1. Advancing Science with DNA Sequence Metatranscriptomics: Challenges and Progress Shaomei He DOE Joint Genome Institute AUG

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

3. Advancing Science with DNA Sequence Evolving of Metatranscriptomics  cDNA clone libraries + Sanger sequencing  Microarrays  RNA-seq enabled by next-generation sequencing technologies. Sorek & Cossart, NRG (2010) 11, 9-16 RNA-seq is superior to microarrays in many ways in microbial community gene expression analysis.

4. Advancing Science with DNA Sequence 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://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

5. Advancing Science with DNA Sequence 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. Advancing Science with DNA Sequence Validation of two rRNA removal kits Hyb Exo Capture Oligo Magnetic Bead rRNA mRNA Subtractive Hybridization MICROBExpress Bacterial mRNA Enrichment (Ambion) Exonuclease Digestion mRNA-ONLY Prokaryotic mRNA Isolation (Epicentre) 5’ Monophosphate Dependent Exonuclease rRNA mRNA 5’ P 5’ PPP

7. Advancing Science with DNA Sequence Objectives Validate the performance of Hyb and Exo kits on “synthetic” microbial communities, using Illumina sequencing to evaluate:  Efficiency of rRNA removal  Fidelity of mRNA relative transcript abundance Hyb 2 x Hyb Exo Hyb + Exo Exo + Hyb Treatments:

8. Advancing Science with DNA Sequence What we learned  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.

9. Advancing Science with DNA Sequence 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. Advancing Science with DNA Sequence Duplex-specific nuclease (DSN) Efficient on E. coli (final rRNA% = 26 ± 11%) Preserved mRNA relative abundance Little reduction of the very abundant mRNA Total RNA RNA-seq library construction Library normalization using DSN  Denature ds-DNA at high temp  Re-anneal to ds-DNA at lower temp.  DSN degrades DNA duplex which is presumably from abundant transcripts. Yi et al, Nucleic Acids Res, 2011, 1-9

11. Advancing Science with DNA Sequence Still efficient and “faithful” for microbial communities? Environmental microbial communities are very diverse, with a long tail of minor community members. Typical species rank abundance

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

13. Advancing Science with DNA Sequence Test on a real sample from cow rumen Effective even on complex metatranscriptome samples. Sample% rRNA% Map (rumen metagenome) % Other No depletion control 82.43.410.5 Ribo-Zero Metabacteria 15.927.755.2 Ribo-Zero Metabacteria + Human/Mouse /Rat 4.926.756.3 - Cindi Hoover, JGI

14. Advancing Science with DNA Sequence What else about RiboZero TM kit 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? Giannoukos et al, Genome Biology 2012, 13:r23

15. Advancing Science with DNA Sequence Termite Hindgut Metatranscriptomics - A case study (Preliminary results)

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

17. Advancing Science with DNA Sequence Overview of sequencing efforts NasutitermesAmitermes (Lab colony) Dry wood (Arizona desert) Cow dung community analysis 16S pyrotag Metagenomics Sanger at a QC level 454-titanium Metatranscriptomics Illumina GAIIx – 1 x 34 bp Illumina GAIIx – 2 x 76 bp Illumina GAIIx – 2 x ll3 bp 1 lane 3 lanes

18. Advancing Science with DNA Sequence Bioinformatics workflow - Edward Kirton, JGI

19. Advancing Science with DNA Sequence Summary  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.

20. Advancing Science with DNA Sequence 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 Omri Wurtzel Rotem Sorek Acknowledgement Jose Escovar-Kousen Rudolph Scheffrahn