26. Metatranscriptomics - Microbial Genome Program

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Metatranscriptomics:
Challenges and Progress
Shaomei He
DOE Joint Genome Institute
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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
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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://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
http://cybernetnews.com/vista-recovery-disc/
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rRNA Removal Methods
Method
rRNA feature used
Input Manipulate
RNA raw RNA
Before cDNA synthesis
Subtractive hybridization
RNase H digestion
Conserved sequence
High
Exonuclease digestion
5’ monophosphate
Gel extraction
Size
Biased poly(A) tailing
2o structure
Low
Sequence feature
Low
No
High abundance
Low
No
Yes
During cDNA synthesis
Not-so-random primers
After cDNA synthesis
Library normalization w/ DSN
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Validation of two rRNA removal kits
Subtractive Hybridization
Exonuclease Digestion
MICROBExpress Bacterial mRNA Enrichment
(Ambion)
mRNA-ONLY Prokaryotic mRNA Isolation
(Epicentre)
mRNA
5’ PPP
mRNA
rRNA
5’ P
rRNA
Capture Oligo
Magnetic Bead
Hyb
5’ Monophosphate
Dependent Exonuclease
Exo
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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
Treatments:
Hyb
2 x Hyb
Exo
Hyb + Exo
Exo + Hyb
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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)
Yi et al, Nucleic Acids Res, 2011, 1-9
Total RNA
RNA-seq library construction

Denature ds-DNA at high temp

Re-anneal to ds-DNA at lower
temp.

DSN degrades DNA duplex
which is presumably from
abundant transcripts.
Library normalization using DSN
• Efficient on E. coli (final rRNA% = 26 ± 11%)
• Preserved mRNA relative abundance
• Little reduction of the very abundant mRNA
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Still efficient and “faithful” for microbial communities?
Relative abundance of OTU (%)
Typical species rank abundance
3
2.5
2
1.5
1
0.5
0
1
101
201
301
401
501
601
701
801
901
1001
Rank of OTU
Environmental microbial communities are very diverse, with
a long tail of minor community members.
Epicentre Ribo-Zero TM Kit
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Another subtractive hybridization-based kit.
Ribo-Zero depletion
100000
R² = 0.9999
10000
High fidelity!
1000
100
10
1
0.1
0.01
0.01
1
100
10000
No Depletion
- Cindi Hoover, JGI
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Test on a real sample from cow rumen
Sample
% rRNA
% Map (rumen
metagenome)
% Other
No depletion
control
82.4
3.4
10.5
Ribo-Zero
Metabacteria
15.9
27.7
55.2
4.9
26.7
56.3
Ribo-Zero
Metabacteria
+
Human/Mouse
/Rat
Effective even on complex metatranscriptome samples.
- Cindi Hoover, JGI
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What else about RiboZeroTM kit
Giannoukos et al, Genome Biology 2012, 13:r23
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•
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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?
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Termite Hindgut Metatranscriptomics
- A case study
(Preliminary results)
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Termite samples in this study
Species:
Family:
Habitat:
Diet:
Nasutitermes corniger
Termitidae
Laboratory colony
Dry wood
Amitermes wheeleri
Termitidae
Subtropical desert
Cow dung
Aim: Determine system-specific differences between termite
species with different diets.
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Overview of sequencing efforts
Nasutitermes
Amitermes
(Lab colony)
Dry wood
(Arizona desert)
Cow dung


Sanger at a QC level


454-titanium


Illumina GAIIx – 1 x 34 bp
1 lane
1 lane
Illumina GAIIx – 2 x 76 bp
1 lane
1 lane
Illumina GAIIx – 2 x ll3 bp
1 lane
3 lanes
community analysis
16S pyrotag
Metagenomics
Metatranscriptomics
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Bioinformatics workflow
- Edward Kirton, JGI
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Summary

Metatranscriptomics is being advanced by nextgeneration 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.
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Acknowledgement
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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!
• Omri Wurtzel
• Rotem Sorek
• Hans Peter Klenk
• Rudolph Scheffrahn
• Jose Escovar-Kousen
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