Metatranscriptomics: Challenges and Progress

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

2. Metatranscriptome The complete collection of transcribed sequences in a microbial community: Protein-coding RNA (mRNA) Non-coding RNA (rRNA, tRNA, regulatory RNA, etc) Metatranscriptomics Metatranscriptomics studies: • Community functions • Response to different environments • Regulation of gene expression

3. cDNA clone libraries + Sanger sequencing Microarrays RNA-seq enabled by next-generation sequencing technologies. Evolving of Metatranscriptomics 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) 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/

5. rRNA Removal Methods

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

8. Validate the performance of Hyb and Exo kits on synthetic five-member microbial communities, using Illumina sequencing to evaluate: Objectives • Efficiency of rRNA removal • Fidelityof mRNA relative transcript abundance Treatments: 2 x Hyb Hyb + Exo Hyb Exo Exo + Hyb

9. Microbial Isolates in the Two Synthetic Communities Community 1 Community 2

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

11. rRNA Removal Efficiency

12. Community 2 Read Distribution Community 1

13. rRNA 85% 97% rRNA mRNA 97 3 Before removal - 80 - 0 17 3 After removal rRNA Observed and Actual rRNA 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. rRNA Removal (%) 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 rRNA Removal (%) Hyb 2 x Hyb Exo Hyb + Exo Exo + Hyb r = 0.945 r = 0.946 r = 0.958 r = 0.874 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. Community 1 Community 2 Hyb > Exo > Hyb+Exo Hyb ≈ 2xHyb > Exo > Hyb+Exo ≈ Exo+Hyb Fidelity of mRNA Relative Abundance

19. 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. Conclusions

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. Efficient on E. coli (final rRNA% = 26 ± 11%) Preserved mRNA relative abundance Little reduction of the very abundant mRNA Duplex-specific nuclease (DSN) Yi et al, Nucleic Acids Res (2011) doi: 10.1093/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

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. 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. Summary

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