGAAA
TCATAATGCT TGCTCTGATA GGAAAATGAG ATCTACTGTT TTCCTTTACT TACTACACCT CAGATATATT
TCTTCATGAA GACCTCACAG TAAAAATAGG TGATGTTGGT AGCTAGGAGT GAAATCTCGA TGGAGTGGGT
CCCATCAGTT TGAACAGTTG TCTGGATCCA TTTTGTGGAT GGTAAGAATT GAGGCTATTT TTCCACTGAT
TAGTTCCCAG TATTCACAAA AATCAGTGTT CTTATTTTTT ATGTAAATAG ATTTTTTAAC TTTTTTCTTT
...
...
22 May 2014 HVP5 Path-OS 4
Peter Mac Curation Scope
•  Automate the processing from sequencer
to draft report
•  Automate curation evidence collection
•  Sanitise data from external sources
•  Automated reporting
•  Best practice software engineering
22 May 2014 HVP5 Path-OS 5
The System
Patient
Sample
Genologics
wet lab LIMS
External
Variant DBs
•  COSMIC
•  Ensembl
•  Annovar
•  UCSC
•  Clinvar
etc
Loader
Pipeline data repository
FASTQ
BAM
VCF
VEP
Pipeline PipeCleaner
PathOS
Web Server
Pipeline
Validation QC
Reporting
Pipeline
configuration
Sequencers
ETL
configuration
Periodic DB download
and integration
Sequencing QC
Clinical Reporting
Read QC
Synthtetic Reads
Known samples
Filtering
configuration
Users
•  molecular scientists
•  clinicians
•  researchers
Export curated variants
to global repositories
Hospital Records
22 May 2014 HVP5 Path-OS 7
Path-OS Overview
Run QC
This run in the context of
past runs of the same panel
Per sample read yield
highlighting below average
Amplicon performance
read distribution
22 May 2014 HVP5 Path-OS 8
Classification
Page
22 May 2014 HVP5 Path-OS 9
Automatically
generated
classification
Justification
free text field
Check boxes for
variant evidence
Evidence type
tool tip
Classifying variants for the clinic
22 May 2014 HVP5 Path-OS 10
C5: Pathogenic
C4: Likely pathogenic
C3: Unknown
pathogenicity
C2: Unlikely pathogenic
C1: Not pathogenic
5 Level Classification
Stand alone
Strong
Supporting
Criteria
or or
Pathogenic
evidence
Stand alone
Strong
Supporting
Benign
evidence
=
or =
or =or
or =
All other combinations =
Software Components
Role Package Overview
Language Groovy Java on steroids, powerful JVM language
Web Framework Grails Rich Groovy based high productivity framework
Code repository GitLab Private GitHub instance
Database MySQL Widely adopted RDB, good performance
User interactivity Javascript plugins Leverage best available js e.g. Jquery, Google Charts
Object Persistence Hibernate Java standard for mapping POJOs to RDB
Searching Lucene Full-featured text search engine
IoC Layer Spring Java standard for inversion of control
IDE IntelliJ Comprehensive developers environment for Java etc
Build Management Gradle Groovy based DSL leverages Ant and CoC
DB Migration Mgmt LiquiBase DSL based data migration tool for schema versioning
Issue Management Jira Best of breed issue management tracker
LIMS GenoLogics User friendly LIMS for NGS
Aligner Primal Peter Mac in-house amplicon aligner, tuned for amplicons
Variant Caller VarScan 2 Suitable for somatic and germline (for now)
Annotation Ensembl, Annovar Rich set of annotations for multiple transcripts
22 May 2014 HVP5 Path-OS 11
The Panels
Somatic Panel
22 May 2014 HVP5 Path-OS 13
Oncogenes
Tumour suppressors
Consequence type
Other
Missense
Frame shift
Splice site
Stop gained
Gene type
6.2$%$
25.5$%$
0$
20$
40$
60$
80$
100$
Single$ Duplicate$
Variant'Allele'Frequency'(%)'
Variant'Allele'Frequency'for'Soma7c'Panel'Replicates'
Somatic
Replicates
22 May 2014 HVP5 Path-OS 14
0"
5"
10"
15"
20"
25"
30"
0'"<10" 10'<20" 20'"<30" 30'"<40" 40'"<50" 50'"<60" 60'"<70" 70'"<80" 80'"<90" 90'100"
Mean%difference%in%variant%frequency%between%replicates%(%)%
Variant%Read%Frequency%%%(Error:%S.E.M.)%
Replicate%Variant%Frequency%Differences%
72% 28% n=14,771
Amplicon artifacts
22 May 2014 HVP5 Path-OS 15
The Filtering
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig
The Curation of Molecular Pathology Cancer Samples - Kenneth Doig

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The Curation of Molecular Pathology Cancer Samples - Kenneth Doig

  • 1. Path-OS: The Curation of Cancer Samples Ken Doig – Bioinformatics Research Core Peter MacCallum Cancer Centre ken.doig@petermac.org
  • 2. Agenda •  Context •  System overview •  Amplicon Panels •  Filtering •  Futures 22 May 2014 HVP5 Path-OS 2
  • 4. What we do •  Peter MacCallum Cancer Centre –  Molecular Pathology Department •  Provide pathology services to the hospital and ext. labs. •  Blood and tumour tissue samples •  Targeted genetic sequencing using amplicon panels •  Between 4-50 cancer specific genes •  Looking for needles in haystacks •  Very sensitive assays ... ... AAAAGCAGGT TATATAGGCT AAATAGAACT AATCATTGTT TTAGACATAC TTATTGACTC TAAGAGGAAA TCATAATGCT TGCTCTGATA GGAAAATGAG ATCTACTGTT TTCCTTTACT TACTACACCT CAGATATATT TCTTCATGAA GACCTCACAG TAAAAATAGG TGATGTTGGT AGCTAGGAGT GAAATCTCGA TGGAGTGGGT CCCATCAGTT TGAACAGTTG TCTGGATCCA TTTTGTGGAT GGTAAGAATT GAGGCTATTT TTCCACTGAT TAGTTCCCAG TATTCACAAA AATCAGTGTT CTTATTTTTT ATGTAAATAG ATTTTTTAAC TTTTTTCTTT ... ... 22 May 2014 HVP5 Path-OS 4
  • 5. Peter Mac Curation Scope •  Automate the processing from sequencer to draft report •  Automate curation evidence collection •  Sanitise data from external sources •  Automated reporting •  Best practice software engineering 22 May 2014 HVP5 Path-OS 5
  • 7. Patient Sample Genologics wet lab LIMS External Variant DBs •  COSMIC •  Ensembl •  Annovar •  UCSC •  Clinvar etc Loader Pipeline data repository FASTQ BAM VCF VEP Pipeline PipeCleaner PathOS Web Server Pipeline Validation QC Reporting Pipeline configuration Sequencers ETL configuration Periodic DB download and integration Sequencing QC Clinical Reporting Read QC Synthtetic Reads Known samples Filtering configuration Users •  molecular scientists •  clinicians •  researchers Export curated variants to global repositories Hospital Records 22 May 2014 HVP5 Path-OS 7 Path-OS Overview
  • 8. Run QC This run in the context of past runs of the same panel Per sample read yield highlighting below average Amplicon performance read distribution 22 May 2014 HVP5 Path-OS 8
  • 9. Classification Page 22 May 2014 HVP5 Path-OS 9 Automatically generated classification Justification free text field Check boxes for variant evidence Evidence type tool tip
  • 10. Classifying variants for the clinic 22 May 2014 HVP5 Path-OS 10 C5: Pathogenic C4: Likely pathogenic C3: Unknown pathogenicity C2: Unlikely pathogenic C1: Not pathogenic 5 Level Classification Stand alone Strong Supporting Criteria or or Pathogenic evidence Stand alone Strong Supporting Benign evidence = or = or =or or = All other combinations =
  • 11. Software Components Role Package Overview Language Groovy Java on steroids, powerful JVM language Web Framework Grails Rich Groovy based high productivity framework Code repository GitLab Private GitHub instance Database MySQL Widely adopted RDB, good performance User interactivity Javascript plugins Leverage best available js e.g. Jquery, Google Charts Object Persistence Hibernate Java standard for mapping POJOs to RDB Searching Lucene Full-featured text search engine IoC Layer Spring Java standard for inversion of control IDE IntelliJ Comprehensive developers environment for Java etc Build Management Gradle Groovy based DSL leverages Ant and CoC DB Migration Mgmt LiquiBase DSL based data migration tool for schema versioning Issue Management Jira Best of breed issue management tracker LIMS GenoLogics User friendly LIMS for NGS Aligner Primal Peter Mac in-house amplicon aligner, tuned for amplicons Variant Caller VarScan 2 Suitable for somatic and germline (for now) Annotation Ensembl, Annovar Rich set of annotations for multiple transcripts 22 May 2014 HVP5 Path-OS 11
  • 13. Somatic Panel 22 May 2014 HVP5 Path-OS 13 Oncogenes Tumour suppressors Consequence type Other Missense Frame shift Splice site Stop gained Gene type
  • 14. 6.2$%$ 25.5$%$ 0$ 20$ 40$ 60$ 80$ 100$ Single$ Duplicate$ Variant'Allele'Frequency'(%)' Variant'Allele'Frequency'for'Soma7c'Panel'Replicates' Somatic Replicates 22 May 2014 HVP5 Path-OS 14 0" 5" 10" 15" 20" 25" 30" 0'"<10" 10'<20" 20'"<30" 30'"<40" 40'"<50" 50'"<60" 60'"<70" 70'"<80" 80'"<90" 90'100" Mean%difference%in%variant%frequency%between%replicates%(%)% Variant%Read%Frequency%%%(Error:%S.E.M.)% Replicate%Variant%Frequency%Differences% 72% 28% n=14,771
  • 15. Amplicon artifacts 22 May 2014 HVP5 Path-OS 15