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

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?

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

Wet lab  Low RNA yield from environmental samples  Instability of RNA  High rRNA content in total RNA  mRNA = 1-5% of total 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?

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

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.

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

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

Synthetic metatranscriptome Both methods tested on sample Mettr_1: OrganismAmount in pool (ug) Prochlorococcus marinus pastoris CMP Pediococcus pentosaceus6.0 Acinetobacter sp. ADP12.5 Cyanobacterium synechocystis PCC Synechococcus elongates PCC Total Pool12 ug

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

Initial Mettr_1 Data SampleTotal reads (million) % rRNA% Map Mettr_1 CONTROL (no depletion) Mettr_1 (+) probe Mettr_1 Ribo-zero A Mettr_1 Ribo-zero B

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

Gene Expression Ribo-Zero vs Probe Method

Gene Expression Correlations Ribo-Zero Replicates

Ribo-Zero & Cow Rumen

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% Ribo-Zero Metabacteria Ribo-Zero Metabacteria + Human/Mouse /Rat

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

Acknowledgements  Cris Kinross  Matt Blow  Jeff Martin  Weibing Shi  Shaomei He  Erika Lindquist  Feng Chen

Questions?