For Research Use Only. Not for use in diagnostics procedures. © Copyright 2018 by Pacific Biosciences of California, Inc. All rights reserved.

Structural Variant Detection in SMRT Link

powered by pbsv

TYPES OF STRUCTURAL VARIATION

deletion insertion duplication

inversion translocation repeat expansion

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.

WGS FOR STRUCTURAL VARIANT DISCOVERY

PacBio Sequel System Short-read NGS

NGMLR BWA

pbsv GATK

IGV IGV

Structural Variants Small Variants

Sequencing

Read Mapping

Variant Calling

Visualization

WGS FOR STRUCTURAL VARIANT DISCOVERY

PacBio Sequel System Short-read NGS

NGMLR BWA

pbsv GATK

IGV IGV

Structural Variants Small Variants

Sequencing

Read Mapping

Variant Calling

Visualization

SEQUENCING

5 µg DNA

20 kb shear

+ damage repair

SMRTbell adapter ligation

15 kb size selection

Library Preparation

SEQUENCING

polymerase binding

Sequel System

(5 Gb per SMRT Cell)

Sequencing

WGS FOR STRUCTURAL VARIANT DISCOVERY

PacBio Sequel System Short-read NGS

NGMLR BWA

pbsv GATK

IGV IGV

Structural Variants Small Variants

Sequencing

Read Mapping

Variant Calling

Visualization

READ MAPPING

Sedlazeck et al. (2017) bioRxiv. doi:10.1101/169557.

reference

biological indel

PacBio read

gap size

penalty convex

(NGMLR)

affine

(BWA)

NGMLRBWA

WGS FOR STRUCTURAL VARIANT DISCOVERY

PacBio Sequel System Short-read NGS

NGMLR BWA

pbsv GATK

IGV IGV

Structural Variants Small Variants

Sequencing

Read Mapping

Variant Calling

Visualization

VARIANT CALLING

http://pacb.com/sv

FIND SV

SIGNATURES

CIGAR D & I

≥50 bp

CLUSTER SV

SIGNATURES

nearby with

similar sequence

FILTER≥2 and ≥20%

reads support

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPEsupporting reads /

covering reads

VARIANT CALLING

http://pacb.com/sv

FIND SV

SIGNATURES

CIGAR D & I

≥50 bp

CLUSTER SV

SIGNATURES

nearby with

similar sequence

FILTER≥2 and ≥20%

reads support

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPEsupporting reads /

covering reads

VARIANT CALLING

http://pacb.com/sv

FIND SV

SIGNATURES

CIGAR D & I

≥50 bp

CLUSTER SV

SIGNATURES

nearby with

similar sequence

FILTER≥2 and ≥20%

reads support

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPEsupporting reads /

covering reads

VARIANT CALLING

http://pacb.com/sv

FIND SV

SIGNATURES

CIGAR D & I

≥50 bp

CLUSTER SV

SIGNATURES

nearby with

similar sequence

FILTER≥2 and ≥20%

reads support

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPEsupporting reads /

covering reads

4 of 101 of 10

VARIANT CALLING

http://pacb.com/sv

FIND SV

SIGNATURES

CIGAR D & I

≥50 bp

CLUSTER SV

SIGNATURES

nearby with

similar sequence

FILTER≥2 and ≥20%

reads support

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPEsupporting reads /

covering reads

329 bp deletion

4 of 101 of 10

VARIANT CALLING

http://pacb.com/sv

FIND SV

SIGNATURES

CIGAR D & I

≥50 bp

CLUSTER SV

SIGNATURES

nearby with

similar sequence

FILTER≥2 and ≥20%

reads support

SUMMARIZE

INTO SV

consensus of

supporting reads

GENOTYPEsupporting reads /

covering reads

329 bp deletion

4 of 101 of 10

heterozygous (4 of 10)

VARIANT CALLING

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 SMRT Analysis

WGS FOR STRUCTURAL VARIANT DISCOVERY

PacBio Sequel System Short-read NGS

NGMLR BWA

pbsv GATK

IGV 2.4 IGV

Structural Variants Small Variants

Sequencing

Read Mapping

Variant Calling

Visualization

VISUALIZATION

Robinson et al. (2011) Nature Biotechnology. doi:10.1038/nbt.1754.

insertion

deletion

WGS FOR STRUCTURAL VARIANT DISCOVERY

PacBio Sequel System Short-read NGS

NGMLR BWA

pbsv GATK

IGV 2.4 IGV

Structural Variants Small Variants

Sequencing

Read Mapping

Variant Calling

Visualization

HOW MUCH TO SEQUENCE?

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 10 20 30 40 50

% S

Vs d

ete

cte

d

Coverage

Het

Hom

short read

30- to 40-fold

saturate discovery

de novo variant discovery

5- to 10-fold

optimal tradeoff of

cost vs. performance

disease gene discovery;

population characterization

Human HG00733

Sequel System

211 Gb (70-fold)

CLINICAL CASE HISTORY

Merker et al. (2017) Genetics in Medicine. doi:10.1038/gim.2017.86.

7 yrsleft atrial myxoma resection,

atrial repair

10 yrstesticular mass,

right orchiectomy

13 yrs pituitary tumor

16 yrsrecurrence of myxomata, resection,

adrenal microadenoma

18 yrsrecurrence of ventricular myxomata,

resection, VT

19 yrsACTH-independent Cushing’s disease,

thyroid nodules

21 yrs transphenoidal resection of pituitary

present

(26 yrs)

recurrence of myxomata, consideration

for heart transplant

genetics suggests Carney complex

PRKAR1A testing negative

short-read whole genome

sequencing negative

EVALUATING STRUCTURAL VARIANTS

Merker et al. (2017) Genetics in Medicine. doi:10.1038/gim.2017.86.

Deletions Insertions

Initial call set 6,971 6,821

Not in segdup 5,893 6,254

Not in NA12878

“healthy” control2,476 3,171

Overlaps RefSeq

coding exon39 16

Gene linked to some

disease in OMIM3 3

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 10 20 30 40 50%

SV

s d

ete

cte

d

Coverage

Het

Hom

8-fold

HETEROZYGOUS 2.2 KB DELETION IN PRKAR1A

Merker et al. (2017) Genetics in Medicine. doi:10.1038/gim.2017.86.

PacBio

discovery

Sanger

confirmation

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,

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