Methods for improved SV detection and interpretation

Fritz J SedlazeckSchatz group @ CSHL

Breast Cancer

75x PacBio coverage (50x >10kbp)

Nattestad et al, In preparation

Breast Cancer PipelinePacBio Sequencing

Alignment with BWA-MEM

Copy number analysis

SV-calling with Lumpy

Graphical genome threading analysis

Assembly with Falcon on DNAnexus

Alignment with MUMmer (nucmer)

Call variants between

consecutive alignments

Call variants within alignments

IsoSeq transcriptome

analysis

...

Detailed analysis of Her2

amplifications

Validation using PCR.

Illumina sequencing

What we learnedIllumina:

– Nested SV are hard to detect – Problems with repetitive regions

PacBio:– Tools are not yet as accurate– Erroneous alignments hide SV

Assembly vs. mapping– Detection of more SV over assembly– Assembly misses low frequent SVs– Comparing the assembly to the genome is still challenging

What we can offer

• Improve alignments:– NextGenMap-LR*

• Improve SV detection:– Survivor (illumina)– Sniffles (PacBio)*– Assembly guided detection (PacBio)

• Interpretation of the SV:– SplitThreader*

NextGenMap-LR

• Novel algorithms to make mapper aware of SV region

– Builds on very fast and robust NextGenMap algorithms (Sedlazeck et al. Bioinformatics. 2013)

• Major benefits from pairwise alignment with improved scoring functions– Accounts for long (SV) and smaller (error) gaps – SVs are better represented, cleaner breakpoints

NextGenMap-LRBWA-MEM NextGenMap-LR

Very stable deletion calls

NextGenMap-LRBWA-MEM NextGenMap-LR

Very stable insertion call

Sniffles

• Predicts: deletion, duplication, insertion, inversion, translocations and nested SV

• Designed to overcome the problems of current SV callers.

• Scans for erroneous regions in the alignment and predict if they overlap.

Sniffles

Sniffles (Inversion)

SplitThreader

• Graph based tool that uses SV calls.

• Identifies the most likely paths that forms the chromosomes.

50 Mb

Chr 8

PacBio

PacBio

Her2

Chr 17

RARA

PKIA

GSDMB

TATDN1

Cancer lesion reconstruction from genomic threads

How we could contribute

1. Calling SV using PacBio

2. Calling SV using Illumina

3. Interpretation of Structural Variants

Thanks to:Schatz group + McCombie group + Hicks group + OICR + PacBio + DNAnexus