RNA-Seq library preparation - Oregon State...
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RNA-Seq library preparation Brian J. Knaus USDA Forest Service Pacific Northwest Research Station 1
Transcript of RNA-Seq library preparation - Oregon State...
RNA-Seq library preparation
Brian J. Knaus
USDA Forest Service
Pacific Northwest Research Station
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Doerge, R.W. 2002. Nature Reviews Genetics 3: 43-53.2
Why is library preparation
important?
•YieldIllumina claims 1-10 μg total RNAPracticalityCost effectiveness
•BiasInference of results
•Strandedness
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Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
Yield from Levin et al. data.
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Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
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How many reads do you need?
I don’t think there is currently a good answer.
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Control: Phi-X
Fc204, lane 8, OSU CGRB
5,386 bp, 44% GC
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RNA Ligation – SRR059162
Saccharomyces cerevisiae
12,156,677 bp, 38% GC
Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
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SMART - SRR059167
Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
GGG
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SMART - SRR059167
Third file?
Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
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Hybrid - SRR059169
Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
GGG
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NNSR - SRR059171
Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
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Bisulfite ‘S’ - SRR059174
Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
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dUTP- SRR059176
Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
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Control - SRR059178
Levin et al. 2010. Comprehensive comparative analysis of strand-
specific RNA sequencing methods. Nature Methods 7(9):709-715.
Random hexamer bias
Random hexamer #2 Oligo dT #2
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Reproducible bias within but not among methods.
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Parkhomchuck et al. 2009. Transcriptome analysis by strand-specific sequencing of
complimentary DNA. Nucleic Acids Research 37(18):e123
Strand specificity
Purify RNAX2
mRNA AAAAAA
TTTTTTParamagnetic
bead
Fragment RNA (Mg + heat; ~200 bp)
AAA
AAAAAA
First strand synthesisRandom hexamers
AAAmRNA AAAAAA
DNA – 5’ DNA-5’ DNA-5’
Second strandsynthesis
mRNA
DNA – 5’ DNA-5’ DNA-5’
5’-DNA 5’-DNA 5’-DNARN
A-S
eq
lib
rary
pre
p
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End repairDNA – 5’ DNA-5’ DNA-5’
5’-DNA 5’-DNA 5’-DNA
‘A’ overhangDNA – 5’ DNA-5’ DNA-5’
5’-DNA 5’-DNA 5’-DNA A
AA
AA
A
Ligateadapters
DNA-5’
5’-DNA
A
A Adapter
T
T
Adapter
Size select electrophoresis
PCR Enrichment
Lib
rary
pre
p
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Lister et al. 2008. Highly integrated single-base resolution maps of the
epigenome in Arabidopsis. Cell 133:523-536.
RNA ligation method
RNA extracted
Poly(A)+ isolation viaoligo dT selection
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Structure of messenger ribonucleic acid
(mRNA) in eukaryotes.
Exon #15’UTR Intron #1 Exon #2 3’UTR
m7G cap Exon #15’UTR Exon #2 3’UTR Poly(A)+ tail
Transcription
Post transcriptional processing
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Illumina mRNA method – not strand specific
First strand synthesis random hexamer (oligo dT)
Second strand synthesisE.coli pol I, RNase H
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FragmentMetal hydrolysis (e.g., Mg, NaOAC)
PCR enrichmentPhusion, 15 cycles
End bluntingT4 DNA polymerase, DNA pol I,
Klenow, T4 polynucleotide kinase
dA TailingKlenow fragment (3’->5’ exo-)
Size selectionGel-based
CleanQIAquick
CleanQIAquick
CleanMinElute
Adapter ligationT4 DNA ligase
CleanMinElute
CleanQIAquick
dUTP mRNA method – strand specific
First strand synthesis random hexamer (oligo dT)
Second strand synthesis+ dUTP
E.coli pol I, RNase H
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FragmentMetal hydrolysis (e.g., Mg, NaOAC)
PCR enrichmentPhusion, 15 cycles
End bluntingT4 DNA polymerase, DNA pol I,
Klenow, T4 polynucleotide kinase
dA TailingKlenow fragment (3’->5’ exo-)
Size selectionGel-based
CleanQIAquick
CleanQIAquick
CleanMinElute
Adapter ligationT4 DNA ligase
CleanMinElute
CleanQIAquick
Create gaps at UracilsUSER enzyme
Poly(A) + RNA100 ng
De-cap (5’ m7G)Tobacco acid pyrophosphatase
Illumina RNA ligation method
CleanPCIA & EtOH
FragmentNa citrate
CleanEtOH precipitation
5’ phosphorylateT4 polynucleotide kinase
3’ dephosphorylateAntarctic phosphatase
Ligate 3’ adapter T4 RNA ligase 2, truncated
Pre-adenylated adapter
Ligate 5’ adapterT4 RNA ligase 1
Reverse transcriptionSuperScript III
Size selectionGel (175-225 nt)
CleanRnase H, PCIA, EtOH
CleanGel extract, EtOH
PCRPhusion, 14 cycles
CleanAMPure beads
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CleanRneasy MinElute
Poly(A) + RNA100 ng
De-cap (5’ m7G)Tobacco acid pyrophosphatase
Illumina RNA ligation method SPRI
CleanPCIA & EtOH
FragmentNa citrate
CleanEtOH precipitation
5’ phosphorylateT4 polynucleotide kinase
3’ dephosphorylateAntarctic phosphatase
Ligate 3’ adapter T4 RNA ligase 2, truncated
Pre-adenylated adapter
Ligate 5’ adapterT4 RNA ligase 1
Reverse transcriptionSuperScript III
Size selectionGel (175-225 nt)
CleanRnase H, PCIA, EtOH
CleanGel extract, EtOH
PCRPhusion, 14 cycles
CleanAMPure beads
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CleanRneasy MinElute
Poly(A) + RNA200 ng
dUTP method
Clean2 X PCIA, EtOH
FragmentNa citrate
First strand synthesisRandom hexamers, Superscript III
PCR enrichmentPhusion, 14 cycles
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Second strand synthesisE.coli ligase, DNA pol I
End bluntingT4 DNA polymerase, DNA pol I,
Klenow, T4 polynucleotide kinase
dA TailingKlenow fragment (3’->5’ exo-)
Size selectionGel-based
CleanQIAquick
CleanQIAquick
CleanQIAquick
Adapter ligationT4 DNA ligase
CleanQIAquick
CleanQIAquick
RNA-Seq library preparation
Brian J. Knaus
USDA Forest Service
Pacific Northwest Research Station
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