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. Wet lab  Low RNA yield from environmental samples  Instability of RNA  High rRNA content in total RNA  mRNA = 1-5% of total http://cybernetnews.com/vista-recovery-disc/ 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 Main Challenges How do you effectively removal rRNA from metatranscriptome samples?

5. rRNA Removal Methods MethodrRNA feature used Input RNA Manipulate raw RNA Before cDNA synthesis Subtractive hybridization Conserved sequence High Yes RNase H digestion Exonuclease digestion5’ monophosphate Gel extractionSize Biased poly(A) tailing2 o structureLow During cDNA synthesis Not-so-random primersSequence feature Low No After cDNA synthesis Library normalization w/ DSNHigh abundanceLowNo

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: OrganismAmount in pool (ug) Prochlorococcus marinus pastoris CMP1986 0.1 Pediococcus pentosaceus6.0 Acinetobacter sp. ADP12.5 Cyanobacterium synechocystis PCC 6803 3.0 Synechococcus elongates PCC 7942 0.5 Total Pool12 ug

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 SampleTotal reads (million) % rRNA% Map Mettr_1 CONTROL (no depletion) 6.0875.44.1 Mettr_1 (+) probe6.7619.324.3 Mettr_1 Ribo-zero A7.964.168.0 Mettr_1 Ribo-zero B6.824.369.5

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. Sample% rRNA% Map (rumen)% Other No depletion control 82.4%3.410.5 Ribo-Zero Metabacteria 15.927.755.2 Ribo-Zero Metabacteria + Human/Mouse /Rat 4.926.756.3

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?