Download - The Curation of Molecular Pathology Cancer Samples - Kenneth Doig

Path-OS: The Curation of Cancer Samples

Ken Doig – Bioinformatics Research Core

Peter MacCallum Cancer Centre

[email protected]

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 ... ...


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


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


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


Classification Page


Automatically generated

classification

Justification free text field

Check boxes for variant evidence

Evidence type tool tip

Classifying variants for the clinic


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


The Panels

Somatic Panel


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$

Varia

nt'Allele'Frequ

ency'(%

)'

Variant'Allele'Frequency'for'Soma7c'Panel'Replicates'

Somatic Replicates


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%diffe

rence%in%variant%freq

uency%be

tween%replicates%(%

)%

Variant%Read%Frequency%%%(Error:%S.E.M.)%

Replicate%Variant%Frequency%Differences%

72% 28% n=14,771

Amplicon artifacts


The Filtering

Filtering Variants


●

●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●

●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●●●●●●●

●●

●●●●●●

●●●●●●●

●

●●

●●●●●●

●●●●

●●●

●●●●●●●●●●●

●●

●●●●●●●●●●●●

●●●●●●●●●●●●

●

●●●●

●

●

●

●

●●●●●

●●●●●

●●●●●●●●●●●●●●

●

●●●●

●●●

●●●●●●

●

●

●

●●●●

●●●●

●

●●●●

●

●●●

●●

●●●●

●●●

●●

●●●●●●●●●

●●●●●●●

●●

●

●●●●●●●●●

●●●●●●●●●●

●

●

●●●●●●●●●●●

●●

●●●●●●●●●

●

●●●●●●●●●●●●●

●●●

●

●

●

●

●

●

●●

●

●●

●

●

●●●●●●●

●

●●

●●●●●●●●●●●●●●●

●●●●

●●●●

●●●

●

●

●

●●●●●●

●

●

●●●●●●●●●●

●●●●●●●

●●

●●●●●

●

●●●●●●●●

●●●

●●

●●●●

●●

●

●

●●●●●●●●●●●

●

●●●●

●

●

●

●

●

●

●●●●

●●●●●●

●

●●●

●

●●

●●●●●●●●●●●●

●●

●●●●●

●

●●

●

●

●

●

●

●●●●●●

●

●●●

●

●●

●

●●●

●

●●●

●●●●●●●

●

●●

●

●

●●

●

●

●

●

●

●●●●

●

●●●●

●

●●●

●●●●●●●

●

●

●●●●

●●●●

●

●

●

●

●●●●●●●●

●

●●

●

●

●●●●●●●●●●

●

●●

●

●

●

●

●

●

●

●●●

●

●●●●●

●

●●

●

●

●●●●

●

●●●●

●

●●

●

●

●

●

●

●

●●

●●●●

●●●●●●●

●●●

●●●

●

●●

●●

●

●●

●

●

●●

●

●●●●●

●●

●●●●●

●

●●●●●●

●

●

●●

●●●●

●

●

●●●●●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●●

●●●

●

●●

●

●●

●

●

●●

●

●●●

●

●●●●

●●●●●●

●●●●

●

●●●●●●

●

●●

●

●

●

●

●

●●

●●

●

●

●

●●

●

●

●●

●●

●●

●●●●

●●

●●

●●●

●●●●●●●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●●

●●●

●●●●●

●

●●●

●

●

●

●

●●

●

●

●●

●

●

●

●●

●

●

●

●●

●●

●

●●

●

●

●

●●

●●

●●

●

●●●●

●●●●●

●●●●

●

●●●●●●

●

●

●

●

●●●●●●

●●

●

●

●●●

●

●

●

●

●●

●●

●●

●●●

●●

●●

●

●

●

●

●●●●●●●

●

●●

●

●

●

●●●●

●

●

●

●●

●●●

●

●●●●●

●●

●●

●

●

●●

●●

●●●

●●●

●●

●●

●

●●

●●●●●

●

●

●●

●

●

●

●

●●

●

●●●

●●●●●●

●●●●

●

●●●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●●●●●

●●●●●●●

●●

●●

●●●

●

●●●●

●

●●

●●●●●●●

●

●●

●

●●

●●

●●

●

●

●

●

●●●

●

●●●

●

●●●●

●

●●●●

●

●●●●

●

●●●●

●●●●●

●

●●

●

●

●

●

●

●

●●●

●

●

●●●

●

●

●

●●

●

●

●

●

●●●

●

●●

●

●

●●

●

●

●●

●●●●●

●●●●●●●●●

●

●●●

●

●●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●●

●●●●

●●

●

●●●●

●●

●●●●

●●

●

●

●

●

●

●

●●●●●●●

●●●●

●

●

●

●●

●

●

●●

●

●●

●

●

●

●●

●

●●●

●

●●

●

●●●●●●●●

●

●●●●●●

●

●

●

●●●

●

●●

●●●

●

●

●

●

●●

●

●

●●

●●

●●●●

●

●

●

●●

●

●●

●●●

●

●●●●

●●

●

●

●

●

●

●●

●

●●

●

●●

●

●●

●

●

●

●●●

●

●

●

●

●

●●

●

●●●●

●

●

●●●

●

●●●●

●

●

●

●

●●

●

●

●●

●

●●●

●●●

●●

●

●●

●

●●

●

●

●

●●

●

●

●

●●●

●

●●

●

●

●

●

●

●

●●

●

●

●●●●

●

●●

●

●

●

●●●

●

●

●●●●●●●●●

●

●

●●●

●

●

●

●

●

●●●

●

●●●●●●●

●

●

●●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●

●●●●

●●●

●●

●

●●

●

●●

●●●●

●

●

●

●

●●●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●●●●

●

●●●

●

●

●

●

●●

●

●

●●●●●●●●●●●●

●

●

●

●●●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●●●

●

●●●●●

●●●

●

●●●

●

●

●●●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●●

●

●

●

●●

●

●●

●●

●●●●●●

●●

●●●

●

●

●●●

●●●●●●●

●

●

●

●●

●

●

●

●

●

●

●●●●

●●●●

●

●

●●

●

●●

●●●●●

●

●

●

●

●

●

●●

●

●

●●●●●●●●●

●●●

●●

●

●

●

●●

●●●

●

●

●

●●●

●

●

●●

●●

●●●●●●●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●●●●

●●

●

●●●●●

●

●

●●●●●●

●

●

●

●●●

●●

●

●●●

●●

●●●●●

●

●●●●●●●

●

●

●

●

●

●●

●

●●●●

●●●

●

●

●

●●

●

●

●

●●

●●

●

●●●

●●

●

●

●●

●●

●●

●●

●

●●

●

●

●

●

●

●●●

●

●

●

●●●

●

●

●

●

●●

●●

●●●●●●●●●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●●

●

●

●●●

●●●

●

●●

●

●●●●●●●

●

●●

●

●

●

●

●●

●

●●●●●●

●

●●

●

●

●

●●

●●●●

●

●

●

●●

●

●●●●

●●

●●●

●

●●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●●

●

●

●●

●●

●●●

●

●

●●●●

●●

●

●

●

●

●●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●●

●●

●

●●

●●

●●

●

●

●

●

●

●

●●●●●●●●

●

●

●

●●●

●●●

●

●

●

●

●

●●●●

●●

●

●●

●●

●●●

●

●●

●

●●

●

●●

●

●

●

●●●●●●●●●

●●●

●

●

●●

●●

●●●

●

●

●●

●●●●●

●

●●●●●●

●

●●●

●●

●●●●●●

●●●●

●

●●

●●●●●●

●

●

●●●●

●

●●●●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●●●●●

●●

●●

●

●

●

●

●

●●

●●

●●●●●●●●●

●

●●●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●

●●

●

●

●●

●

●●

●

●

●

●

●●●●●●

●●

●

●

●

●

●

●●●●●

●●

●

●●

●●●●●

●

●●

●

●●●●●●●●●

●

●●

●

●●

●

●

●

●

●

●●●

●●●

●●

●

●

●●

●●

●

●

●●

●

●●●

●

●●

●●●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●●

●●

●●●

●●●●

●

●

●●

●

●●

●

●

●

●

●●●●

●●

●●●

●

●●

●

●

●●●●●●●

●

●●

●

●

●

●

●

●

●●●

●

●●●●●●●

●

●

●

●●

●●

●

●●●●●

●

●

●

●

●

●

●

●

●●●

●

●●●●●

●

●

●●●

●

●●

●

●

●●●

●

●

●●

●

●●●●

●●

●

●●

●●●●●

●●●●

●●

●

●●

●

●

●

●

●●●●

●

●

●

●●●●●

●●

●●

●

●●●●●●

●

●

●

●●●●●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●

●●●

●●●

●●●

●

●●●●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●●

●

●

●●●

●●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●●●●●●●●

●

●

●

●●

●

●

●●

●

●●●

●●

●●●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●

●●

●●●●●

●●

●●

●

●●

●●

●

●

●

●

●

●●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●●●

●

●

●●●●●

●

●

●

●●

●

●●

●●●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●●●●

●●●

●●

●

●●

●

●●

●●

●●●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●●

●●●

●

●

●

●●●

●

●

●

●●

●

●●●

●

●●●

●●

●●●

●

●●●

●

●●

●

●●●

●

●●

●

●

●

●●●●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●

●●

●

●●

●

●●

●

●●

●●●●

●●

●

●

●

●

●●

●

●

●

●●●●●

●

●●

●

●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●●●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●●

●●●

●

●●●●●●

●

●

●●●

●

●

●

●●●●

●●

●●●●●

●●●

●●●

●●●

●

●

●

●

●

●●

●●●

●●●●

●

●

●●

●●

●●●●

●

●●

●

●

●

●

●●

●●●

●●●

●

●

●●

●

●●●●●●

●●●●●●

●

●●●●

●

●●

●

●

●●

●

●

●

●●

●●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●●

●

●●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●●

●

●

●

●

●●

●●

●

●

●

●

●●

●●●

●●

●

●

●

●

●

●●●●

●●

●

●

●

●

●

●●●●●

●●●●

●

●

●●

●

●

●

●

●●●●

●●●

●●●

●

●

●

●

●

●●●

●

●

●

●

●

●●

●●

●

●●●

●●●

●

●

●●●●

●●

●

●

●

●●●●●

●

●

●●

●

●

●●●

●

●

●

●

●●●

●

●

●●●

●

●

●●

●●●

●

●●

●●

●●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●●●

●

●●

●

●

●●●

●

●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●●

●●

●●

●

●●

●

●

●●●

●

●

●●

●

●

●●

●

●●●●●●

●

●

●

●

●

●●

●

●

●

●

●

●●

●●

●

●●

●●

●●●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●●

●

●

●

●

●

●●

●

●

●

●●●

●●

●

●

●

●●

●●

●

●

●●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●●

●

●●

●

●●●

●●

●●

●

●●●

●

●

●●●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●●

●●

●

●

●

●●

●

●

●●●

●●

●

●●

●●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●●

●●

●

●●

●●

●

●●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●

●●●

●

●

●●●

●

●

●

●●●

●

●●●●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●●●●●

●

●

●●

●

●●

●●

●●

●●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●●

●

●●

●

●

●

●

●●●

●●

●

●●●●●

●●

●

●

●●●

●●●

●●

●

●●●●

●

●

●

●

●●

●●

●

●

●

●

●●●●

●

●

●●●●

●

●

●●●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●●●

●●

●

●

●●●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●

●●●●●

●●●

●●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●

●●

●●●●●●

●

●

●●

●●

●●●

●

●

●

●●

●●●

●

●●●●●●

●●

●

●

●

●

●

●

●

●●●

●●●

●

●●

●●

●●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●●

●

●●●

●●●

●●

●●●●●●

●

●

●

●

●

●

●●

●●●●

●

●

●

●

●

●

●

●

●●

●●●

●

●●●

●

●

●

●

●

●●

●●●●●

●

●

●●

●

●●●●●

●

●●

●●●●

●

●

●

●●

●

●

●●

●

●

●

●●●●●●

●

●

●●

●●●

●

●

●

●

●●

●●

●

●

●●●●

●

●●

●

●●

●●●

●

●●●●●●

●

●

●

●●●●

●

●

●●

●

●

●

●●●

●

●

●●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●●●●●●●

●●

●●

●

●●

●

●●●

●

●

●●

●●

●

●●

●●

●

●

●

●

●●

●

●

●●●●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●●

●

●

●●●

●

●

●●

●●

●

●

●

●●

●

●

●

●

●●●●

●

●●●●

●●

●

●

●●●

●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●●

●●

●●

●

●

●●●●●

●

●

●

●

●

●

●

●●●●●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●●●●

●

●

●●●

●●●

●

●

●●

●

●●

●●

●

●

●

●●

●

●

●

●

●●●

●●

●

●

●

●

●

●●

●●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●●●

●

●

●

●

●●●

●

●

●●

●

●

●●●●

●●●●●●●

●●●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●●

●

●

●

●●●

●

●●

●

●●●●

●

●

●

●

●

●●●●●

●

●

●

●

●

●

●

●●●●●●

●

●●●●●

●●

●

●

●

●

●

●

●●●●

●●

●

●●

●

●●

●●●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●●●

●

●●

●●

●

●

●

●●●●●●

●

●●●

●

●●●

●●●●

●

●●

●

●●

●●

●●

●●

●

●

●●

●

●●●●●

●

●

●

●

●

●●

●

●

●●

●●

●●●

●●

●

●

●●●●

●

●●

●

●●●●

●

●

●●

●

●●

●

●●

●

●●●

●●●

●

●

●

●

●

●●●

●

●●●●●

●

●

●●●●

●●●

●

●●

●

●

●●

●●●

●●

●●

●

●●

●

●

●●●

●

●

●●

●

●

●●●●

●

●●●

●●●●●

●

●●●●●●

●●●

●●●

●

●

●●

●●●●●●●

●●●●●●●●●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●●●

●

●●●●●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●●

●

●

●●●

●

●

●●

●●

●

●

●

●

●●

●●●

●●

●

●

●

●

●

●●●

●

●

●

●

●

●●●

●

●

●●

●

●●

●●●●

●

●

●

●●

●●

●

●●

●

●

●

●

●●●

●●

●●

●●●●

●●●●

●

●●

●

●●●

●●

●

●

●

●

●●

●

●

●●●●●

●

●

●●●●

●

●

●●

●

●

●

●

●●

●●

●●

●●●

●

●

●●

●●

●

●

●●●●●●

●

●●●●

●

●●●●●●

●●

●●

●

●

●

●●

●

●

●●

●●

●

●●●●

●

●●●

●●

●

●

●●●●

●●●●●

●

●●

●

●

●●●●

●●

●

●

●

●●

●

●

●

●●●●

●

●

●

●●

●

●●●

●●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●●

●

●●

●

●

●

●

●●

●

●●●

●

●●

●

●

●●●

●●●

●

●

●●

●

●●

●●●●●

●

●

●

●

●●

●

●

●

●

●

●●

●

●●●

●●

●

●

●

●●

●

●

●

●

●

●●●

●●●●●

●●●

●

●

●●

●

●

●

●●

●

●

●

●

●

●●

●

●●●

●

●●●

●

●●

●●●

●

●

●

●

●

●

●●●

●

●

●

●

●

●●●●●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●

●

●

●●

●

●●●

●●●

●

●

●

●

●●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●●●●●●●

●●●●

●

●

●

●

●

●●

●●●

●●●

●

●●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●●

●

●

●

●●

●

●●

●●

●

●●

●●●●

●

●●●

●

●

●●●

●●●

●●●

●

●

●

●●

●

●●

●

●

●

●●●●●

●●

●●

●

●

●

●●●●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●●

●●

●

●

●●●

●●

●●

●

●●●●

●●●●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●●

●

●

●●

●

●

●

●

●

●

●●

●

●●●

●

●

●

●●

●●

●

●●●

●●●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●●

●●

●

●

●

●

●

●●●●

●

●

●

●●

●●●●

●

●

●

●

●●

●

●●

●

●

●●●

●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●●●

●

●●●●

●

●●

●

●

●●

●

●

●

●

●●●

●

●●

●

●

●●●

●

●●●

●●

●

●●

●

●

●

●

●

●●●

●

●

●●

●

●●●

●

●●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●●

●

●

●

●

●●

●

●●●

●

●

●

●

●

●●●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●●●

●

●

●●●

●●●

●

●

●

●●

●●

●

●

●

●

●

●●

●●●●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●●

●

●

●

●

●

●●●●●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●●

●

●

●

●●

●●

●

●

●

●●

●

●

●

●●●

●

●●

●

●

●●●

●

●

●●

●

●

●

●●

●●

●●

●

●

●

●●●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●●

●●

●

●

●●●

●

●

●

●●●

●

●●●●●●●●

●

●

●●●

●●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●●

●●

●

●

●

●

●

●●●

●●●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●●

●

●●●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●●

●

●

●●

●●

●●

●

●●

●

●●●

●

●●●

●

●●●●

●

●

●

●

●●●

●

●

●

●

●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●●●●●●●

●●

●

●

●

●

●

●

●

●

●●

●

●

●●

●●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●●

●

●

●

●

●

●●●●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●●●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●●

●

●

●

●●

●

●●●

●

●●●

●●●

●●●

●●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●●●

●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●●●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●

●●●

●

●●●

●

●

●

●

●

●

●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●

●●●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●●●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●●●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●

●●

●

●

●●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●●

●

●●

●

●

●●

●

●●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●●

●

●●

●●●●

●

●

●

●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●●

●●

●

●

●

●●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●●●

●

●●

●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●●

●

●●●

●

●

●

●

●

●

●●●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●●

●●

●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●●

●

●●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●●●

●●

●

●

●

●

●●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●●

●

●

●●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●●

●

●

●

●

●

●●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●●●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●

●●

●●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●●●

●●

●

●

●

●

●

●

●●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●

●

●●●●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●●

●

●

●●

●

●

●●●

●●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●●●

●

●

●●

●

●●

●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●●

●●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●●●

●●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●●

●

●●●

●

●

●

●●

●●

●●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●●

●

●●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●

●●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●●

●

●

●●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●●

●

●

●

●

●

●

●●●●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●

●

●

●●

●●

●

●

●

●

●

●

●

●

●●●●●●

●

●●●

●

●

●

●●●

●

●

●●●

●

●

●●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●●

●●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●●

●

●●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●●●●●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●●●●●

●●●

●

●●

●●

●

●●●●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●●

●

●●

●

●

●

●●●

●●

●

●

●

●

●●●

●

●

●●

●

●

●●

●

●

●

●

●

●

●●●

●

●●●●●

●

●●

●●

●●

●

●

●●

●●●

●

●●

●

●●●

●

●●●●●●

●●

●●●●●●●●●●●●●●●

●●●

●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●

●

●

0 20 40 60 80 100

1e+0

01e

+02

1e+0

41e

+06

Raw Variants

Allele Frequency

Varia

nt C

ount

●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●●

●

●

●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●

●●

●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●

●●

●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●

●

●●●●

●

●●●●●●

●●

●●●●●●●●●●●

●

●●●

●

●

●

●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●

●

●●●●

●

●

●

●●●●●●●●●●●●●

●

●●

●

●●●●●

●

●●●●●●

●

●●●●●

●

●●●●●

●●

●●●●

●

●

●

●●●●

●●

●●●●●

●

●

●

●●●●●●

●

●●●●

●●

●

●

●●●●

●●

●●●

●●

●

●

●●●

●

●

●●●

●●

●

●●

●

●

●●

●●●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●●

●

●

●●●●●

●

●

●

●

●

●

●●●●

●

●●

●

●●●

●

●

●

●

●

●●●

●

●●●

●

●

●

●●●●●●●

●

●●

●

●●

●

●

●●

●

●

●

●●●●●

●

●●●

●

●●●●●

●

●●●●●●●

●

●●●●●

●●

●●

●

●

●

●

●

●●●●

●

●●●●●●●●

●

●●●●●

●

●

●

●●●●●

●●

●

●

●●●●

●

●

●

●

●

●

●

●●●●

●

●

●●

●

●●

●●

●

●●

●

●●●●

●

●●●●

●

●●●●

●●

●

●

●

●●

●●●●●

●

●●●●●●

●

●●●●

●

●●

●

●●

●

●●●

●

●●●●

●

●●●●●●

●

●

●●●

●●●●●

●●

●

●●●

●●

●●●●●●

●

●

●

●●

●

●●

●

●●●●

●

●

●

●●●●●●

●

●●●●●

●

●●

●

●●●

●●●

●●●●●●

●

●●

●

●

●

●●●●

●

●●

●

●●●●●

●

●

●

●●●●●

●

●●

●

●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ●●●●●● ● ● ●●●● ●●●●●●●●●●●●●●●

●

0 20 40 60 80 100

02

46

810

Filtered Variants

Allele Frequency

Varia

nt C

ount

●

●

●

●●●●●

●

●

●

●

●

●●

●

●●

●

●

●

●●●

●●

●●

●

●●●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●●

●●

●

●●●

●

●

●

●●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●●●

●

●

●

●

●

●●

●

●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●●

●

●

●●

●

●

●

●●

●

●

●●●●

●

●

●●●●●●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●

●●

●●●

●●●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●●

●●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●●

●●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●●●●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●●

●

●

●●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●●

●●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●●

●

●

●

●

●●

●

●

●●

●

●●

●●

●

●

●

●

●

●

●

●

●●●●

●

●

●●

●

●

●

●

●●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●

●●

●

●●●

●

●

●

●●

●

●●

●●●

●

●

●

●

●●●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●

●●

●●

●

●●●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●●●

●

●

●●

●

●●

●

●

●

●●

●

●●

●●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●●

●●●●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●●●●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●

●●

●●●

●●

●

●

●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●

●●●●

●

●●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●●

●

●●

●

●●

●

●

●●

●

●

●●

●●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●●

●

●

●

●

●●●

●

●

●●●

●

●

●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●●

●

●●

●

●

●●●

●

●

●

●

●

●●

●●

●

●

●

●

●

●●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●●

●●

●

●

●

●●

●

●

●

●

●

●●

●●●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●●

●

●

●●

●

●

●●●

●

●

●●

●

●●●

●

●

●

●

●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●●

●●

●●●

●

●

●

●

●

●●

●

●●

●

●

●●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●

●

●●

●●

●

●●

●

●●

●

●

●

●●

●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●●

●●●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●●

●●

●

●●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●

●●●

●

●

●●●

●

●●●

●

●●

●

●

●

●●●

●

●

●

●

●

●●

●●●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●●

●

●●

●

●

●

●●

●●●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●●●

●

●●

●

●

●

●

●

●

●●

●

●●

●●

●

●

●●

●●

●

●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●

●

●●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●●●●

●●

●

●●●

●

●●

●

●

●

●

●●

●●●

●

●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●●●

●

●

●

●

●●

●

●

●

●

●●

●●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●●

●●

●

●●●

●

●●

●

●

●

●

●●

●

●●●

●

●

●●

●

●

●

●

●

●●●●

●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●●

●

●

●

●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●●●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●

●●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●●

●●

●

●●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●●

●

●●●●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●

●

●

●

●●

●

●

●

●

●

●●

●●

●

●

●

●

●

●●

●

●

●●

●

●

●●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●●●●

●

●●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●●

●

●

●

●

●

●●●

●

●●●●

●●

●

●

●●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●●●

●

●●

●●

●

●

●●●

●●

●●●

●

●

●●●●

●

●●

●

●

●●●

●●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●●●

●

●●

●

●●

●

●●

●

●

●●

●

●

●

●●●

●

●

●

●

●●●

●

●●

●

●●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●●●●

●

●●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●●

●

●●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●●

●

●●

●

●●●●

●

●●

●

●

●●

●●

●

●●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●●

●●●●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●●

●●●

●

●

●●

●●●●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●●

●●

●

●

●

●●●●

●

●

●

●

●

●

●

●●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●●

●

●

●

●●

●

●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●●●●

●

●●

●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●

●●

●

●●

●

●●●

●

●

●

●

●●

●

●●

●

●

●●

●

●●

●●●

●

●

●●●●●●

●

●

●

●

●

●

●●

●●

●●

●

●●●

●

●●

●

●●

●

●

●●●

●●●

●

●●●●

●

●●●●●●

●

●●

●

●

●

●●

●

●

●●●

●●●●●

●

●●●●

●

●

●●●

●

●●●

●

●●

●●●

●●

●

●

●●

●●

●

●●

●

●●

●●●●

●

●

●

●●●

●●

●

●●

●●●●

●

●

●

●

●●●●

●

●●●●●●

●

●

●●

●●

●

●

●●

●●

●

●

●

●●●●

●

●

●

●

●●

●

●

●●●

●●●●

●

●

●

●

●●

●

●●

●

●●●

●

●

●

●●●●●●

●●

●●

●

●

●●

●●

●●●

●●

●●

●●●

●

●●

●●●

●

●

●●

●

●

●●●●●●

●●

●

●●●

●

●

●

●

●●●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●●

●

●

●

●●

●

●

●

●

●

●●●

●

●

●

●●●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●●●●●

●●

●

●

●

●

●

●

●

●

●●●

●●

●●

●

●●

●

●

●

●

●●●●

●

●●●●●●●●●

●●

●

●●

●●

●●

●●

●

●●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●●●●●

●●

●●●●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●●●●

●

●

●●

●

●

●●

●●●

●●

●

●

●●●●

●

●●●

●

●

●●

●●

●

●●●●

●●

●

●●

●

●

●●●

●●

●

●●●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●●●

●

●●●

●

●

●

●●

●●●●●

●

●●

●●

●●●●●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●●●

●●

●●

●

●●●

●

●

●

●

●

●

●

●●

●

●●●

●

●●

●●●●●

●

●●

●

●

●

●

●

●

●

●

●●●●

●●

●

●

●●●●●

●

●

●

●

●

●

●●●

●

●

●

●●●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●●

●

●

●●●

●●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●●

●

●

●

●●●

●

●

●

●●

●

●●●●●

●

●

●

●

●

●

●

●

●

●

●●

●●●●●●●●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●●

●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●●

●

●

●●●●●●

●

●

●●

●

●

●

●

●●

●

●

●

●●

●●

●

●●

●●

●

●

●

●●

●

●●●

●

●●

●

●

●

●

●●●●●

●

●

●

●

●

●

●

●

●●

●●●

●●

●

●●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●●●●

●●

●

●

●

●

●

●●

●

●

●

●●

●●

●

●

●

●

●

●●●

●

●

●

●

●

●●●●

●●

●●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●●●

●

●●●●●

●●

●●●

●

●

●

●

●

●●●●

●●

●●●

●

●

●●

●●

●●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●

●●

●●

●●

●

●

●

●

●●

●

●

●

●

●●●●

●

●●●●●●●

●

●

●●

●

●

●

●

●

●

●●●

●

●●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●●

●

●

●●

●●

●●

●

●

●

●●

●●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●●●

●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●

●

●

●

●

●

●

●●

●

●●

●●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●●●

●

●

●

●●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●

●

●

●●

●●●

●

●

●●

●

●●

●

●

●●

●

●●

●

●

●

●

●●

●●

●

●●

●

●

●●

●

●

●

●●

●

●

●

●●

●●

●

●

●

●●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●●●●

●

●

●

●

●●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●●

●

●●

●

●

●●

●

●

●

●

●

●●

●

●●●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●●●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●●●●

●

●●

●

●

●●●●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●●

●

●

●●

●

●

●

●●●

●

●

●●

●

●●

●

●

●●●●

●

●●

●

●

●

●●●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●●

●●●●

●●

●

●

●

●●●●

●●

●●

●●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●●

●●

●●●●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●●●

●

●

●●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●●●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●●

●●

●

●

●

●

●●

●

●●

●

●

●●●●●

●

●

●

●

●

●

●●

●●

●

●●●

●

●●

●

●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●

●●

●

●

●

●●●

●

●

●

●

●

●●

●●●

●

●

●

●

●●●

●●

●

●

●

●●

●●

●

●

●●

●●

●

●

●

●●●●●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●●●

●●●

●●●

●●

●●

●●●●●

●●●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●●●●●

●●

●

●

●●

●

●●

●

●●

●

●●

●

●●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●●●

●

●●

●

●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●

●●

●

●●

●

●

●

●●

●

●●●●●

●

●

●

●●

●

●

●

●●

●●

●●

●●●

●

●

●

●

●●●●

●

●●

●

●

●

●●●

●

●●●

●

●

●●●●

●

●●●●

●●●

●

●●

●

●

●

●

●●

●●●

●

●

●●●

●

●

●

●

●●

●

●

●●

●

●

●●●

●

●

●

●●

●

●●

●●

●

●●●●●

●

●

●●

●●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●●

●●

●

●

●●

●

●

●

●

●●●

●

●

●

●●

●

●

●

●

●

●

●

●●●●●●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●●

●

●

●

●●

●

●

●

●●●

●

●

●●

●

●

●

●

●

●

●●●●

●●●

●

●

●●

●●●●

●

●●●

●●●●

●

●●

●

●●●

●

●

●

●

●

●

●

●●●●●

●●

●

●

●

●

●

●

●

●

●

●●

●●●

●

●●

●

●●●

●

●

●●

●●

●

●

●

●

●●●

●

●

●●

●●

●

●

●

●

●●

●●

●

●

●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●●

●

●

●

●●●●

●●

●

●

●

●

●

●●●

●

●●●●●

●

●●

●

●

●

●●

●●

●●●●●

●

●●●

●

●

●

●●

●

●

●●●●

●●

●●●

●●

●

●

●●

●

●

●●

●●

●

●●

●

●●●

●

●

●

●●

●●

●●

●●●●

●

●

●

●

●

●●

●●

●

●

●●●●

●

●●●

●

●●●

●

●

●

●

●

●

●●●

●

●●

●

●

●

●●●

●

●●●

●

●●

●

●

●

●

●

●●

●

●

●

●

●●●●●●●●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●

●

●

●●

●

●●

●●●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●●

●

●

●

●●

●

●●

●

●

●

●●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●●●

●

●

●●

●●

●

●

●

●

●●

●

●●

●●

●

●

●●

●●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●●

●●●

●

●

●

●●

●

●●

●●●●●

●

●

●

●

●●●

●●●●

●●

●

●

●

●●

●●●●

●

●

●

●●●

●

●

●

●

●●●●●●●

●●●●

●●●

●

●

●

●●●

●

●●

●

●

●

●●●

●

●●

●

●●

●

●

●●

●

●●

●

●

●●●

●

●●●●

●

●●

●

●●●

●

●

●●

●●●●

●●

●●

●●

●

●

●

●●

●

●

●

●●●

●

●

●●

●

●

●

●●

●●●

●●

●

●

●

●●

●●●●●

●

●

●

●

●

●

●

●●

●

●

●

●●●●●

●●●

●●●●

●●

●●

●

●●●

●

●●●●

●●●

●

●

●

●●●

●

●●●

●

●●

●

●

●

●●●●●

●

●

●●●

●●

●

●●●

●

●●

●

●

●●●

●

●●

●

●●●●

●

●

●

●

●

●

●●●●

●

●●

●

●●

●

●●●

●

●●

●

●●●

●

●

●

●●●

●

●

●

●

●●●

●

●●●

●

●

●

●●●●●

●

●●●

●

●

●

●●●

●

●

●

●

●

●

●

●●

●

●●●●●●●●●●

●

●●●●●●

●

●●

●

●

●

●●●

●

●

●●

●●●●●●

●

●

●●●

●

●●●●

●

●●

●

●●

●●

●●

●

●●

●

●

●●●●●●

●

●●

●●●

●●●●

●●

●

●●●●

●

●

●●●●●●

●●

●

●●

●

●

●

●

●

●

●

●

●●●●●●

●●

●

●

●

●

●

●●●●●●●

●

●

●

●

●

●

●

●●●●

●

●

●

●●

●

●●

●

●●●●

●

●

●●

●

●●

●

●

●

●●●●●●

●

●●●●

●

●

●●●●●●●

●

●

●

●

●

●●

●

●●●

●

●●●●●●

●

●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●●

●

●●

●

●

●

●●

●●

●

●

●●●

●

●

●

●

●●

●

●

●

●●

●

●●

●

●●

●

●

●

●●●●●●●●●●●●●●

●

●

●●●

●

●

●

●

●

●

●

●●

●●●

●

●

●

●

●

●●

●

●●

●●●

●

●

●●

●

●●

●

●●

●

●

●

●●

●

●

●●●●

●●

●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●●●

●

●

●

●

●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●●

●●

●●

●

●

●

●●●●●

●

●

●●●

●

●

●●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●●

●●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●●●●

●●

●●●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●●

●●

●

●

●

●●

●●●

●●

●

●

●

●●

●

●●

●

●

●

●●●●●

●

●●

●

●

●●

●

●

●

●●

●●●●

●

●●●●●

●

●●

●●

●●

●

●

●

●●●●

●

●●

●

●●●

●

●●●●●●

●●

●●●●●●●●●●●●●●●

●●●

●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●

●

●

0 20 40 60 80 100

1e+0

01e

+02

1e+0

41e

+06

dbSNP Variants

Allele Frequency

Varia

nt C

ount

●

●

●●●●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●●

●●

●

●

●●●●

●●●

●

●●

●●●●

●●●●●

●

●

●●

●

●

●

●

●●

●

●●

●●●

●●●

●●

●●●

●

●

●

●

●●●

●

●●

●●●●

●

●●

●

●

●

●

●

●

●●

●●

●

●●●●●●

●

●

●●●

●

●●

●

●●

●●

●●

●

●●

●

●

●

●●●

●

●●

●●

●●●

●

●●●●●●

●

●

●

●

●●

●

●

●●

●

●

●●

●

●

●

●●

●●●

●

●

●

●●●●

●

●

●

●

●●

●

●

●

●●

●●●

●●●●

●

●

●●

●

●

●●

●

●●

●●●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●●●

●●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●●

●

●

●●

●●

●

●

●

●●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●●

●

●●●●

●

●

●

●

●

●

●

●●●

●

●●

●●

●

●

●●

●

●

●●

●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●

●

●●

●

●

●●

●

●

●

●●●

●

●

●●

●

●

●●

●●●

●

●

●●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●●

●●

●

●

●●

●●●

●

●●

●

●●

●●

●●

●

●

●

●●

●●●●●

●●

●

●

●

●

●

●

●

●

●

●

●●●●●

●

●

●

●

●●

●

●

●

●●●●

●

●

●

●

●

●

●●

●●

●

●●●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●●●

●

●●

●

●●

●

●

●

●

●

●●●

●●

●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●

●

●●

●●

●

●●●●

●●

●

●●

●

●

●●●●

●●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●●●

●

●●

●

●

●

●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●●

●●

●

●

●

●

●

●●

●

●●

●●

●

●

●

●

●●●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●●

●

●

●

●

●●

●

●●

●

●

●●●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●●●

●●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●●

●

●●

●

●●

●●

●

●

●●●●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●

●

●

●

●●

●

●●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●●

●

●●●●

●

●●

●

●

●

●

●

●●

●

●

●●

●●

●

●●

●

●

●

●●

●

●●

●●

●

●●

●●●

●

●

●

●

●●

●

●●

●

●

●●

●●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●●●●

●

●

●

●

●

●

●

●●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●●

●●

●●

●●

●

●

●●

●●

●

●●●●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●●

●●

●

●

●

●●●

●

●

●

●●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●●

●

●

●●

●

●

●●●

●●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●●●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●●●

●

●

●

●

●

●

●●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●●

●●

●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●●

●●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●●●

●

●

●

●●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●●●●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●

●●

●

●●

●●●

●

●●

●●●

●

●●

●

●●●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●●

●●

●

●

●●

●

●●

●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●●●

●

●

●

●

●

●

●

●●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●●

●●●●

●

●●

●●●

●●

●

●

●●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●●

●

●

●●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●●●

●●●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●●●

●

●

●

●●●

●

●

●

●●

●●●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●●

●

●

●●●●

●

●

●●

●

●

●●

●●

●

●●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●●

●●

●●

●

●

●

●

●●

●

●●●●●●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●

●

●●

●

●

●●

●

●●●

●

●●●

●

●

●●

●

●

●

●

●

●●

●

●

●

●●

●

●●

●

●

●

●●

●●●

●

●

●●●●

●

●●

●

●

●

●

●

●●●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●●●

●

●

●●●

●●●

●●●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●●

●●

●

●

●

●

●

●

●

●

●

●●●●●●●●

●●

●●●●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●●●●●

●

●●

●

●

●●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●●

●

●

●●●

●●

●

●

●

●

●●●

●●

●

●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●●

●

●●

●●●

●

●●●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●●

●

●

●●●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●●●●●●●

●

●

●●

●

●

●

●●●

●●

●●

●

●

●●

●●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●●●

●

●

●

●●●

●

●

●

●

●

●

●●

●●

●

●

●●

●

●

●●●

●●●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●●●

●

●

●●●●

●

●

●●●

●

●

●●

●

●●

●

●●

●

●●

●

●●

●●●●

●

●

●●

●●●

●

●

●●●●

●

●●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●

●●●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●

●●

●●●

●●●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●●

●●●

●●

●

●●

●

●●

●

●●

●●

●

●●●●

●

●

●

●

●●●

●

●●●

●●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●●●

●

●●

●●●

●●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●●

●

●

●

●●

●

●

●

●●

●●

●

●●

●

●

●

●

●●●

●●

●●●

●

●

●

●●●

●●

●

●

●

●

●

●

●

●●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●●

●●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●●

●●

●

●

●●

●●

●

●●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●●●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●●●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●●

●●

●

●

●

●

●

●

●

●

●●

●●●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●

●●

●●●

●

●

●●●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●●

●●●

●●

●●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●●

●●●●

●

●●

●

●

●●

●

●

●

●

●

●

●●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●

●●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●●

●

●●

●

●

●

●

●

●

●●

●

●

●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●●●

●

●

●

●●

●●

●●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●●

●

●●●

●●

●●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●●

●●

●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●●

●

●●

●

●

●

●

●

●●●

●

●

●

●●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●●

●

●

●●●●

●

●

●●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●

●●

●●

●

●

●

●

●

●●●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●●

●

●

●●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●●●

●●

●

●

●

●●

●

●

●●●

●

●

●

●

●

●

●

●

●●

●●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●

●●●

●

●

●

●●

●

●●

●

●●●

●●

●

●●

●

●●

●

●●●

●

●●

●●

●

●

●

●

●

●

●

●

●

●●

●

●●●

●

●

●●

●

●

●●

●

●

●●

●

●

●

●

●●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●●

●

●

●

●●

●●

●

●

●

●

●

●

●

●

●

●●

●

●

●●●

●

●

●

●●

●

●

●

●●●

●●

●

●●●●●●

●

●

●

●

●

●●

●

●●●

●

●

●

●

●

●●●●●●●●

●

●

●

●●

●

●●

●

●●●

●

●

●●

●●

●

●

●●

●

●

●

●●

●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●●●

●

●

●●●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●

●

●●

●

●●

●

●

●

●

●

●

●●●

●

●●

●●●

●●

●●●●

●●●

●●●

●

●

●

●●

●

●●

●

●

●●

●

●

●

●●●●●●●

●

●

●

●●●

●●

●

●●●●

●

●

●

●●

●

●

●

●●

●

●●

●

●

●●

●

●

●

●●

●

●

●

●

●●

●●

●●

●●

●

●●

●

●●

●

●

●

●

●●

●

●

●

●●●●●

●

●●●●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●●●●●

●

●

●

●●

●

●

●

●

●

●

●●●●●●●●●●●

●●

●●

●

●●

●

●

●

●●

●

●●

●

●●

●

●

●●●

●

●

●●

●●

●

●●●●●●

●

●●

●

●

●

●●●

●

●●

●

●

●

●

●

●

●●●

●

●

●

●●

●

●

●●

●

●●●

●

●●

●

●

●

●●

●

●

●

●●●

●●

●●●●

●●

●

●

●●●

●

●

●

●●●

●

●

●●●●●●●

●

●

●●

●

●●●●●●●●●●●●

●

●

●

●

●●

●

●

●●●

●

●●●

●

●●●●

●

●

●

●●●

●

●●

●

●

●●

●●●

●

●●●

●

●●

●●

●

●

●●●

●

●●

●

●

●●

●

●●●

●

●

●

●

●

●●●●●●●

●

●●

●

●●●●●

●

●

●

●

●

●

●

●

●

●

●●●●

●

●

●

●●

●

●

●●●●●●●●

●

●

●●●

●

●

●●●●●●

●●

●

●●●

●

●

●●●●

●

●●●●●

●

●●●●●●

●

●●●●●●

●

●●●

●

●●●●

●

●●●

●

●●●

●

●●●●

●

●

●

●●

●

●●●

●

●●●

●

●●●

●

●

●

●

●

●

●

●

●

●●●●●●●●

●●

●

●

●

●

●

●

●●

●●

●

●●●●●●

●

●●●

●

●

●●

●●

●

●●●●●●●●●

●●

●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●●

●

●●●

●

●●●●●

●●

●

●●●●●●●●

●

●●●

●●

●●●●

●●

●●●

●

●●

●

●●●

●

●●●●●●

●●●

●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●

●

●

●

●

●

●●●

●

●

●

●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●

●

●

●●

●●●●●

●●●●

●●●

●

●●●●●

●

●●●●●●●

●

●●●

●●

●●

●

●●●●

●

●●

●

●●

●

●●●

●

●●●●●●●●●

●

●

●●

●

●●●●

●

●●●●●●●●●●●●●●●

●

●●●

●

●

●

●

●

●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●

●●

●●●●

●

●

●●

●

●●●●●

●

●●●●

●

●●

●

●●●●●●●●

●

●●

●

●

●●●

●●

●●●●●●●●●●●

●●

●●●●●●●●●●●

●

●

●

●●

●

●

●●

●●●●●

●●

●

●

●●

●●

●●●●●

●●

●

●

●

●

●●

●

●●●●●●●●●

●●

●

●

●●●●●

●

●

●

●●●●●●

●

●●●●

●

●●●

●

●●●●●

●

●●●●●●●●●●

●●

●

●

●●●

●

●●●●●●●

●

●●

●

●

●

●●●

●●

●

●●●●●●

●●●

●●

●

●

●

●●

●

●

●

●●●

●

●●●

●

●

●●●●●

●●

●

●

●

●

●

●

●

●●

●●

●●●●●●●●

●

●●

●●

●●

●

●

●

●●

●

●●●

●●

●●●

●

●

●●

●●●●●

●

●●

●●

●

●

●●●●●

●

●●●

●

●●

●

●

●

●●●

●

●●●●●●●

●

●●●●

●

●

●

●●●

●

●

●

●●●●●●●●●●●

●●

●●

●

●●

●

●

●

●

●●●●●●●●●

●

●●●●

●

●●

●

●●

●

●

●

●●●●

●

●●●

●

●

●

●●

●

●●

●

●●●●●

●

●

●

●

●●

●●

●●●●

●

●

●●

●●●●

●●

●●●●●●

●

●●●●●●●

●

●

●

●●●●●●

●

●●●●●●●

●

●●●●●●●●●

●

●●●

●

●

●

●●

●●

●

●

●●●●●

●

●

●

●●●●●●●●●●●●●●●●

●

●●

●

●

●

●●●●●●●●

●

●●

●

●●

●

●●

●

●●●●

●

●●

●

●●●●●

●●

●●●●

●

●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●

●

●

●

●●●

●

●●

●

●●

●

●●●●

●

●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●

●

●●●

●

●

●●●●●●●●

●●

●●●●●●●●●●

●

●

●●●●●

●●

●●

●

●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●●●

●

●

●●●●

●

●

●

●●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●

●●

●●

●

●●

●

●

●

●

●

●

●

●

●

●●●

●

●

●

●

●

●

●

●

●

●

●

●●●

●●

●

●

●

●

●●

●●

●

●●

●

●

●

●●

●

●

●

●

●

●

●●●●

●

●

●

●

●

●

●

●●

●

●●●●

●

●●

●●

●

●●●●

●

●

●●●●

●●●

●●●●●●●●●●●●

●●

●●●

●

●

●

●

●

●

●

●

0 20 40 60 80 100

1e+0

01e

+02

1e+0

41e

+06

COSMIC Variants

Allele Frequency

Varia

nt C

ount

Raw Polymorphisms

Somatic Passed

Sample Curation Page


In-silico and DB classifiers

Filter Flags

Variant Searching and Filtering


“OR” clause

Template searches

Variant attribute

Predicate

Value

Variant Filter Attributes

Filter Flag Description Filter Criteria pass Passed all filters no filters match

vaf Low Variant Allele Frequency < 8% Somatic <15% Germline

vrd Low Total Read Depth < 100 vad Low Variant Read Depth < 50 blk Assay panel specific black list user defined con Inferred Benign Consequences system defined gmaf High Global Minor Allele Frequency > 1% pnl High Variant Frequency in Panel > 50% sin Singleton variant in duplicate sample for run Not in both samples amp Uneven read distribution across amplicons Single amp. bias


Multiple filter flags may be assigned per variant

Filter effects


0.0#

1.0#

2.0#

3.0#

4.0#

5.0#

6.0#

GMAF#>#1%# Benign#Consequences# Passed#all#filters# Reportable#

Mean%CA

DD%sc

ores%(scaled%scores%0%log)%

Filtering%Criteria%(all%S.E.M.%<%0.1)%

Effect%of%filtering%on%CADD%scores% Without#Filter#

With#Filter#

Average#CADD#scores#for#variants#(top#20%)#

The Future

In the Pipeline…

•  Pluggable modular pipelines (using bpipe)

•  Upload existing FASTQ, BAM and VCF files

•  PipeCleaner for Pipeline validation

•  Include CNV and fusion modules

•  Drugability of variants and clinical trials

•  Write paper…


Acknowledgements

•  Molecular Pathology Department –  Andrew Fellowes –  Anthony Bell –  Stephen Wong

•  Peter Mac Bioinformatics –  Jason Ellul –  Jason Li –  Maria Doyle –  Franco Caramia

•  Cancer 2015 –  John Parisot


Email: [email protected]

Additional Slides

Column Chooser & Ordering


Download - The Curation of Molecular Pathology Cancer Samples - Kenneth Doig

Top Related