1. Single Molecule, Real-Time Sequencing of
Full-length cDNA Transcripts
FIND MEANING IN COMPLEXITY
© Copyright 2012 by Pacific Biosciences of California, Inc. All rights reserved.
Tyson Clark 7/11/14

2. Single Molecule, Real-Time (SMRT) DNA Sequencing
PacBio RS II

3. P5-C3 Sequencing Chemistry

4. Transcript Diversity

5. Current State of Transcript Assembly
“The way we do RNA-seq now
is… you take the transcriptome,
you blow it up into pieces and
then you try to figure out how
they all go back together
again… If you think about it, it’s
kind of a crazy way to do
things”.
Michael Snyder
Stanford University
Tal Nawy (2013) End-to-end RNA sequencing,
Nature Methods 10: 1144–1145
Ian Korf (2013) Genomics: the state of the art in
RNA-seq analysis. Nature Methods 10: 1165-1166.

6. PacBio Iso-Seq for High-quality, Full-length Transcripts
1 2 3 4 5
PolyA mRNA
AAAAA
AAAAA
AAAAA
AAAAA
cDNA synthesis
with adapters
AAAAA
TTTTT
AAAAA
TTTTT
AAAAA
TTTTT
AAAAA
TTTTT
a b
AAAAA
TTTTT
AAAAA
TTTTT
AAAAA
TTTTT
AAAAA
TTTTT
Size partitioning &
PCR amplification
SMRTbell
ligation
PacBio RS II
Sequencing
Experimental Pipeline
(AAA)n
Reads of Insert Informatics Pipeline
Clean
sequence
reads
Remove adapters
Remove artifacts
Reads
clustering
5’
UTR Coding sequence 3’
UTR
Isoform
clusters
Experimental pipeline Informatics pipeline
AAAAA
polyA
tail
(TTT)n
Nonredundant
transcript
isoforms
Consensus
calling
AAAAA
cDNA synthesis
with adapters
Quality
filtering
Final isoforms
PacBio raw
sequence
reads
Raw
5’ primer 3’ primer
Map to
PacBio raw
sequence reads
sequence reads
Isoform clusters
Nonredundant
transcript isoforms
reference genome
Figure 1
AAAA
AAAA
AAAAA
AAAAA
AAAAA
Size partitioning &
PCR amplification
SMRTbell ligation
RS sequencing
Remove adapters
Remove artifacts
Clean
Reads clustering
Quality filtering
Final isoforms
TTTT
TTTT
Consensus calling
Map to reference genome
Evidence-based gene models
polyA mRNA
AAAA
AAAA
TTTT
TTTT
AAAA
TTTT
AAAA
TTTT
AAAA
TTTT
AAAA
TTTT
Evidenced-based
gene models
(AAA)n
(TTT)n
SMRT adapter
6 7 8 9 10
SMRT adapter
https://github.com/PacificBiosciences/cDNA_primer/

7. Detailed Clontech workflow for conversion of cDNA into SMRTbell libraries
7
Total RNA
Optional Poly-A Selection
polyA+ RNA
Reverse Transcription
Full Length
1st Strand cDNA
PCR
Optimization
Large Scale Amplification
Amplified cDNA
1-2
kb
2-3
kb
3-6
kb
Size Selection
(Blue Pippin or Gel)
1-2
kb
2-3
kb
3-6
kb
Re-Amplification
1-2
kb
2-3
kb
3-6
kb
SMRTbell Template Preparation
1-2
kb
2-3
kb
3-6
kb
SMRT Sequencing
3-6
kb
Optional Size Selection
(Blue Pippin)

8. Brain Amplified cDNA – Testing PCR Enzymes
8
Phusion
Kapa Hifi
SeqAmp

9. Brain Amplified cDNA (zoom)
9
Phusion
Kapa Hifi
SeqAmp

10. 2nd Amplification (after Blue Pippin size selection)
10
4000
2000
1250
800
500
Brain
1-2 kb
2-3 kb
3-6 kb
5-10 kb
6-10 kb
8-12 kb
10-15 kb
Kapa Polymerase

11. 2nd Amplification (after Blue Pippin size selection)
11
4000
2000
1250
800
500
Heart
1-2 kb
2-3 kb
3-6 kb
5-10 kb
8-12 kb
Liver 1-2 kb
2-3 kb
3-6 kb
5-10 kb
Kapa Polymerase

12. Amplified cDNA from Multiple Human Tissues
12
Brain
Heart
Liver

13. SageELF
13

14. Brain Amplifed cDNA – Size Selected
14
M 12 11 10 9 8 7 6 5 4 3 2 1
800-1600
1600-2700
2700-4800
4800-8000
3000
1500
800
500
300
100
SageELF BluePippin
Kapa Polymerase

15. 15
SageELF – 12 size bins (Amplified cDNA)

16. SageELF – 12 size bins (Amplified cDNA)
16

17. Brain cDNA – ELF Size Selected – 2nd Amplification
17

18. Actual FL Lengths from each ELF Fraction
18
ELF 12 (400 bp)
Actual:
181 - 266 bp
ELF 11 (550 bp)
Actual:
370 - 480 bp
ELF 10 (800 bp)
Actual:
617 – 727 bp
(25 percentile – 75 percentile)

19. Actual FL Lengths from each ELF Fraction
19
ELF 9 (1.2 kb)
Actual:
955 – 1113 bp
ELF 8 (1.5 kb)
Actual:
1355 – 1544 bp
ELF 7 (1.8 kb)
Actual:
1800 – 2033 bp

20. Actual FL Lengths from each ELF Fraction
20
ELF 6 (2.5 kb)
Actual:
2398 – 2737 bp
ELF 5 (3 kb)
Actual:
3193 – 3574 bp
ELF 4 (4 kb)
Actual:
2127 – 4664 bp

21. Actual FL Lengths from each ELF Fraction
21
ELF 3 (5.5 kb)
Actual:
1342 – 6075 bp
ELF 2 (7 kb)
Actual:
1229 – 7446 bp

22. Actual FL Lengths from each ELF Fraction
ELF 1 (9 kb)
180 min
Actual:
1295 – 1814 bp

23. Summarizing ELF for Size Selection
ELF Lane # Actual FL range
ELF12-400bp 181 - 266 bp
ELF11-500bp 370 - 480 bp
ELF10-800bp 617 - 727 bp
ELF9-1.2kb 955 - 1113 bp
ELF8-1.5kb 1355 - 1544 bp
ELF7-1.8kb 1800 - 2033 bp
ELF6-2.5kb 2398 - 2737 bp
ELF5-3kb 3193 - 3574 bp
ELF4-4kb 2127 - 4664 bp
ELF3-5.5kb 1342 - 6075 bp
ELF2-7kb 1229 - 7446 bp
ELF1-9kb 1295 - 1814 bp
The Good:
1. One run, 12 fractions
2. Finer size fractions (~ 200 bp)
3. 100 bp – 10 kb spread
The Not-Good-Yet:
1. > 4 kb gets small inserts competing
To Work On:
1. New beta machine
2. Combining fractions

24. Targeted Sequencing
24

25. Targeted Sequencing
25

26. Targeted Sequencing
26

27. ERCC 2.0 Controls (from the PacBio perspective)
• Long Transcripts (>10kb, if possible)
• Transcript Isoforms that span size bins
• Complex alternative splicing patterns
• Diversity of GC contents
27

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the United States and/or other countries. All other trademarks are the sole property of their respective owners.