Pacific Biosciences Of California, Inc. · NASDAQ:PACB
Pacific Biosciences Of California — Technical Presentation (June 2017)
35 slides from Pacific Biosciences Of California's technical presentation, Jun 27, 2017
Slide 1: Iso-Seq Method Updates and Target Enrichment Without Amplification for SMRT Sequencing
Iso-Seq Method Updates and Target Enrichment Without Amplification for SMRT Sequencing PacBio Americas User Group Meeting Sample Prep Workshop June.27.2017 Tyson Clark, Ph.D. For Research Use Only. Not for use in diagnostics procedures. © Copyright 2017 by Pacific Biosciences of California, Inc. All
Slide 2: Agenda
AGENDA - Iso-Seq Method Updates -Overview of New Iso-Seq Method Workflow for Sequel Systems -Example Sequel System Iso-Seq Method Datasets -Size Selection Option for Iso-Seq Method on the Sequel System -Sequencing and Experimental Design Recommendations -Summary - Target Enrichment R&D Updates -Targ
Slide 3: Iso-Seq Method Updates
PACBIO’ Iso-Seq Method Updates
Slide 4: Determination of Transcript Isoforms
DETERMINATION OF TRANSCRIPT ISOFORMS Gene mRNA isoforms Short-read technologies: Insufficient Connectivity Reads spanning Splice Isoform Uncertainty splice junctions PacBio’s Iso-Seq Analysis solution: Full-length cDNA Sequence Reads Splice Isoform Certainty – No Assembly Required
Slide 5: Sequel System Iso-Seq Procedure Overview
Sequel System Iso-Seq Procedure Overview
Slide 6: Existing Iso-Seq Sample Prep Workflow on the PacBio RS II Involves Extensive Size Selection
E X I S T I N G I S O - S E Q S AM P L E P R E P W O R K F L O W O N T H E PAC B I O R S I I I N V O LV E S E X T E N S I V E S I Z E S E L E C T I O N 1-2 2-3 3-6 5-10 Total RNA kb kb kb kb Optional Poly-A Selection Re-Amplification polyA+ RNA 1-2 2-3 3-6 5-10 kb kb kb kb Reverse Transcription SMRT
Slide 7: Decreased Loading Bias in Sequel System Reduces Need for Size Selection
DECREASED LOADING BIAS IN SEQUEL SYSTEM REDUCES NEED FOR SIZE SELECTION Sequel SYSTEM output correlates with input SMRTbell library size distribution BioAnalyzer trace of a non-size selected Iso-Seq Library Full-length Transcript Sizes - Histogram plot of number of full-length sequences by - The ful
Slide 8: New Streamlined Iso-Seq Workflow for Sequel
NEW STREAMLINED ISO-SEQ WORKFLOW FOR SEQUEL
Slide 9: Example Sequel System Iso-Seq Method Datasets
Example Sequel System Iso-Seq Method Datasets
Slide 10: Example Sequel System Iso-Seq Method Dataset: Primary Sequencing Metrics
EXAMPLE SEQUEL SYSTEM ISO-SEQ METHOD DATASET: PRIMARY SEQUENCING METRICS Sample [On- Total Gb Movie Pol RL (bp) Longest P0 (%) P1 (%) P2 (%) Plate] Subread 1x + 0.4x 50 pM 4.89 360 11766 2816 339905 415685 281207 (32.8%) (40.1%) (27.1%) 1x + 0.4x 40 pM 7.57 600 13845 2855 172510 546423 317867 (17%)
Slide 11: Example Sequel System Iso-Seq Method Dataset: Secondary Analysis Metrics
EXAMPLE SEQUEL SYSTEM ISO-SEQ METHOD DATASET: SECONDARY ANALYSIS METRICS ( ISO-SEQ PROTOCOL) Sample [On-Plate] Movie #CCS CCS RL #FLNC (%) FLNC RL # Polished # Polished HQ Isoforms LQ Isoforms 1x + 0.4x 50 pM 360 415,539 2602 202,328 2892 14,722 96,755 (48.7%) 1x + 0.4x 40 pM 600 545,724 2535 264,77
Slide 12: Size Selection Option for Iso-Seq Method on the Sequel System
Size Selection Option for Iso-Seq Method on the Sequel System
Slide 13: New Streamlined Iso-Seq Workflow for Sequel
NEW STREAMLINED ISO-SEQ WORKFLOW FOR SEQUEL
Slide 14: Effect of Using Size Selection Option with Iso-Seq Method on the Sequel System
EFFECT OF USING SIZE SELECTION OPTION WITH ISO-SEQ METHOD ON THE SEQUEL SYSTEM Non-size selected - BluePippin (or SageELF) size-selected Non-size selected library (4.5 – 10 kb) can be pooled with plus >4 kb size selected library non-size selected library and co-loaded (co-loaded) together onto a sin
Slide 15: Recommendations for Existing Size-Selected Fractions
RECOMMENDATIONS FOR EXISTING SIZE -SELECTED FRACTIONS 0.5-2 kb 1.5-3 kb 2.5-6 kb Transcript Count 4.5-10 kb Full-Length Transcript Size - Anneal and Bind size-selected fractions separately - Pool fractions by equimolar pooling (Make adjustments where necessary) - Data shown for 4 size bins pooled to
Slide 16: Available Technical Resources for Iso-Seq Analysis
AVAILABLE TECHNICAL RESOURCES FOR ISO-SEQ ANALYSIS Iso-Seq Best Practices NimbleGen Targeted Iso-Seq Barcoding Iso-Seq Iso-Seq on Sequel IDT Targeted Iso-Seq
Slide 17: Summary
SUMMARY - Prepare full-length transcripts using the Clontech® SMARTer® PCR cDNA Synthesis Kit with as little as 1 ng of poly A+ RNA or 2 ng of total RNA - Sequel System loading protocols reduce need for size selection for transcripts <4 kb - Optional size-selection protocols to enrich for transcript
Slide 18: Target Enrichment R&D Updates
Target Enrichment R&D Updates
Slide 19: Targeted SMRT Sequencing of Repeat-Expansion Disease Regions Using CRISPR/Cas9
Targeted SMRT Sequencing of Repeat - Expansion Disease Causative Genomic Regions Using a CRISPR/Cas9, Non -Amplification Based Method For Research Use Only. Not for use in diagnostics procedures. © Copyright 2015 by Pacific Biosciences of California, Inc. All rights reserved.
Slide 20: Repeat Expansion Diseases
R EPEAT EXPA N SIO N D ISEA SES 23
Slide 21: CRISPR/Cas9 System
C R ISPR /C A S9 SYST EM • 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 cell
Slide 22: PCR-Free Target Enrichment via Cas9 Digestion: Method Overview
PC R - F R EE TA R G ET EN R IC H MEN T VIA C A S9 D IG EST ION: MET H O D O VERVIEW ( C U R R EN TLY IN D EVEL O PM EN T )
Slide 23: Using CRISPR/Cas9 to Enrich for Repeat Expansion Disorders
U SIN G C R ISPR /C A S9 TO EN R IC H F O R R EPEAT EXPA N SIO N D ISO R D ER S * * *Restriction Enzyme Number of individual molecules sequenced - Improved on-target rate with complexity reduction: - Restriction enzymes are used to degrade unwanted SMRTbell templates - Additional starting DNA is req
Slide 24: Coverage Across the Genome
COVERAGE ACROSS THE GENOME ED pacsio ~ Chromosome Hy 7 8 9 10 W 12 13 14 15 16 «17 «18 19 20 21 22 x Y s @ 2000+ SCA10 3 = rom © 1500+ 5 HTT FMR1 rs E 10004 € a gw 2004 C9orf72 3 S 0 | 1 rn Genomic Position
Slide 25: Huntington's Disease Tomorrow
RICARDO MOURO PINTO WILL BE DISCUSSING HUNTINGTON’S DISEASE TOMORROW
Slide 26: Fragile X Syndrome
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
Slide 27: >700 CGG Repeats Sequenced from the FMR1 Gene
>700 CGG REPEATS SEQUENCED FROM THE FMR1 GENE
Slide 28: AGG Interruptions Reduce the Chances of Pre- to Full-Mutation Transmission
AGG “INTERRUPTIONS” REDUCE THE CHANCES OF PRE- TO FULL- MUTATION TRANSMISSION …CGG CGG CGG CGG AGG CGG… 80% • Difference in risk is greatest 60% near 75-80 CGG repeats • Having full sequence information is medically relevant Yrigollen et al. (2012) Genet Med 15% Maternal CGG repeat number 2 …CGG CGG
Slide 29: FMR1 Repeat Read Number
Read Number 800 &® csi" NA20241 FMR1 Repeat Unit Repeat Context ® CGG AGG
Slide 30: CGG Repeat Counts
Counts CAG Repeats 120 250 350 FMR1 Control (293) NA03620 NA06903 119 NA20241 321 it NA07537 80 120 160 200 240 CGG Repeats 280 320 360 400
Slide 31: Direct Detection of Methylation
DIRECT DETECTION OF METHYLATION
Slide 32: Methylation Detection of FMR1 Sample
MET H YL AT ION D ET EC T IO N O F F M R 1 SA M PL E
Slide 33: Methylation Detection of FMR1 Sample
MET H YL AT ION D ET EC T IO N O F F M R 1 SA M PL E • CGG repeat region appears to be heavily methylated (5mC)
Slide 34: Conclusion
C O N C L U SION 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 error
Slide 35: For Research Use Only. Not for use in diagnostics procedures. © Copyright 2017 by
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
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