Metatranscriptomics: Challenges and Progress Cindi Hoover DOE Joint Genome Institute May 17, 2012.
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Transcript of Metatranscriptomics: Challenges and Progress Cindi Hoover DOE Joint Genome Institute May 17, 2012.
Metatranscriptomics:Challenges and Progress
Cindi Hoover
DOE Joint Genome Institute
May 17, 2012
MetatranscriptomicsMetatranscriptomics
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 MethodsEvolution 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
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 ChallengesMain Challenges
How do you effectively removal rRNA from metatranscriptome samples?
rRNA Removal MethodsrRNA Removal Methods
Method rRNA feature usedInput RNA
Manipulate raw RNA
Before cDNA synthesis
Subtractive hybridization Conserved sequence
HighYes
RNase H digestion
Exonuclease digestion 5’ monophosphate
Gel extraction Size
Biased poly(A) tailing 2o structure Low
During cDNA synthesis
Not-so-random primers Sequence feature Low No
After cDNA synthesis
Library normalization w/ DSN High abundance Low No
Sample-specific probe method 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 Epicentre: Ribo-Zero TMTM
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 TypesRibo-Zero Types
Metabacteria: handles Gram (-) and Gram (+)
Human/Mouse/Rat: also works on fungal samples
Plant LeafPlant Seed/Root
Synthetic metatranscriptomeSynthetic metatranscriptome
Both methods tested on sample Mettr_1:
Organism Amount in pool (ug)
Prochlorococcus marinus pastoris CMP1986
0.1
Pediococcus pentosaceus 6.0
Acinetobacter sp. ADP1 2.5
Cyanobacterium synechocystis PCC 6803
3.0
Synechococcus elongates PCC 7942
0.5
Total Pool 12 ug
Example of Depletion QCExample 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 DataInitial Mettr_1 Data
Sample Total reads
(million)
% rRNA % Map
Mettr_1 CONTROL (no depletion)
6.08 75.4 4.1
Mettr_1 (+) probe 6.76 19.3 24.3
Mettr_1 Ribo-zero A 7.96 4.1 68.0
Mettr_1 Ribo-zero B 6.82 4.3 69.5
Gene Expression Correlations Ribo-Zero vs. No Depletion
Ribo-Zero does not appear cause bias in gene expression.
Gene Expression Ribo-Zero vs Probe Gene Expression Ribo-Zero vs Probe MethodMethod
Gene Expression Correlations Ribo-Zero Gene Expression Correlations Ribo-Zero ReplicatesReplicates
Ribo-Zero & Cow RumenRibo-Zero & Cow Rumen
Cow Rumen DataCow Rumen Data
Ribo-Zero is effective, even on complex metatranscriptome samples like cow rumen.
Sample % rRNA % Map (rumen) % Other
No depletion control
82.4% 3.4 10.5
Ribo-Zero Metabacteria
15.9 27.7 55.2
Ribo-Zero Metabacteria + Human/Mouse/Rat
4.9 26.7 56.3
SummarySummary
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
AcknowledgementsAcknowledgements
Cris Kinross
Matt Blow Jeff Martin Weibing Shi Shaomei He Erika Lindquist Feng Chen
Questions?