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Metatranscriptomics: Challenges and Progress

Shaomei He

DOE Joint Genome Institute

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

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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.

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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://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 http://cybernetnews.com/vista-recovery-disc/

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rRNA Removal Methods

Method rRNA feature used Input RNA Manipulate raw RNA

Before cDNA synthesis

Subtractive hybridization RNase H digestion Exonuclease digestion Gel extraction Biased poly(A) tailing

During cDNA synthesis

Not-so-random primers

After cDNA synthesis

Library normalization w/ DSN Conserved sequence 5’ monophosphate Size 2 o structure Sequence feature High abundance High Low Low Low Yes No No

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Validation of two rRNA removal kits

Subtractive Hybridization

MICROBExpress Bacterial mRNA Enrichment (Ambion) mRNA Capture Oligo rRNA Magnetic Bead

Hyb Exonuclease Digestion

mRNA-ONLY Prokaryotic mRNA Isolation (Epicentre) 5’ PPP 5’ P 5’ Monophosphate Dependent Exonuclease mRNA rRNA

Exo

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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

Treatments: Hyb 2 x Hyb Exo Hyb + Exo Exo + Hyb

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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.

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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.

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Duplex-specific nuclease (DSN)

Yi et al, Nucleic Acids Res, 2011, 1-9

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.

• Efficient on

E. coli

(final rRNA% = 26 ± 11%) • Preserved mRNA relative abundance • Little reduction of the very abundant mRNA

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Still efficient and “faithful” for microbial communities?

Typical species rank abundance

3 2.5

2 1.5

1 0.5

0 1 101 201 301 401 501 601

Rank of OTU

701 801 901 1001 Environmental microbial communities are very diverse, with a long tail of minor community members.

Epicentre Ribo-Zero

TM

Kit

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Another subtractive hybridization-based kit. 100000 10000 1000 100 10 1 0,1 0,01 0,01

R² = 0,9999

1 100

No Depletion

High fidelity!

10000

- Cindi Hoover, JGI

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Test on a real sample from cow rumen

Sample

No depletion control Ribo-Zero Metabacteria Ribo-Zero Metabacteria + Human/Mouse /Rat

% rRNA

82.4

15.9

4.9

% Map (rumen metagenome)

3.4

% Other

10.5

27.7

26.7

55.2

56.3

Effective even on complex metatranscriptome samples.

- Cindi Hoover, JGI

What else about RiboZero

TM

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kit

Giannoukos et al, Genome Biology 2012, 13:r23

• 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?

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Termite Hindgut Metatranscriptomics

- A case study

(Preliminary results)

Termite samples in this study

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Species: Family: Habitat: Diet:

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.

Overview of sequencing efforts

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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

Nasutitermes

(Lab colony) Dry wood    1 lane 1 lane 1 lane

Amitermes

(Arizona desert) Cow dung    1 lane 1 lane 3 lanes

Bioinformatics workflow

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- Edward Kirton, JGI

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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.

• • • • • • • • • • • • • • • •

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!

Acknowledgement

Advancing Science with DNA Sequence

• •

Omri Wurtzel Rotem Sorek

•

Hans Peter Klenk

•

Rudolph Scheffrahn

•

Jose Escovar-Kousen