Detecting Structural Variants in PacBio Reads Tools and Applications

Transcription

1 Detecting Structural Variants in PacBio Reads Tools and Applications Aaron Wenger For Research Use Only. Not for use in diagnostics procedures. Copyright 2017 by Pacific Biosciences of California, Inc. All rights reserved.

2 STRUCTURAL VARIANT = DIFFERENCE 50 BP Deletion Insertion Duplication Inversion Tandem Repeat Translocation

3 ASSAYS TO DETECT STRUCTURAL VARIANTS Cytogenetics Microarray Sequencing technologies karyotype, FISH oligo acgh, SNP acgh short read, PacBio long read resolution >5 Mb, >100 kb >50 kb basepair limitation marker density, microscope resolution probe density read length (mappability and spanning), systematic bias

4 STRUCTURAL VARIANTS DETECTED IN A HUMAN GENOME PacBio 20,000 Short reads 4,000 repeats + GC-rich + large insertions Huddleston et al. (2017) Genome Research 27(5): Seo et al. (2016) Nature 538: Sudmant et al. (2016) Nature 526:75-81.

5 VARIATION BETWEEN TWO HUMAN GENOMES vs variants basepairs affected 5 Mb 3 Mb 10 Mb SNVs indels structural variants Huddleston et al. (2017) Genome Research 27(5):

6 THE HUMAN GENOME IS COMPARATIVELY EASY structural variants per Mb percent of genome affected by SV reported short-read sensitivity for SV 7 0.3% 20% human fruit fly % 23% zebrafish % 1% Huddleston et al. (2017) Genome Research 27(5): Zichner et al. (2013) Genome Research 23(3): Patowary et al. (2013) Zebrafish 10(1): Zebrafish image courtesy of Lizzy Griffiths

7 EACH CLADE HAS ITS OWN STRUCTURAL VARIANT PROFILE human fruit fly zebrafish

8 Any study of genetic variation is incomplete until structural variation is detected with PacBio sequencing.

9 Any study of genetic variation is incomplete until structural variation is detected with PacBio sequencing. rare disease diagnosis GWAS evodevo

10 Any study of genetic variation is incomplete until structural variation is detected with PacBio sequencing. rare disease diagnosis GWAS evodevo

11 CLINICAL CASE HISTORY 7 yrs 10 yrs left atrial myxoma resection, atrial repair testicular mass, right orchiectomy 13 yrs pituitary tumor 16 yrs 18 yrs 19 yrs 21 yrs present (26 yrs) recurrence of myxomata, resection, adrenal microadenoma recurrence of ventricular myxomata, resection, VT ACTH-independent Cushing s disease, thyroid nodules transphenoidal resection of pituitary recurrence of myxomata, consideration for heart transplant genetics suggests Carney complex PRKAR1A testing negative short-read whole genome sequencing negative Merker et al. (2017) Genetics in Medicine. doi: /gim

12 EVALUATING STRUCTURAL VARIANTS Sequel System SEQUENCE (8-FOLD) Deletions 50 bp NGM-LR MAP READS PBHoney CALL VARIANTS Insertions 50 bp Initial call set 6,971 6,821 Merker et al. (2017) Genetics in Medicine. doi: /gim

13 EVALUATING STRUCTURAL VARIANTS Sequel System SEQUENCE (8-FOLD) Deletions 50 bp NGM-LR MAP READS PBHoney CALL VARIANTS Insertions 50 bp Initial call set 6,971 6,821 Not in segdup 5,893 6,254 Not in NA12878 healthy control Overlaps RefSeq coding exon Gene linked to some disease in OMIM Merker et al. (2017) Genetics in Medicine. doi: /gim Rescheneder, Sedlazeck, and Schatz. 2,476 3, English et al. (2014) BMC Bioinformatics 15:180.

14 HETEROZYGOUS 2.2 KB DELETION IN PRKAR1A Merker et al. (2017) Genetics in Medicine. doi: /gim

15 RNA-SEQ SHOWS REDUCED EXPRESSION AND NOVEL EXON-EXON JUNCTION normalized read counts control (n=16) case Merker et al. (2017) Genetics in Medicine. doi: /gim

16 DELETION CLASSIFIED AS PATHOGENIC PVS1 null variant (nonsense, frameshift, canonical splice sites, initiation codon, single or multiexon deletion) in a gene where loss of function is a known mechanism of disease. PS2 de novo (both maternity and paternity confirmed) in a patient with the disease and no family history Merker et al. (2017) Genetics in Medicine. doi: /gim

17 CLINICAL CASE HISTORY 7 yrs 10 yrs left atrial myxoma resection, atrial repair testicular mass, right orchiectomy 13 yrs pituitary tumor 16 yrs 18 yrs 19 yrs recurrence of myxomata, resection, adrenal microadenoma recurrence of ventricular myxomata, resection, VT ACTH-independent Cushing s disease, thyroid nodules genetics suggests Carney complex PRKAR1A testing negative 21 yrs present (26 yrs) transphenoidal resection of pituitary recurrence of myxomata, consideration for heart transplant Merker et al. (2017) Genetics in Medicine. doi: /gim short-read whole genome sequencing negative PacBio sequencing identifies causative structural variant in PRKAR1A

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

19 Any study of genetic variation is incomplete until structural variation is detected with PacBio sequencing. rare disease diagnosis GWAS evodevo

20 SMRT LINK 5.0 INTRODUCES PBSV pbsv command line utility for top-level commands pbsvutil command line utility for detailed commands SMRT Link web interface

21 PBSV: SMRT LINK STRUCTURAL VARIANT CALLER SMRT Analysis

22 PBSV: SMRT LINK STRUCTURAL VARIANT CALLER SMRT Analysis

23 PBSV: SMRT LINK STRUCTURAL VARIANT CALLER SMRT Analysis chr ACGCGGCCGCCTCCTCCTCCGAACGTGGCCTCCTCCGAACGCGGCCGCCTCCTCCTCCGAACGCGGCCGCCTCCTCCTCCGA A PASS IMPRECISE;SVTYPE=DEL;END=904587;SVLEN=-97;SVANN=TANDEM GT:AD:DP 0/1:9:15

24 PBSV: SMRT LINK STRUCTURAL VARIANT CALLER SMRT Analysis chr Deletion -97. GT:AD:DP 0/1:9:15 SVANN=TANDEM

25 PBSV: SMRT LINK STRUCTURAL VARIANT CALLER SMRT Analysis

26 PBSV: SMRT LINK STRUCTURAL VARIANT CALLER SMRT Analysis

27 PBSV: SMRT LINK STRUCTURAL VARIANT CALLER SMRT Analysis

28 PBSV: SMRT LINK STRUCTURAL VARIANT CALLER SMRT Analysis

29 HOW MUCH TO SEQUENCE? Human HG00733 Sequel System 211 Gb (70-fold) Standard is full dataset Subsample / titrate to lower coverage Evaluate overlap with standard call set 100% 90% % SVs detected 80% 70% 60% 50% 40% 30% 20% Het Hom Het Hom short read 10% 0% Coverage Coverage

30 SUMMARY vs. 5 Mb 3 Mb 10 Mb PacBio 20,000 SNVs indels structural variants Short reads 4,000 repeats + GC-rich + large insertions

31 ACKNOWLEDGMENTS Stanford Univ PacBio Schatz Lab Euan Ashley Jason Merker Sowmi Utiramerur Yuan Li Chris Dunn Ben Lerch Jim Drake Mary Budagyan Jason Chin Sarah Kingan Greg Concepcion Michael Schatz Philipp Rescheneder Fritz Sedlazeck Nat Echols Jonas Korlach Aaron Klammer Paul Peluso Christine Lambert Kevin Eng NGM-LR penalty convex errors indels gap size

32 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.