1. Metatranscriptomics: Challenges and Progress
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Metatranscriptomics: Challenges and Progress
Shaomei He and Edward Kirton
DOE Joint Genome Institute

2. 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

3. 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.

4. 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)
Bioinformatics
General challenges with short reads and large data size
Small overlap between metagenome and metatranscriptome, or complete lack of metagenome reference

5. rRNA Removal Methods Method rRNA feature used Input RNA
Manipulate raw RNA
Before cDNA synthesis
Subtractive hybridization
Conserved sequence
High
Yes
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 No
After cDNA synthesis
Library normalization w/ DSN
High abundance

6. Validation of Two Ribosomal RNA Removal Methods for Microbial Metatranscriptomics
Shaomei He, Omri Wurtzel, Kanwar Singh, Jeff L. Froula, Suzan Yilmaz, Susannah G. Tringe, Zhong Wang, Feng Chen, Erika A. Lindquist, Rotem Sorek and Philip Hugenholtz

7. Subtractive Hybridization & Exonuclease Digestion
MICROBExpress Bacterial mRNA Enrichment
(Ambion)
Exonuclease Digestion
mRNA-ONLY Prokaryotic mRNA Isolation
(Epicentre)
Capture Oligo
Magnetic Bead
rRNA
mRNA
5’ Monophosphate
Dependent Exonuclease
rRNA
mRNA
5’ P
5’ PPP
Hyb
Exo

8. Objectives
Validate the performance of Hyb and Exo kits on synthetic five-member 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

9. Microbial Isolates in the Two Synthetic Communities
Organism
Genome size (Mbp)
%GC
Phylum
Match Hyb target sites
Desulfovibrio vulgaris
3.7 63
Proteobacteria
Yes 
Streptomyces sp.
8-10 71
Actinobacteria
Lactococcus lactis
2.53 35
Firmicutes
Spirochaeta aurantia
4.3 65
Spirochaeta
Yes
Lactobacillus brevis
2.3 46
Kangiella koreensis
2.9 43
Catenulispora acidiphila
10.5 70
Halorhabdus utahensis
3.1
Euryarchaeota No
Community 1
Community 2

10. Technical Reproducibility
Hyb
Exo
Hyb, rep2
Exo, rep2
Hyb, rep1
Exo, rep1
All treatments exhibited good technical reproducibility.

11. rRNA Removal Efficiency

12. Read Distribution
Community 1
Community 2

13. Observed and Actual rRNA Removal
97%
rRNA 97 3
Before removal
rRNA
mRNA
- 80
- 0
85%
rRNA 17 3
After removal
Observed rRNA reduction = 97% - 85% = 12%
Actual percent removal = 80/97 = 82.5%
Actual removal is much higher than what appears, due to the very high original rRNA content.

14. Community rRNA Removal
Community 1: Hyb + Exo > Hyb > Exo
Community 2: Hyb + Exo > Exo + Hyb > Exo > 2 x Hyb ≈ Hyb

15. rRNA Removal and RNA Integrity
Hyb x Hyb Exo Hyb + Exo Exo + Hyb
r = 0.946
r = 0.958
r = 0.874
r = 0.945
RIN: RNA integrity number
RNA Integrity Number (RIN)
More intact RNA  Higher rRNA removal efficiency

16. Enrichment of mRNA & Increase of Detection Sensitivity

17. Fidelity of mRNA Relative Abundance

18. Fidelity of mRNA Relative Abundance
Hyb > Exo > Hyb+Exo
Community 1
Hyb ≈ 2xHyb > Exo > Hyb+Exo ≈ Exo+Hyb
Community 2

19. Conclusions
rRNA removal efficiency 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.

20. 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.

21. Duplex-specific nuclease (DSN)
Yi et al, Nucleic Acids Res (2011) doi: /nar/gkr617
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

22. Still efficient and “faithful” for microbial communities?
Typical species rank abundance
Environmental microbial communities are very diverse, with a long tail of minor community members.

23. Termite Hindgut Metatranscriptomics
A case study
(Preliminary results)

24. Summary
Metatranscriptomics is being advanced by next-generation sequencing technologies.
Currently, high rRNA content is still a major bottleneck of metatranscriptomics projects.
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 sensitive and computationally efficient to do this for large datasets.

25. Acknowledgement Omri Wurtzel Rotem Sorek Phil Hugenholtz
Susannah Tringe
Edward Kirton
Kanwar Singh
Erika Lindquist
Feng Chen
Falk Warnecke
Natalia Ivanova
Martin Allgaier
Steve Lowry
Jeff Froula
Zhong Wang
R&D group
Production group
Many others!
Hans Peter Klenk
Rudolph Scheffrahn
Jose Escovar-Kousen