For Research Use Only. Not for use in diagnostics procedures. © Copyright 2017 by Pacific Biosciences of California, Inc. All rights reserved.

Iso-Seq Method Updates and Target Enrichment

Without Amplification for SMRT SequencingPacBio Americas User Group Meeting Sample Prep Workshop

June.27.2017

Tyson Clark, Ph.D.

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

-Targeted Sequencing Using a CRISPR/Cas9-based Non-Amplification

Method

-Conclusion

Iso-Seq Method Updates

Gene

DETERMINATION OF TRANSCRIPT ISOFORMS

Short-read technologies:

Reads spanning

splice junctions

Insufficient ConnectivitySplice Isoform Uncertainty

PacBio’sIso-Seq Analysissolution:

Full-length cDNA Sequence ReadsSplice Isoform Certainty – No Assembly Required

mRNA isoforms

Sequel System Iso-Seq Procedure

Overview

EXISTING ISO-SEQ SAMPLE PREP WORKFLOW ON THE

PACBIO RS I I INVOLVES EXTENSIVE SIZE SELECTION

polyA+ RNA

Total RNA

Optional Poly-A Selection

Reverse Transcription

Full Length1st Strand cDNA

PCR

Optimization

Large Scale Amplification

Amplified cDNA

Size Selection

(BluePippin™,

SageELF™, or gel)

Re-Amplification

SMRTbell™

Template Preparation

SMRT® Sequencing

Optional Size Selection

(BluePippin or SageELF)

5-10kb

1-2kb

2-3kb

3-6kb

5-10kb

1-2kb

2-3kb

3-6kb

5-10kb

1-2kb

2-3kb

3-6kb

5-10kb

1-2kb

2-3kb

3-6kb

5-10kb

3-6kb

Optional 5-10 kb

size fraction

Full-length Transcript SizesBioAnalyzer trace of a non-size selected Iso-Seq Library

DECREASED LOADING BIAS IN SEQUEL SYSTEM REDUCES

NEED FOR SIZE SELECTION

- Histogram plot of number of full-length sequences by

transcript length for a Magbead-loaded, non-size selected

Iso-Seq library sequenced on both the PacBio RS II and

the Sequel System.

- The full-length cDNA sequences run on the Sequel System

closely resemble the size distribution of the input SMRTbell

library

Sequel SYSTEM output correlates with input SMRTbell library size distribution

NEW STREAMLINED ISO-SEQ WORKFLOW FOR SEQUEL

Example Sequel System Iso-Seq

Method Datasets

EXAMPLE SEQUEL SYSTEM ISO-SEQ METHOD DATASET:

PRIMARY SEQUENCING METRICS

Sample [On-Plate]

Total Gb Movie Pol RL (bp) Longest Subread

P0 (%) P1 (%) P2 (%)

1x + 0.4x 50 pM 4.89 360 11766 2816 339905 (32.8%)

415685 (40.1%)

281207 (27.1%)

1x + 0.4x 40 pM 7.57 600 13845 2855 172510 (17%)

546423 (53%)

317867 (31%)

1x + 0.4x 50 pM 7.17 600 12015 2928 102676 (10%)

596453 (58%)

337671 (33%)

- Target P1 ≥ ~50%, P0 ≥ 10%

-MagBead loading

- Pre-extension = 120 mins

- Polymerase Read Length increases with increasing movie time

- Obtained ≥4.5 Gb for a 6-h movie and ≥7 Gb for a 10-h movie

EXAMPLE SEQUEL SYSTEM ISO-SEQ METHOD DATASET:

SECONDARY ANALYSIS METRICS ( ISO-SEQ PROTOCOL)

Sample [On-Plate] Movie #CCS CCS RL #FLNC (%) FLNC RL # Polished HQ Isoforms

# Polished LQ Isoforms

1x + 0.4x 50 pM 360 415,539 2602 202,328(48.7%)

2892 14,722 96,755

1x + 0.4x 40 pM 600 545,724 2535 264,779(48.5%)

2867 20,386 132,119

1x + 0.4x 50 pM 600 595,533 2597 244,521(41.1%)

3019 17,765 125,467

- 1 Sequel SMRT Cell typically yields ≥200 K Full-length non-chimeric (FLNC) reads

- Number of FLNC and HQ isoforms drops with increased loading concentration (40 pM

to 50 pM on-plate) even though the # CCS increases

Size Selection Option for Iso-Seq

Method on the Sequel System

NEW STREAMLINED ISO-SEQ WORKFLOW FOR SEQUEL

EFFECT OF USING SIZE SELECTION OPTION WITH ISO-SEQ

METHOD ON THE SEQUEL SYSTEM

Full-length Transcript Sizes

- BluePippin (or SageELF) size-selected

library (4.5 – 10 kb) can be pooled with

non-size selected library and co-loaded

together onto a single Sequel SMRT Cell

Non-size selected

Non-size selected

plus >4 kb size

selected library

(co-loaded)

Tran

scri

pt

Co

un

t

Full-Length Transcript Size

0.5-2 kb1.5-3 kb2.5-6 kb4.5-10 kb

RECOMMENDATIONS FOR EXISTING SIZE-SELECTED

FRACTIONS

- Anneal and Bind size-selected fractions separately

- Pool fractions by equimolar pooling (Make adjustments where necessary)

- Data shown for 4 size bins pooled together and sequenced on a single Sequel SMRT Cell

AVAILABLE TECHNICAL RESOURCES FOR ISO-SEQ

ANALYSIS

Iso-Seq Best Practices

Iso-Seq on Sequel

Barcoding Iso-Seq

NimbleGen Targeted Iso-Seq

IDT Targeted Iso-Seq

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 transcripts >4 kb

- Survey transcriptomes in 1–2 SMRT Cells on the Sequel System

- Increase sequencing depth for more comprehensive transcriptome characterization

- Compatible with standard target enrichment methods, such as NimbleGen SeqCap EZ or

IDT xGen Lockdown Probes

-Multiplex transcripts or full transcriptomes with sample barcoding

- Profile transcripts from multiplexed samples in a single Sequel SMRT Cell 1M

- Data analysis protocols and tools available through SMRT Analysis and PacBio DevNet

to generate high-quality, full-length transcript sequences with no assembly required

- Run Iso-Seq analysis in either de novo (no genome reference required) or reference-based

mode

- Run Iso-Seq with Mapping analysis (map isoforms to GMAP) to enable studying gene families,

gene fusion, accurate identification of unique isoforms.

Target Enrichment R&D Updates

For Research Use Only. Not for use in diagnostics procedures. © Copyright 2015 by Pacific Biosciences of California, Inc. All rights reserved.

Targeted SMRT Sequencing of Repeat -

Expansion Disease Causative Genomic Regions

Using a CRISPR/Cas9, Non-Amplification Based

Method

REPEAT EXPANSION DISEASES

23

CRISPR/CAS9 SYSTEM

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

• Bacterial Adaptive Immunity• RNA-guided DNA Endonuclease

PCR-FREE TARGET ENRICHMENT VIA CAS9 DIGESTION:

METHOD OVERVIEW (CURRENTLY IN DEVELOPMENT)

USING CRISPR/CAS9 TO ENRICH FOR REPEAT

EXPANSION DISORDERS

- Improved on-target rate with complexity reduction:

- Restriction enzymes are used to degrade unwanted SMRTbell templates

- Additional starting DNA is required to maintain input into Cas9 digestion step

-Multiplexing:

-Multiple regions can be targeted in the same reaction

- Patient samples could be barcoded during initial SMRTbell library preparation

Number of individual molecules sequenced

*

*

*Restriction Enzyme

COVERAGE ACROSS THE GENOME

RICARDO MOURO PINTO WILL BE DISCUSSING

HUNTINGTON’S DISEASE TOMORROW

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

>700 CGG REPEATS SEQUENCED FROM THE FMR1 GENE

Maternal CGG repeat number

• Difference in risk is greatest

near 75-80 CGG repeats

• Having full sequence

information is medically

relevantYrigollen et al. (2012) Genet Med

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 …

…CGG CGG CGG CGG AGG CGG…

AGG “INTERRUPTIONS” REDUCE THE CHANCES OF PRE- TO FULL-

MUTATION TRANSMISSION

Yrigollen et al. (2012) Genet Med 14:729–736

80%

60%

15%

DIRECT DETECTION OF METHYLATION

METHYLATION DETECTION OF FMR1 SAMPLE

METHYLATION DETECTION OF FMR1 SAMPLE

• CGG repeat region appears to be heavily methylated (5mC)

CONCLUSION

-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 and characterize epigenetic modification signals

- Detect sample mosaicism

Amplification-free enrichment with CRISPR/Cas9 and SMRT Sequencing achieves the base-level resolution required to understand the underlying biology of repeat expansion disorders

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.

All other trademarks are the sole property of their respective owners.