LONG-READ RNA SEQUENCING
BEST PRACTICES
PLANT AND ANIMAL
SCIENCES
HUMAN BIOMEDICAL
RESEARCH
With Single Molecule, Real-Time (SMRT®) Sequencing and the Sequel™ System, you can easily and affordably sequence
transcript isoforms of up to 10 kb in their entirety. The Iso-Seq™ method allows users to generate full-length cDNA
sequences - with no assembly required - in order to confidently characterize the full complement of transcript isoforms
within targeted genes, or across an entire transcriptome.
SAMPLE PREPARATION RECOMMENDATIONS
FROM RNA TO ACCURATE GENE MODELS
-- Prepare full-length transcripts using the Clontech®
Total RNA
---
Optional Poly-A Selection
--
poly A+ RNA
Reverse Transcription
--
Full Length
1st Strand cDNA
--
Large-scale Amplification
Amplified cDNA
>4 kb
Optional
Size Selection
Combined SMRTbell Library
SMARTer® PCR cDNA Synthesis Kit with as little as 1 ng
of poly A+ RNA or 2 ng of total RNA1
Sequel System loading protocols reduce need for size
selection for transcripts <4 kb2
Optional size-selection protocols to enrich for
transcripts >4 kb3
Compatible with standard target enrichment methods,
such as NimbleGen SeqCap EZ4 or IDT xGen Lockdown
Probes5
Multiplex transcripts or full transcriptomes with sample
barcoding6
Scalable throughput
-- Profile transcripts from multiplexed samples in a
single SMRT Cell
-- Survey transcriptomes in 1–2 SMRT Cells on the
Sequel System
-- Increase sequencing depth for more comprehensive
transcriptome characterization
MORE CONSISTENT LOADING ON SEQUEL SYSTEM
REDUCES NEED FOR SIZE SELECTION
SMRT Sequencing on Sequel System
Non-size Selected
Iso-Seq Libraries
Non-SizeSelected
SMRTbell
Library
PacBio
RS II
Sequel
System
Analyze with SMRT Analysis
Software Suite
Full-length Transcript Size
Depicted on the left is a 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 (shown on the right).
w w w. p a c b . c o m / i s o s e q
tal pipeline
DATA ANALYSIS SOLUTIONS WITH PACBIO SMRT ANALYSIS
-- Use the Iso-Seq Algorithm in SMRT Analysis to output high quality, full-length transcript sequences, with no assembly
required, to characterize transcripts and splice variants and map transcripts back to a reference genome
-- Run Iso-Seq analysis in either de novo (no genome reference required) or reference-based mode
-- Install SMRT Analysis locally7 or access it via Amazon Cloud8
-- View tutorial9 for running the Iso-Seq Algorithm in SMRT Analysis via SMRT Link
INFORMATICS PIPELINE FOR ISO-SEQ ANALYSIS
1
Gene
PacBio raw
sequence reads
Remove adapters
Remove artifacts
2
b
partitioning &
amplification
Tbell ligation
equencing
Insufficient Connectivity
Splice Isoform Uncertainty
Informatics pipeline Reads clustering
3
Reads spanning
splice junctions
Iso-Seq
solution:
Full-length cDNA Sequence Reads
Splice Isoform Certainty – No Assembly Required
PacBio raw
sequence
Isoformreads
clusters
Remove adapters
Remove artifacts
Clean
mRNA
isoforms
Short-read
technologies:
Classify
sequence reads
A
synthesis
dapters
DETERMINATION OF TRANSCRIPT ISOFORMS
Consensus calling
The Iso-Seq method allows you to make evidence-based genome
annotations, discover novel genes and isoforms, identify promoters and
splice sites to understand gene regulation, improve accuracy of RNA-seq
quantification for gene expression studies, and distinguish important
stress response, developmental, or tissue-specific isoforms.
4sequence reads
SIGNIFICANTLY IMPROVE EXISTING GENOME
ANNOTATIONS
Nonredundant
Reads clustering
transcript
isoforms
Isoform clusters
Quality filtering
5
Consensus calling
Nonredundant
Final isoforms
transcript isoforms
Quality filtering
Final isoforms
Map to
reference genome
Evidenced-based
gene models
Map to reference genome
Evidence-based gene models
The Iso-Seq informatics pipeline, available in SMRT Analysis, generates
consensus sequences and determines those transcripts that are full-length
by detecting and identifying the 5’ primer, Poly-A sequence, and the 3’
primer of the reads.
Splice isoform analysis in the sorghum transcriptome using the Iso-Seq
method greatly improved genome annotation, with >11,000 novel splice
isoforms and >2,100 novel genes identified. In this example, a gene was
discovered to produce 13 novel alternatively spliced isoforms, where the
previous gene model contained only a single isoform10.
KEY
REFERENCES
Figure
1
1. Procedure & Checklist – Isoform Sequencing (Iso-Seq) using the Clontech® SMARTer® PCR cDNA Synthesis Kit and No Size Selection
2. Clark, T. et al. (2017) Full-Length cDNA Sequencing on the PacBio Sequel Platform. Poster presented at Plant and Animal Genome Conference. San
Diego, CA.
3. User Bulletin: Guidelines for Preparing cDNA Libraries for Isoform Sequencing (Iso-Seq)
4. Full-length cDNA Target Sequence Capture Using SeqCap® EZ Libraries
5. Full-length cDNA Target Sequence Capture Using IDT xGen® Lockdown® Probes
6. Barcoding Samples for Isoform Sequencing (Iso-Seq Analysis)
7. SMRT Analysis Software Installation (v2.3.0)
8. Running SMRT Analysis on Amazon
9. Tutorial: Iso-Seq Analysis Application
10.Abdel-Ghany, S.E. et al. (2016) A survey of the sorghum transcriptome using single-molecule long reads. Nature Communications. 7, e11706.
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2017, Pacific Biosciences of California, Inc. All rights reserved. Information in
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