Metatranscriptomics: Challenges and Progress

1. Metatranscriptomics:Challenges and Progress Cindi Hoover DOE Joint Genome Institute May 17, 2012

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) • Metagenome = who’s there? • Metatranscriptome = function? • What genes are active in environment? • How does gene expression change in response to particular conditions?

3. Evolution of Metatranscriptome Methods • cDNA clone libraries + Sanger sequencing (low throughput) • Microarrays (medium throughput) • RNA-seq enabled by next-generation sequencing technologies (high throughput) • Influenced by presence of rRNA

4. Main Challenges Wet lab • Low RNA yield from environmental samples • Instability of RNA • High rRNA content in total RNA • mRNA = 1-5% of total http://www.nwfsc.noaa.gov/index.cfm How do you effectively removal rRNA from metatranscriptome samples? 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. Sample-specific probe method Stewart et al, ISME J (2010) 4, 896–907 • One of the first to successfully tackle the rRNA in metatranscriptome problem • PRO: Customized probes are specific to communities of interest • CONS: Very time consuming process; requires >3ug RNA or matched DNA samples • Different batches of probe may give different results Method has been applied on marine metatranscriptome samples to substantially reduce rRNA.

7. Epicentre: Ribo-Zero TM • Essentially a subtractive hybridization • rRNA removal reagent contains oligo probes complementary to rRNA sequences • Magnetic beads bind rRNA-probe complexes and remove them from solution • Process takes ~1-1.5 hours; requires 1ug total RNA

8. Ribo-Zero Types • Metabacteria: handles Gram (-) and Gram (+) • Human/Mouse/Rat: also works on fungal samples • Plant Leaf • Plant Seed/Root

9. Synthetic metatranscriptome Both methods tested on sample Mettr_1:

10. Example of Depletion QC Red = total RNA Blue = (+) Ribo-Zero A Green = (+) Ribo-Zero B Agilent Nano chip: total RNA vs depletion with beta test kit

11. Initial Mettr_1 Data

12. Gene Expression Correlations Ribo-Zero vs. No Depletion Ribo-Zero does not appear cause bias in gene expression.

13. Gene Expression Ribo-Zero vs Probe Method

14. Gene Expression Correlations Ribo-Zero Replicates

15. Ribo-Zero & Cow Rumen

16. Cow Rumen Data Ribo-Zero is effective, even on complex metatranscriptome samples like cow rumen.

17. Summary • rRNA removal technique is critical to metatranscriptome sequencing success! • Ribo-Zero = efficient rRNA removal method • Highly effective on complex metatranscriptome samples • Ability to customize by mixing rRNA removal solutions

18. Acknowledgements • Cris Kinross • Matt Blow • Jeff Martin • Weibing Shi • Shaomei He • Erika Lindquist • Feng Chen

19. Questions?