structuarl variant detection in smrt link powered by pbsv · 2019. 4. 10. · structural variant...
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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
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TYPES OF STRUCTURAL VARIATION
deletion insertion duplication
inversion translocation repeat expansion
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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
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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.
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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
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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
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SEQUENCING
5 µg DNA
20 kb shear
+ damage repair
SMRTbell adapter ligation
15 kb size selection
Library Preparation
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SEQUENCING
polymerase binding
Sequel System
(5 Gb per SMRT Cell)
Sequencing
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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
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READ MAPPING
Sedlazeck et al. (2017) bioRxiv. doi:10.1101/169557.
reference
biological indel
PacBio read
gap size
penalty convex
(NGMLR)
affine
(BWA)
NGMLRBWA
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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VISUALIZATION
Robinson et al. (2011) Nature Biotechnology. doi:10.1038/nbt.1754.
insertion
deletion
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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
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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)
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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
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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
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HETEROZYGOUS 2.2 KB DELETION IN PRKAR1A
Merker et al. (2017) Genetics in Medicine. doi:10.1038/gim.2017.86.
PacBio
discovery
Sanger
confirmation
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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
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