advancing science with dna sequence metatranscriptomics: challenges and progress shaomei he doe...
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Advancing Science with DNA Sequence
Metatranscriptomics:Challenges and Progress
Shaomei HeDOE Joint Genome Institute
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Advancing Science with DNA Sequence
Metatranscriptomics
Metatranscriptome
The complete collection of transcribed sequences in a microbial community:
Protein-coding RNA (mRNA) Non-coding RNA (rRNA, tRNA, regulatory RNA, etc)
Metatranscriptomics studies: Community functions Response to different
environments Regulation of gene expression
Advancing Science with DNA Sequence
Evolving of Metatranscriptomics
cDNA clone libraries + Sanger sequencing
Microarrays
RNA-seq enabled by next-generation sequencing technologies.
Sorek & Cossart, NRG (2010) 11, 9-16
RNA-seq is superior to microarrays in many ways in microbial community gene expression analysis.
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Challenges in Metatranscriptomics
Wet lab Low RNA yield from environmental samples Instability of RNA (half-lives on the order of
minutes) High rRNA content in total RNA (mRNA
accounts for 1-5% of total RNA)
http://cybernetnews.com/vista-recovery-disc/
http://www.nwfsc.noaa.gov/index.cfm
Bioinformatics General challenges with short reads and large
data size Small overlap between metagenome and
metatranscriptome, or complete lack of metagenome reference
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rRNA Removal Methods
Method rRNA feature usedInput RNA
Manipulate raw RNA
Before cDNA synthesis
Subtractive hybridization Conserved sequence
HighYes
RNase H digestion
Exonuclease digestion 5’ monophosphate
Gel extraction Size
Biased poly(A) tailing 2o structure Low
During cDNA synthesis
Not-so-random primers Sequence feature Low No
After cDNA synthesis
Library normalization w/ DSN High abundance Low No
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Validation of two rRNA removal kits
Hyb Exo
Capture Oligo
Magnetic Bead
rRNA
mRNA
Subtractive Hybridization
MICROBExpress Bacterial mRNA Enrichment(Ambion)
Exonuclease Digestion
mRNA-ONLY Prokaryotic mRNA Isolation(Epicentre)
5’ Monophosphate Dependent Exonuclease
rRNA
mRNA
5’ P
5’ PPP
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Objectives
Validate the performance of Hyb and Exo kits on
“synthetic” microbial communities, using Illumina
sequencing to evaluate:
Efficiency of rRNA removal
Fidelity of mRNA relative transcript abundance
Hyb 2 x Hyb Exo Hyb + Exo Exo + Hyb
Treatments:
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What we learned
rRNA removal efficiency for both kits was community composition and RNA integrity dependent.
Exo degraded some mRNA, introducing larger variation than Hyb.
Combining Hyb and Exo provided higher rRNA removal than used alone, but the fidelity was significantly compromised.
Hyb had high fidelity, but its performance was limited by rRNA probe target range and RNA integrity.
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Customized subtractive hybridization
Stewart et al, ISME J (2010) 4, 896–907
Customized probes specific to communities of interest
Probes cover near-full-length rRNA, and should also capture partially degraded (fragmented) rRNA
It has been applied on marine metatranscriptome samples to substantially reduce rRNA.
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Duplex-specific nuclease (DSN)
• Efficient on E. coli (final rRNA% = 26 ± 11%)• Preserved mRNA relative abundance• Little reduction of the very abundant mRNA
Total RNA
RNA-seq library construction
Library normalization using DSN
Denature ds-DNA at high temp
Re-anneal to ds-DNA at lower temp.
DSN degrades DNA duplex which is presumably from abundant transcripts.
Yi et al, Nucleic Acids Res, 2011, 1-9
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Still efficient and “faithful” for microbial communities?
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1 101 201 301 401 501 601 701 801 901 1001
Rank of OTU
Rel
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Environmental microbial communities are very diverse, with a long tail of minor community members.
Typical species rank abundance
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Epicentre Ribo-Zero Epicentre Ribo-Zero TM TM Kit Kit
- Cindi Hoover, JGI
Another subtractive hybridization-based kit.
High fidelity!
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Test on a real sample from cow rumenTest on a real sample from cow rumen
Effective even on complex metatranscriptome samples.
Sample % rRNA % Map (rumen metagenome)
% Other
No depletion control
82.4 3.4 10.5
Ribo-Zero Metabacteria
15.9 27.7 55.2
Ribo-Zero Metabacteria + Human/Mouse/Rat
4.9 26.7 56.3
- Cindi Hoover, JGI
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What else about RiboZeroTM kit
• Outperformed other four tested kits/methods• Effective even on highly fragmented RNA sample • But needs sufficient input RNA (e.g. > 1 ug)• For environmental samples with very low RNA yield, no
rRNA depletion is the recommendation.
How about Archaea?
Giannoukos et al, Genome Biology 2012, 13:r23
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Termite Hindgut Metatranscriptomics
- A case study
(Preliminary results)
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Nasutitermes cornigerTermitidaeLaboratory colonyDry wood
Amitermes wheeleriTermitidaeSubtropical desertCow dung
Aim: Determine system-specific differences between termite species with different diets.
Termite samples in this study
Species:Family:Habitat:
Diet:
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Overview of sequencing efforts
Nasutitermes Amitermes
(Lab colony)Dry wood
(Arizona desert)Cow dung
community analysis
16S pyrotag Metagenomics
Sanger at a QC level
454-titanium Metatranscriptomics
Illumina GAIIx – 1 x 34 bp
Illumina GAIIx – 2 x 76 bp
Illumina GAIIx – 2 x ll3 bp
1 lane
1 lane
1 lane
1 lane
1 lane
3 lanes
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Bioinformatics workflow
- Edward Kirton, JGI
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Summary
Metatranscriptomics is being advanced by next-generation sequencing technologies.
RiboZero kit is promising to knock down high rRNA content for more effective RNA-seq.
Bioinformatically removing rRNA reads should increase computational speed in de novo assembly, and improve the assembly of low-abundance mRNAs. Need to investigate algorithm that is more sensitive and computationally efficient to do this for large datasets.
Advancing Science with DNA Sequence
• Phil Hugenholtz• Susannah Tringe• Edward Kirton• Kanwar Singh• Erika Lindquist• Feng Chen• Jeff Froula• Falk Warnecke• Natalia Ivanova• Martin Allgaier• Zhong Wang• Tao Zhang• Cindi Hoover• R&D group• Production group• Many others!
• Hans Peter Klenk
• Omri Wurtzel• Rotem Sorek
Acknowledgement
• Jose Escovar-Kousen
• Rudolph Scheffrahn