CRISPR/Cas9 Enrichment and Long-read WGS for Structural Variant Discovery

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1 CRISPR/Cas9 Enrichment and Long-read WGS for Structural Variant Discovery PacBio CoLab Session October 20, 2017 For Research Use Only. Not for use in diagnostics procedures. Copyright 2017 by Pacific Biosciences of California, Inc. All rights reserved.

2 PACBIO SMRT SEQUENCING Long Reads average 10 to 15 kb High Consensus Accuracy random errors produce QV50 consensus Uniform, Unbiased Coverage no GC% or sequence complexity bias Sequel System Epigenetic Characterization simultaneous detection of DNA methylation

3 APPLICATIONS OF SMRT SEQUENCING De novo genome assembly Full isoform sequencing Epigenetic characterization Minor variant discovery Structural variant discovery Sequel System Targeted sequencing

4 APPLICATIONS OF SMRT SEQUENCING De novo genome assembly Full isoform sequencing Epigenetic characterization Minor variant discovery Structural variant discovery Sequel System Targeted sequencing

5 cumulative percent VARIATION IN A HUMAN GENOME HG00733 SNVs 100% indels 50 bp structural variants (SVs) 80% 60% 60% 40% base pairs 20% count 0.5% 0% ,000 10, ,000 variant size (bp) Chaisson et al. (2017) biorxiv. doi: /

6 TYPES OF STRUCTURAL VARIATION deletion insertion duplication inversion translocation repeat expansion

7 STRUCTURAL VARIANTS AND DISEASE Schizophrenia Carney complex repeat expansion disorders Poor drug metabolism Breast & ovarian cancer Neurofibromatosis Chronic myeloid leukemia Richards et al. (2013) Front Mol Neurosci. doi: /fnmol

8 TECHNOLOGY TO DETECT STRUCTURAL VARIANTS Deletions Insertions Missed PacBio Illumina repeats + large insertions Bionano variants 1.5 kb 0 5,000 10,000 15,000 20,000 25,000 structural variants Chaisson et al. (2017) biorxiv. doi: /

9 A move forward to full-spectrum SV detection... will increase the diagnostic yield in patients with genetic disease, SV-mediated mutation, and repeat expansions. Chaisson et al. (2017) biorxiv. doi: /

10 PacBio Long-Read WGS for Structural Variant Discovery Targeted Enrichment without Amplification and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

11 PacBio Long-Read WGS for Structural Variant Discovery Targeted Enrichment without Amplification and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

12 FOR MORE INFORMATION PACB.COM/SV

13 WGS FOR STRUCTURAL VARIANT DISCOVERY Structural Variants Small Variants Sequencing PacBio Sequel System Short-read NGS Read Mapping NGMLR BWA Variant Calling pbsv GATK Visualization IGV 2.4 IGV

14 WGS FOR STRUCTURAL VARIANT DISCOVERY Structural Variants Small Variants Sequencing PacBio Sequel System Short-read NGS Read Mapping NGMLR BWA Variant Calling pbsv GATK Visualization IGV 2.4 IGV

15 SEQUENCING Library Preparation 5 µg DNA 20 kb shear + damage repair SMRTbell adapter ligation 15 kb size selection

16 SEQUENCING Sequencing polymerase binding Sequel System (5 Gb per SMRT Cell)

17 WGS FOR STRUCTURAL VARIANT DISCOVERY Structural Variants Small Variants Sequencing PacBio Sequel System Short-read NGS Read Mapping NGMLR BWA Variant Calling pbsv GATK Visualization IGV 2.4 IGV

18 penalty READ MAPPING reference biological indel affine (BWA) convex (NGMLR) PacBio read gap size BWA NGMLR Sedlazeck et al. (2017) biorxiv. doi: /

19 WGS FOR STRUCTURAL VARIANT DISCOVERY Structural Variants Small Variants Sequencing PacBio Sequel System Short-read NGS Read Mapping NGMLR BWA Variant Calling pbsv GATK Visualization IGV 2.4 IGV

20 VARIANT CALLING FIND SV SIGNATURES CLUSTER SV SIGNATURES FILTER SUMMARIZE INTO SV GENOTYPE CIGAR D & I 50 bp nearby with similar sequence 2 and 20% reads support consensus of supporting reads supporting reads / covering reads

21 VARIANT CALLING FIND SV SIGNATURES CLUSTER SV SIGNATURES FILTER SUMMARIZE INTO SV GENOTYPE CIGAR D & I 50 bp nearby with similar sequence 2 and 20% reads support consensus of supporting reads supporting reads / covering reads

22 VARIANT CALLING FIND SV SIGNATURES CLUSTER SV SIGNATURES FILTER SUMMARIZE INTO SV GENOTYPE CIGAR D & I 50 bp nearby with similar sequence 2 and 20% reads support consensus of supporting reads supporting reads / covering reads

23 VARIANT CALLING FIND SV SIGNATURES CIGAR D & I 50 bp CLUSTER SV SIGNATURES nearby with similar sequence FILTER 2 and 20% reads support 1 of 10 4 of 10 SUMMARIZE INTO SV consensus of supporting reads GENOTYPE supporting reads / covering reads

24 VARIANT CALLING FIND SV SIGNATURES CIGAR D & I 50 bp CLUSTER SV SIGNATURES nearby with similar sequence FILTER 2 and 20% reads support 1 of 10 4 of 10 SUMMARIZE INTO SV consensus of supporting reads 329 bp deletion GENOTYPE supporting reads / covering reads

25 VARIANT CALLING FIND SV SIGNATURES CIGAR D & I 50 bp CLUSTER SV SIGNATURES nearby with similar sequence FILTER 2 and 20% reads support 1 of 10 4 of 10 SUMMARIZE INTO SV consensus of supporting reads 329 bp deletion GENOTYPE supporting reads / covering reads heterozygous (4 of 10)

26 VARIANT CALLING SMRT Analysis SMRT Analysis chr ACGCGGCCGCCTCC TCCTCCGAACGTGGCCTCCTCCGAACGCGGCCGCCTCCTCCTCCGAACGCGGCCGCCTCCTCCTCCGA A PASS IMPRECISE;SVTY PE=DEL;END=904587;SVLEN=-97;SVANN=TANDEM GT:AD:DP 0/1:9:15 SMRT Analysis SMRT Analysis

27 WGS FOR STRUCTURAL VARIANT DISCOVERY Structural Variants Small Variants Sequencing PacBio Sequel System Short-read NGS Read Mapping NGMLR BWA Variant Calling pbsv GATK Visualization IGV 2.4 IGV

28 VISUALIZATION insertion deletion Robinson et al. (2011) Nature Biotechnology. doi: /nbt.1754.

29 WGS FOR STRUCTURAL VARIANT DISCOVERY Structural Variants Small Variants Sequencing PacBio Sequel System Short-read NGS Read Mapping NGMLR BWA Variant Calling pbsv GATK Visualization IGV 2.4 IGV

30 % SVs detected HOW MUCH TO SEQUENCE? 100% 90% 80% 70% 60% 50% 40% Het Hom Human HG00733 Sequel System 211 Gb (70-fold) 30% 20% short read 10% 0% Coverage 5- to 10-fold optimal tradeoff of cost vs. performance disease gene discovery; population characterization 30- to 40-fold saturate discovery de novo variant discovery

31 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 transphenoidal resection of pituitary present (26 yrs) recurrence of myxomata, consideration for heart transplant short-read whole genome sequencing negative Merker et al. (2017) Genetics in Medicine. doi: /gim

32 % SVs detected EVALUATING STRUCTURAL VARIANTS Deletions Insertions Initial call set 6,971 6, % 90% 8-fold 80% 70% 60% 50% 40% 30% 20% 10% Het Hom Merker et al. (2017) Genetics in Medicine. doi: /gim % Coverage

33 % SVs detected EVALUATING STRUCTURAL VARIANTS Deletions Insertions Initial call set 6,971 6,821 Not in segdup 5,893 6, % 8-fold Not in NA12878 healthy control Overlaps RefSeq coding exon 2,476 3, % 80% 70% 60% 50% 40% Het Hom Gene linked to some disease in OMIM % 20% 10% Merker et al. (2017) Genetics in Medicine. doi: /gim % Coverage

34 HETEROZYGOUS 2.2 KB DELETION IN PRKAR1A PacBio discovery Sanger confirmation Merker et al. (2017) Genetics in Medicine. doi: /gim

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36 PacBio Long-Read WGS for Structural Variant Discovery Targeted Enrichment without Amplification and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

37 REPEAT EXPANSION DISEASES

38 CRISPR/CAS9 SYSTEM Bacterial Adaptive Immunity RNA-guided DNA Endonuclease Some in vivo applications: - Gene silencing - Homology-directed repair - Transient gene silencing or transcriptional repression - Transient activation of endogenous genes - Transgenic animals and embryonic stem cells

39 PCR-FREE TARGET ENRICHMENT VIA CAS9

40 COVERAGE ACROSS THE GENOME 1 SMRT Cell (PacBio RS II)

41 HUNTINGTON S DISEASE (HD) - Autosomal dominant neurodegenerative genetic disorder - Caused by an expansion of a CAG triplet repeat stretch in the Huntingtin (HTT) gene - polyglutamine tract

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43 CAG REPEAT COUNTS

44 CAG REPEAT COUNTS IN HD PATIENTS Widening repeat number distribution at the mutated allele is biological Obtained roughly equal number of sequenced molecules for normal and mutated alleles Samples obtained from Vanessa Wheeler (Harvard Medical School)

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46 FRAGILE X SYNDROME - Most common heritable form of cognitive impairment - Caused by expansion of a CGG trinucleotide repeat in the 5 UTR of the FMR1 gene fraxa.org

47 AGG INTERRUPTIONS REDUCE THE CHANCES OF PRE- TO FULL MUTATION TRANSMISSION CGG CGG CGG CGG AGG CGG Difference in risk is greatest near CGG repeats Having full sequence information is medically relevant 80% 60% 15% Yrigollen et al. (2012) Genet Med Maternal CGG repeat number 2 CGG CGG CGG CGG AGG CGG CGG CGG CGG CGG CGG CGG CGG CGG AGG CGG 1 CGG CGG CGG CGG AGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG 0 CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG Yrigollen et al. (2012) Genet Med 14:

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50 SUBREAD COVERAGE ON THE SEQUEL SYSTEM 1 Sequel SMRT Cell 1M

51 MULTIPLEXED SAMPLES ON THE SEQUEL SYSTEM CAG Repeat Counts from 3 Controls and 3 HD Patients

52 CONCLUSION Amplification-free enrichment with CRISPR/Cas9 and SMRT Sequencing achieves the base-level resolution required to understand the underlying biology of repeat expansion disorders - Target any hard-to-amplify genomic region regardless of sequence context - Avoid PCR bias and PCR errors - Accurately sequence through long repetitive and low-complexity regions - Count repeats and identify sequence interruptions - Detect sample mosaicism

53 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.