20140711 3 t_clark_ercc2.0_workshop
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Transcript of 20140711 3 t_clark_ercc2.0_workshop
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FIND MEANING IN COMPLEXITY © Copyright 2012 by Pacific Biosciences of California, Inc. All rights reserved.
Tyson Clark 7/11/14
Single Molecule, Real-Time Sequencing of Full-length cDNA Transcripts
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Single Molecule, Real-Time (SMRT) DNA Sequencing
PacBio RS II
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P5-C3 Sequencing Chemistry
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Transcript Diversity
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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.
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PacBio Iso-Seq for High-quality, Full-length Transcripts
PolyA mRNA AAAAA
AAAAA
AAAAA
AAAAA
cDNA synthesis with adapters
AAAAA TTTTT
AAAAA TTTTT
AAAAA TTTTT
AAAAA TTTTT
AAAAA TTTTT
AAAAA TTTTT
AAAAA TTTTT
AAAAA TTTTT
Size partitioning & PCR amplification
SMRTbell ligation
PacBio RS II Sequencing
Experimental Pipeline
Informatics Pipeline
Remove adapters Remove artifacts
Clean sequence
reads
Reads clustering
Isoform clusters
Consensus calling
Nonredundant transcript isoforms
Quality filtering
Final isoforms PacBio raw sequence
reads
Raw 5’ primer 3’ primer
Map to reference genome
Experimental pipeline Informatics pipeline
PacBio raw sequence reads
Figure 1
a b
AAAA
AAAA
AAAAAAAAAA
AAAAAAAAAAAAAAA
Size partitioning &PCR amplification
cDNA synthesiswith adapters
SMRTbell ligation
RS sequencing
Remove adaptersRemove artifacts
Reads clustering
Quality filtering
Cleansequence reads
Nonredundant transcript isoforms
Final isoforms
TTTT
TTTT
Consensus calling
Isoform clusters
Map to reference genome
Evidence-based gene models
polyA mRNA
AAAA
AAAA
TTTT
TTTT
AAAATTTT
AAAATTTT
AAAATTTT
AAAATTTT
Evidenced-based gene models
(AAA)n
(TTT)n
SMRT adapter
1 2 3 4 5
6 7 8 9 10
(TTT)n
(AAA)n
5’ UTR Coding sequence 3’
UTR polyA
tail
SMRT adapter
https://github.com/PacificBiosciences/cDNA_primer/
(AAA)n Reads of Insert (AAA)n
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Detailed Clontech workflow for conversion of cDNA into SMRTbell libraries
7
polyA+ RNA
Total RNA
Optional Poly-A Selection
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)
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Brain Amplified cDNA – Testing PCR Enzymes
8
Phusion Kapa Hifi SeqAmp
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Brain Amplified cDNA (zoom)
9
Phusion Kapa Hifi SeqAmp
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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
10-1
5 kb
Kapa Polymerase
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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
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Amplified cDNA from Multiple Human Tissues
12
Brain Heart Liver
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SageELF
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Brain Amplifed cDNA – Size Selected
14
M 12 11 10 9 8 7 6 5 4 3 2 1 800-
1600
1600
-270
0
2700
-480
0
4800
-800
0
3000 1500
800 500 300
100
SageELF BluePippin
Kapa Polymerase
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15
SageELF – 12 size bins (Amplified cDNA)
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SageELF – 12 size bins (Amplified cDNA)
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Brain cDNA – ELF Size Selected – 2nd Amplification
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Actual FL Lengths from each ELF Fraction
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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)
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Actual FL Lengths from each ELF Fraction
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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
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Actual FL Lengths from each ELF Fraction
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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
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Actual FL Lengths from each ELF Fraction
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ELF 3 (5.5 kb) Actual: 1342 – 6075 bp
ELF 2 (7 kb) Actual: 1229 – 7446 bp
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ELF 1 (9 kb) 180 min Actual: 1295 – 1814 bp
Actual FL Lengths from each ELF Fraction
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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
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Targeted Sequencing
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Targeted Sequencing
25
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Targeted Sequencing
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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
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Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, and SMRTbell are trademarks of Pacific Biosciences in the United States and/or other countries. All other trademarks are the sole property of their respective owners.