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Metatranscriptomics: Challenges and Progress

AUG. AUG. AUG. AUG. AUG. AUG. AUG. Metatranscriptomics: Challenges and Progress. Shaomei He DOE Joint Genome Institute. Metatranscriptome The complete collection of transcribed sequences in a microbial community: Protein-coding RNA (mRNA)

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Metatranscriptomics: Challenges and Progress

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  1. AUG AUG AUG AUG AUG AUG AUG Metatranscriptomics:Challenges and Progress Shaomei He 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. 5’ PPP mRNA mRNA rRNA 5’ P rRNA Capture Oligo 5’ Monophosphate Dependent Exonuclease Magnetic Bead Validation of two rRNA removal kits Exonuclease Digestion mRNA-ONLY Prokaryotic mRNA Isolation (Epicentre) Subtractive Hybridization MICROBExpress Bacterial mRNA Enrichment (Ambion) Exo Hyb

  7. Validate the performance of Hyb and Exo kits on “synthetic” 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

  8. 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. What we learned

  9. 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.

  10. 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, 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

  11. 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.

  12. Epicentre Ribo-Zero TM Kit Another subtractive hybridization-based kit. High fidelity! - Cindi Hoover, JGI

  13. Test on a real sample from cow rumen Effective even on complex metatranscriptome samples. - Cindi Hoover, JGI

  14. What else about RiboZeroTM kit Giannoukos et al, Genome Biology 2012, 13:r23 • 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?

  15. Termite Hindgut Metatranscriptomics • A case study • (Preliminary results)

  16. Nasutitermes corniger Termitidae Laboratory colony Dry wood Amitermes wheeleri Termitidae Subtropical desert Cow dung Termite samples in this study Species: Family: Habitat: Diet: Aim: Determine system-specific differences between termite species with different diets.

  17. Overview of sequencing efforts

  18. Bioinformatics workflow - Edward Kirton, JGI

  19. 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. Summary

  20. Acknowledgement • Omri Wurtzel • Rotem Sorek • 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 • Rudolph Scheffrahn • Jose Escovar-Kousen

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