Structural Variant Detection in SMRT Link 5 with pbsv€¦ · STRUCTURAL VARIANTS DETECTED IN A HUMAN GENOME 4,000 20,000 Short reads PacBio repeats + GC-rich + large insertions Huddleston

For Research Use Only. Not for use in diagnostics procedures. © Copyright 2017 by Pacific Biosciences of California, Inc. All rights reserved.

Structural Variant Detection in

SMRT Link 5 with pbsv

Aaron Wenger 2017-06-27

STRUCTURAL VARIANT = DIFFERENCE ≥50 BP

Insertion Duplication

Inversion Tandem Repeat Translocation

Deletion

VARIATION BETWEEN TWO HUMAN GENOMES

Huddleston et al. (2017) Genome Research 27(5):677-85.

vs.

5×106

5 Mb 3 Mb 10 Mb

variants

basepairs

affected

SNVs

4×105

structural variantsindels

2×104

STRUCTURAL VARIANTS DETECTED IN A HUMAN GENOME

4,000

20,000

Short reads

PacBio

repeats + GC-rich +

large insertions

Huddleston et al. (2017) Genome Research 27(5):677-85.

Seo et al. (2016) Nature 538:243-7.

Sudmant et al. (2016) Nature 526:75-81.

SEQUENCING + ANALYSIS

Li and Durbin (2009) Bioinformatics 25:1754-60.

McKenna et al. (2010) Genome Research 20:1297-303.

Structural

Variants

BWASNVs +

Indels

Short

reads

?pbsv

3 COMPONENTS TO PBSV

pbsv command line utility for top-level commands

pbsvutil command line utility for detailed commands

SMRT Link web interface

TOP-LEVEL PBSV COMMANDS

pbsv generate-config [-h] [-o sv.cfg]

(optional) Generate a configuration file to specify options for other stages.

pbsv align [-h] [--cfg_fn sv.cfg]

ref.fa subreads.bam ref.align.bam

Map reads to a reference genome with a “structural variant aware” aligner.

pbsv call [-h] [--cfg_fn sv.cfg]

ref.fa ref.align.bam ref.sv.bed|vcf

Call structural variants from aligned reads.










PBSV ALIGN UTILIZES NGM-LR

Rescheneder, Sedlazeck, and Schatz. https://github.com/philres/ngmlr/.

gap size

pe

na

lty

sequencing errors

(frequent & independent)

structural variants

(infrequent & correlated)

pbsvutil ngmlr

PBSV ALIGN UTILIZES NGM-LR

NGM-LRBWA

gap size

penalty

gap size

penalty

sequencing errors

structural variants

sequencing errors

structural variants

pbsvutil ngmlr

X

PBSV ALIGN CHAINS CO-LINEAR ALIGNMENTS

Reference

Read

ZY

XZ

W W

W

pbsvutil chain

X ZYW W










PBSV CALL: STAGED STRUCTURAL VARIANT CALLER

FIND SV

SIGNATURES

CIGAR D & I

≥ 50 bp

CLUSTER SV

SIGNATURES

nearby with similar

sequence

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPE

SV

supporting reads /

covering reads

ANNOTATE SV

Alu, LINE, SVA,

tandem repeat

FILTER SV

≥ 2 and ≥ 20%

reads support


FIND SV

SIGNATURES

CIGAR D & I

≥ 50 bp

CLUSTER SV

SIGNATURES

nearby with similar

sequence

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPE

SV

supporting reads /

covering reads

ANNOTATE SV

Alu, LINE, SVA,

tandem repeat

FILTER SV

≥ 2 and ≥ 20%

reads support


FIND SV

SIGNATURES

CIGAR D & I

≥ 50 bp

CLUSTER SV

SIGNATURES

nearby with similar

sequence

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPE

SV

supporting reads /

covering reads

ANNOTATE SV

Alu, LINE, SVA,

tandem repeat

FILTER SV

≥ 2 and ≥ 20%

reads support


FIND SV

SIGNATURES

CIGAR D & I

≥ 50 bp

CLUSTER SV

SIGNATURES

nearby with similar

sequence

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPE

SV

supporting reads /

covering reads

ANNOTATE SV

Alu, LINE, SVA,

tandem repeat

FILTER SV

≥ 2 and ≥ 20%

reads support

329 bp

deletion

63 bp

insertion


FIND SV

SIGNATURES

CIGAR D & I

≥ 50 bp

CLUSTER SV

SIGNATURES

nearby with similar

sequence

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPE

SV

supporting reads /

covering reads

ANNOTATE SV

Alu, LINE, SVA,

tandem repeat

FILTER SV

≥ 2 and ≥ 20%

reads support

heterozygous

(4 of 10)

heterozygous

(1 of 10)

329 bp

deletion

63 bp

insertion


FIND SV

SIGNATURES

CIGAR D & I

≥ 50 bp

CLUSTER SV

SIGNATURES

nearby with similar

sequence

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPE

SV

supporting reads /

covering reads

ANNOTATE SV

Alu, LINE, SVA,

tandem repeat

FILTER SV

≥ 2 and ≥ 20%

reads support

Alu-

heterozygous

(4 of 10)

heterozygous

(1 of 10)

329 bp

deletion

63 bp

insertion


FIND SV

SIGNATURES

CIGAR D & I

≥ 50 bp

CLUSTER SV

SIGNATURES

nearby with similar

sequence

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPE

SV

supporting reads /

covering reads

ANNOTATE SV

Alu, LINE, SVA,

tandem repeat

FILTER SV

≥ 2 and ≥ 20%

reads support

Alu-

heterozygous

(4 of 10)

heterozygous

(1 of 10)

329 bp

deletion

63 bp

insertion

PBSV: SMRT LINK STRUCTURAL VARIANT CALLER

SMRT Analysis


SMRT Analysis


SMRT Analysis

chr1

904490

ACGCGGCCGCCTCCTCCTCCGAACGTGGCCTCCTCCGAACGCGGCCGCCTCCTCCTCCGAACGCGGCCGCCTCCTCCTCCGA

A

PASS

IMPRECISE;SVTYPE=DEL;END=904587;SVLEN=-97;SVANN=TANDEM

GT:AD:DP

0/1:9:15


SMRT Analysis

chr1 904490 904587 Deletion -97 . GT:AD:DP 0/1:9:15 SVANN=TANDEM


SMRT Analysis


SMRT Analysis


SMRT Analysis


SMRT Analysis

3 COMPONENTS TO PBSV

pbsv command line utility for top-level commands

pbsvutil command line utility for detailed commands

SMRT Link web interface

PacBio

ACKNOWLEDGMENTS

Schatz LabMichael Schatz

Philipp Rescheneder

Fritz Sedlazeck

gap size

penalty

convexerrorsindels

NGM-LR

Yuan Li

Chris Dunn

Ben Lerch

Jim Drake

Nat Echols

Aaron Klammer

Mary Budagyan

For Research Use Only. Not for use in diagnostics procedures. © Copyright 2017 by Pacific Biosciences of California, Inc. All rights reserved. Pacific Biosciences, the Pacific Biosciences logo,

PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx.

FEMTO Pulse and Fragment Analyzer are trademarks of Advanced Analytical Technologies.

All other trademarks are the sole property of their respective owners.

www.pacb.com

Documents

Structural Variant Detection in SMRT Link 5 with pbsv€¦ · STRUCTURAL VARIANTS DETECTED IN A HUMAN GENOME 4,000 20,000 Short reads PacBio repeats + GC-rich + large insertions Huddleston