Introduction

Methods

Results

Translocation detection in lung cancer
using mate-pair sequencing and iVIGS
Richard Meier ...
Introduction

Methods

Results

Content

Introduction
Methods
Results

Translocation detection in lung cancer using mate-p...
Introduction

Methods

Results

Introduction

Translocation detection in lung cancer using mate-pair sequencing and iVIGS
...
Introduction

Methods

Results

Structural variations

Chromosome 1

Chromosome 17

No variation
Deletion
Insertion
Transl...
Introduction

Methods

Results

Structural variations in mate pair mapping
Insertion

Deletion
reference genome

reference...
Introduction

Methods

Results

Breakpoint resolution with split reads
Where are the breakpoints ?

known reference

clust...
Introduction

Methods

Results

Methods

Translocation detection in lung cancer using mate-pair sequencing and iVIGS

Rich...
Introduction

Methods

Results

Data

A set of mate-pair sequencing data from lung cancer patients was analysed.

35 sampl...
Introduction

Methods

Results

Mate-pair preprocessing

Translocation detection in lung cancer using mate-pair sequencing...
Introduction

Methods

Results

Translocation analysis: general strategy

Translocation detection in lung cancer using mat...
Introduction

Methods

Results

Translocation analysis: tools and workflow

Translocation detection in lung cancer using ma...
Introduction

Methods

Results

Comparison of the tools
Both tools
• cluster paired reads to find potential translocation r...
Introduction

Methods

Results

Results

Translocation detection in lung cancer using mate-pair sequencing and iVIGS

Rich...
Introduction

Methods

Results

Effect of iVIGS quality control filtering
The separation distance distribution was similar f...
Introduction

Methods

Results

Problems with delly
• Applying iVIGS filter resulted in delly not reporting any translocati...
Introduction

Methods

Results

Selection of breakpoint distributions calculated by iVIGS
Kernel Density Estimation

Kerne...
Introduction

Methods

Results

Model of translocation divergence due to cancer proliferation
chromosomes with highly
acti...
Introduction

Methods

Results

Error sources
• Adapter contamination

(before filtering approximately 15% of all reads are...
Introduction

Methods

Results

General information
• Estimated breakpoint positions were highly variable (spanning up to
...
Introduction

Methods

Results

Typical translocation distribution observed in samples
CHRY

CHR1

CHRX

CH

R2

R2

2

1
...
Introduction

Methods

Results

Occuring genes altered by a translocation

Genes used for the diagram were altered in one ...
Introduction

Methods

Results

Potential gene fusions

Genes used for the diagram were altered in one or more samples
del...
Introduction

Methods

Results

Potential gene to intergenic fusions

Genes used for the diagram were altered in one or mo...
Introduction

Methods

Results

Reproducibility: delly

Gene alterations
sample_A1

13

1

2

sample_A2

Translocation det...
Introduction

Methods

Results

Reproducibility: iVIGS

Gene alterations
sample_A1

11

15

10

sample_A2

Translocation d...
Introduction

Methods

Results

Conclusion
• Results varied significantly between delly and iVIGS

• Reproducibility of iVI...
Introduction

Methods

Results

Plans for the future
• Apply adapter removal in preprocessing to improve mapping yield

• ...
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Translocation detection in lung cancer using mate-pair sequencing and iVIGS

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Translocation detection in lung cancer using mate-pair sequencing and iVIGS

  1. 1. Introduction Methods Results Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw University of Kansas Medical Center February 3, 2014 Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  2. 2. Introduction Methods Results Content Introduction Methods Results Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  3. 3. Introduction Methods Results Introduction Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  4. 4. Introduction Methods Results Structural variations Chromosome 1 Chromosome 17 No variation Deletion Insertion Translocation Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  5. 5. Introduction Methods Results Structural variations in mate pair mapping Insertion Deletion reference genome reference genome cancer genome cancer genome reads map closer than expected reads map farther away than expected Translocation reference genome cancer genome reads map to different chromosomes Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  6. 6. Introduction Methods Results Breakpoint resolution with split reads Where are the breakpoints ? known reference cluster cluster Looking at soft clipping reads known reference cluster cluster reads unknown sample Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  7. 7. Introduction Methods Results Methods Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  8. 8. Introduction Methods Results Data A set of mate-pair sequencing data from lung cancer patients was analysed. 35 samples were processed with the sv tool iVIGS 32 samples were processed with the sv tool delly Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  9. 9. Introduction Methods Results Mate-pair preprocessing Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  10. 10. Introduction Methods Results Translocation analysis: general strategy Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  11. 11. Introduction Methods Results Translocation analysis: tools and workflow Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  12. 12. Introduction Methods Results Comparison of the tools Both tools • cluster paired reads to find potential translocation regions • use split reads to find potential breakpoint positions delly • re-assembles split reads • re-maps the assembly to the cluster region iVIGS (tool for identification of variations in genomic structure) • is developed in our lab and currently still a work in progress • performs Kernel Density Estimation on split read mapping positions • estimates propability distribution of breakpoint positions Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  13. 13. Introduction Methods Results Results Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  14. 14. Introduction Methods Results Effect of iVIGS quality control filtering The separation distance distribution was similar for all samples 4e+06 Molina−Dataset 2e+06 0e+00 1e+06 counts 3e+06 unfiltered filtered 0 1000 2000 3000 4000 5000 6000 distance between mate−reads Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  15. 15. Introduction Methods Results Problems with delly • Applying iVIGS filter resulted in delly not reporting any translocations • Taking all reads and applying delly internal filtering resulted in finding translocations • Coverage for reads after strict iVIGS filtering was probably too inconsistent for assembly. The type of used assembly is also important. (see next slides) • Thus the following results for delly are refering to a workflow that uses the internal filtering method. Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  16. 16. Introduction Methods Results Selection of breakpoint distributions calculated by iVIGS Kernel Density Estimation Kernel Density Estimation 27735000 27740000 || | | | 135500000 | 0.00010 breakpoint density | 135496000 0.00000 0.0000 ||||||||||||||||||| ||||||||||| |||||||||||||||| ||||| || ||||||||||||||| ||||||||| ||||||||||| ||||||||| || |||| | | | | | | 27730000 0.00005 0.0010 breakpoint density 0.0005 2e−04 0e+00 1e−04 breakpoint density 3e−04 0.0015 0.00015 4e−04 Kernel Density Estimation | 135504000 135508000 | | 33970000 | || || || ||| | || | | || | 33975000 base position Kernel Density Estimation Kernel Density Estimation | 121484000 121486000 base position 121488000 0.015 breakpoint density 0.010 || 190194000 190198000 | || || | || 190202000 base position Translocation detection in lung cancer using mate-pair sequencing and iVIGS 0.000 0.005 0.0005 0.0000 |||||| | || |||||||||| | | | 121482000 0.020 0.025 0.0020 0.0015 breakpoint density 0.0010 1e−03 8e−04 6e−04 4e−04 2e−04 breakpoint density | 33980000 Kernel Density Estimation 0e+00 121480000 || | | || base position 0.030 base position | 61792000 |||| | 61794000 61796000 61798000 base position Richard Meier & Stefan Graw
  17. 17. Introduction Methods Results Model of translocation divergence due to cancer proliferation chromosomes with highly active regions breaking and translocation subsequent variations in daughter cells breakpoint and cluster distribution density position Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  18. 18. Introduction Methods Results Error sources • Adapter contamination (before filtering approximately 15% of all reads are contaminated.) • Ligation errors • PCR bias • Sequencing errors Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  19. 19. Introduction Methods Results General information • Estimated breakpoint positions were highly variable (spanning up to several thousand base positions in difference) • Translocations were found to almost always overlap with potential deletion or insertion cluster regions (estimated by iVIGS). • In most cases around 35 translocations per sample were estimated 0.02 0.00 0.01 Density 0.03 0.04 translocation discovery of iVIGS 20 30 40 50 60 70 80 number of estimated translocations per sample Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  20. 20. Introduction Methods Results Typical translocation distribution observed in samples CHRY CHR1 CHRX CH R2 R2 2 1 CH CH R2 0 R2 CH R1 8 CH R1 9 CH CH CHR16 CHR1 7 R3 CHR4 CHR15 4 CHR5 CHR1 R1 CH CH R6 3 CH R1 2 CH R1 R7 CH 1 CHR1 CHR8 0 CHR9 Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  21. 21. Introduction Methods Results Occuring genes altered by a translocation Genes used for the diagram were altered in one or more samples delly 108 41 158 iVIGS Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  22. 22. Introduction Methods Results Potential gene fusions Genes used for the diagram were altered in one or more samples delly 32 11 73 iVIGS Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  23. 23. Introduction Methods Results Potential gene to intergenic fusions Genes used for the diagram were altered in one or more samples delly 106 27 109 iVIGS Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  24. 24. Introduction Methods Results Reproducibility: delly Gene alterations sample_A1 13 1 2 sample_A2 Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  25. 25. Introduction Methods Results Reproducibility: iVIGS Gene alterations sample_A1 11 15 10 sample_A2 Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  26. 26. Introduction Methods Results Conclusion • Results varied significantly between delly and iVIGS • Reproducibility of iVIGS seems promising • It is still unclear how strong the influences of diversity in close related cancer cells and library preparation errors are in respect to the results. • It is thus still difficult to determine whether predictions are FP or TP. Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw
  27. 27. Introduction Methods Results Plans for the future • Apply adapter removal in preprocessing to improve mapping yield • Pick a subset of potential gene fusions and validate them • Examine other structural variation types (insertions, deletions, inversions) • Find and use additional sv tools Translocation detection in lung cancer using mate-pair sequencing and iVIGS Richard Meier & Stefan Graw

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