© 2010 Nature America, Inc. All rights reserved. P.H. ([email protected]). The Biosciences, University of Queensland, Brisbane, Australia. USA. 1 bands non-rRNA of extraction and separation digestion H hybrids RNase rRNA:DNA by degrade to followed primers rRNA-specific with rRNA against discriminate to addition tail poly(A) rRNA, on acts preferentially that exonuclease with probes rRNA-specific with hybridization tractive sub including rRNA, prokaryotic eliminate to used been have methods Different sensitivity. detection mRNA improve to ing Therefore, prokaryotic rRNAs are often removed before sequenc sequences rRNA predominantly are cDNAs archaeal and rial tails poly(A) their of virtue by cDNA into synthesized and tRNAs ref. (1–5%; RNA cellular total in mRNAs of abundance relative low the is sequencing scriptome and communities isolates microbial of analysis microarray to alternative viable a ing are making sequenc advances in ultra-high-throughput Rapid technological analysis by introducing a G+C bias between runs. sequencing itself also can compromise quantitative data greatly compromised m whereas exonuclease and in particular combined treatments alone introduced the least bias in relative transcript abundance, RN of r massively parallel sequencing. We found that the effectiveness two synthetic five-microorganismmetatranscriptomes using digestion, as well as combinations of these treatments, on used approaches, subtractive hybridization and exonuclease the effectiveness and fidelity ofthe two most commonly any of these approaches has been reported. no systematic investigation of potential biases introduced by been applied to deplete r microbial isolates and communities. technical challenge in analysis sequence-based of c T Zhong Wang Shaomei He microbial metatranscriptomics Validation of two ribosomal Received Received 12 Ap Department Department of JointEnergy Genome Institute, Walnut Creek, USA. California, he he predominance of r A A integrity for these treatments. RN 3 Department Department of Molecular A A removal was a function of community composition and 3 . Unlike eukaryotic mRNAs, which can be selectively mRNAs, can be selectively which . Unlike eukaryotic r il; il; 1 1 , de de novo a 2 , , Feng Chen ccepted ccepted 30 August; published online 19 , 5 1 , , Omri Wurtzel . A major technical challenge for challenge . A major technical RN sequencing of transcriptomes (RNA-seq) of transcriptomes sequencing RN As in the transcriptome is a major

RN Genetics, WeizmannGenetics, Institute of Rehovot,Science, Israel. A A abundance fidelity. As from (meta)transcriptomes, but 1 , Erika , A Erika Lindquist 1 1 2), the bulk of which is rRNAs is which of bulk the 2), , and gel size size electrophoresis gel and , S 3 S ubtractive hybridization , 5 everal approaches have 9,10 , , Kanwar Singh , reverse transcription transcription reverse , H ere we validated 1 2 I . llumina llumina 5 These These authors contributed equally to this work. Correspondence should be addressed to S 7,8 de de novo eptembe DN , digestion digestion , 1 As from , , Rotem Sorek 4 , bacte , 1 , , Jeff L Froula r tran 2010; 5,6 2 Energy Biosciences Institute,Energy University of California-Berkeley, Berkeley, California, - - - - - .

doi:10.1038/nmeth.150 tive hybridization with capture oligonucleotides specific to 16S 16S to specific oligonucleotides capture with hybridization tive a subtrac uses kit former The kit). mRNA Isolation Prokaryotic (mRNA-ONLY Epicentre and kit) Enrichment mRNA Bacterial (MICROBExpress Ambion from kits commercial of availability the to owing popular most the become have digestion nuclease removal using RNA electropherograms (Agilent 2100 Bioanalyzer) used total RNA without rRNA removal. We initially assessed( Hyb) rRNA + (Exo Hyb by followed Exo as well of as rounds Hyb two (2Hyb) include also to tested methods depletion the expanded and 1 experiment of results the confirm Then we conducted experiment 2 using synthetic community 2 to 1. community synthetic using Exo) + (Hyb Exo by followed Hyb Hyb, and Exo tested we 1, In experiment communities. synthetic (Exo) and combined treatments in two experiments using the two We (Hyb), digestion exonuclease hybridization tested subtractive ( communities both to common were species five the of content range. (G+C) Two and genome-size phylogenetic, a wide span that genomes sequenced with isolates microbial from ently by pooling equimolar amounts of total extracted independ We constructed two five-member synthetic microbial communities E RESULTS analyses. quantitative for relative hybridization adequately preserved transcript abundance and that only subtractive and munity- RNA integrity–dependent and were found com that sequencing rRNA removal efficiencies two synthetic five-member microbial communities using Illumina with kits these of performance the validated we Here anecdotal. are data existing and approach, best the on however, consensus, to improve rRNA removal combination in used rRNAs) be to RNAs with 5 rounds Epicentre two kit uses exonuclease to or preferentially degrade processed one in isolates samples, bacterial environmental both and to applied been has It rRNAs. 23S and xperimental design xperimental RN 4 mn tee ehd, utatv hbiiain n exo and hybridization subtractive methods, these Among Australian Centre for Ecogenomics, School ofand Chemistry Molecular 3 & Philip Hugenholtz 1 , , Suzan Yilmaz A A removal methods for ′ monophosphate (the majority of which are believed 19,20 7 n a . In some instances, these methods have been been have methods these instances, some In . ture methods 1 , , Susannah G Tringe 1 , 2

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© 2010 Nature America, Inc. All rights reserved. Resource Center (NBRC)13350(8,545,929bp)and mega–base pairs(Mbp),which isinthe range of the sizeof genomes of thisorganism’s closerelatives, such as number (RIN)were determined using the Agilent 2100Bioanalyzer. RINvaluesrange from 1(most degraded) to10(most intact). a Halorhabdus utahensis acidiphila Catenulispora Kangiella koreensis Lactobacillus brevis Lactobacillus Spirochaeta aurantia rate from lanes included ineachruntoindicate runquality. failure. digestion (mRNA-ONLY Prokaryotic mRNAIsolation Kit,Epicentre); Hyb +Exo =Hyb followed byExo; Exo +Hyb =Exo followed by Hyb. enrichment a Cluster pass rate Lane 7 Lane 6 Lane 5 Lane 3 Lane 2 Lane 1 Lactococcus lactis Lane 4 T ( organisms individual to correspond that distinct regressions multiple to owing reproducible less were 4 and 3 runs between replicates technical interrun the contrast, By runs. two ( ii; 1 Exo and i 1 (Exo 2 and 1 runs between replicate technical interrun sole the tion, were variation minor.and ­variation sequencing intrarun In addi 0.001; coefficient, correlation (Pearson’sproduct-moment and controls in runs 1, 3 and 4; (Hyb runs in 1, in run independent replicates three technical run per gene normalized by total mapped mRNA reads. The four intra tive transcript abundance of technical replicates calculated as reads We first evaluated technical reproducibility by comparing the rela T Note 2 Supplementary and negligible contamination ( quality read good of to 99% a mapped reads indicating reference, we sample one but all for and sample, each for reads single-end ( variation interrun and intra- evaluate to sequencer II Analyzer Genome Illumina an in on 2) 2 experiment in 4 and 3 and runs and 1, 1 experiment runs (flowcells; runs four between and within uted distrib RNApooling) (after replicates technical sequenced then ( Streptomyces Desulfovibrio vulgaris O T  Information about16Sand 23SrRNAs matching tothe capture oligos inthe MICROBExpress mRNAEnrichment Kitwasobtained from Ambion. For completeness, there are twoother metatranscriptomic studies not listedinthe tablethatusedaddition of poly(A)tailswhich preferentially ligate tomRNAs providing the basisfor mRNA Supplementary Figs. 1 1 Figs. Supplementary Fig. Fig. 1 echnical reproducibility echnical able able 2 able 1

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) was also highly reproducible reproducible highly also was ) 1 d n M1 a S. avermitilis b Supplementary Note 1 Note Supplementary ture methods 2 ) ) were reproducible highly c Hyb, subtractive hybridization (MICROBExpress Bacterial mRNA Enrichment Kit, Ambion); 2Hyb, two rounds of Hyb; Exo = Exonuclease size ( Genome 88% Exo (ii) 1 1 R 8–10 10.5 2.53 un 3.1 2.9 2.3 4.3 3.7 MA-468 (9,119,895bp). M 2 bp) c

G+C content Exo+Hyb (i) Hyb+Exo (i) Hyb (i) Control (ii) Control (i) 53 53 Exo (i) 2Hyb (i) r – (%) = 0.997 0.997 = associated 63 70 43 46 65 35 71 63 and R ± Fig. 1 Fig. un 3 6% 2 ). We ). and E Community xperiment d c ATCC, American Type Culture Collection; DSM,Deutsche Sammlung vonMikrorganismen. Euryarchaeota Actinobacteria Proteobacteria Firmicutes Spirochaeta Firmicutes Actinobacteria Proteobacteria ). ). ± - - - -

Phylum content ( G+C gene or organism source the from counts mRNA total the ( quality run in variability to due was believe we sequences which 4, run to relative (G+C)-rich underrepresented systematically 3 run ( content G+C genomic their with correlated positively organism each for slopes regression Linear Exo Exo (‘ Hyb was it 2, for experiment Exo; + Hyb < Hyb < Exo was efficiencies removal rRNA of order rank the 1, experiment For treatment. the to owing rRNA non- to relative rRNA of redistribution the reflected RNA total in percentage rRNA in decrease This Exo). + (Hyb 19.9% to up and (Exo) 3.6% as little as by decreased content rRNA observed ( controls the in represented equally approximately being member was community controls the of each with archaea, and bacteria content of typical RNA, total of rRNA 95–97% relative the expected, As E comparison. intrarun the performed we after average interrun ( 2 experiment in run same the to pairs treatment-control of comparisons and restricted we fidelity, mRNA efficiency depletion rRNA assess accurately to Therefore, with G+C was largely content. variation associated indeed to-run ( replicates technical between 71 71 Exo+Hyb (ii) Hyb+Exo (ii) Exo (ii) 2Hyb (ii) Hyb (ii) Control (ii) Control (i) fficiency of r fficiency 2 2 R ± un 4 3% ≈ ≥ d ’ indicates no statistically significant difference, difference, significant no ’ statistically indicates 0.6)divided bytotalnumber of identified clusters. We usedaverage clusterpass Table S. coelicolor Supplementary Supplementary Note 3 target sites M atch Yes Yes Yes Yes Yes Yes Yes No 2 Ocean Ocean water Bacterial isolates Soil Ocean water RN H ). Two simple normalization strategies based on based strategies normalization Two ). simple A3(2)(9,054,847bp), Published (meta)transcriptomic studiesusingr yb i. 2a Fig. 1 A A depletion 7 a c Draft assembly;genome sizewasestimated tobeeight toten depletion methodslistedinrun4 d 2 , 21–23 13,14, Lanes were excluded from analysis owing totechnical b 3 ≈ ). After the various treatments, the the treatments, various the After ). S 1.9 1.3 1.4 1.4 2.1 1.6 1.4 0.9 2Hyb < Exo < Exo + Hyb < Hyb + Hyb < Hyb + Exo < Exo < 2Hyb / 1 , , bioreactor 19,20 Fig. 1 Fig. 6 b The ratio of 23S/16SrRNAs and RNAintegrity S ) improved the overall correlation b S. griseus Fig. 1 Fig. d ), confirming that the run- the that confirming ), 1 subspecies 5 and bacterial isolates 10 10 RIN Table c 8.6 9.9 9.8 9.8 5.5 7.4 ). This indicates that that indicates This ). b griseus 2 ). We took the the took We ). NITEBiological Community 1 1 and 2 1 1 and 2 a P > 0.05). 2 2 2 1 1 1 RN 6,16,18 A

© 2010 Nature America, Inc. All rights reserved. Intact rRNA is important for the success of the Hyb treatment Hyb the of success the for important is rRNA Intact RNA had the lowest RNA integrity number ( Hyb by geted tar poorly generally is rRNA actinobacterial that possibility the out ruled Actinobacteria, phylum the of member anotherrRNA, of removal Efficient removal. rRNA observed 0.2% and actual 15% only Hyb, with was organism this experiment 1 ( in treatmentsapplied the of three all foraverage than lower edly methods. tested the using by archaea dominated communities rRNAs microbial in deplete solfataricus 5 exposed an requiring adapters because rather monophosphate groups a than hydroxyl exposed have may rRNAs archaeal of ( Halorhabdus ( kit Hyb are Ambion the by archaea targeted not because treatments Hyb the for this expected We lowest percentage removal for all five treatments in 2. experiment 3 Tables mentary ( organisms individual for non-rRNA and rRNA of proportion relative the on based removal rRNA actual 4 Note 20% removal until was actual > 80% ( exceeding not removal rRNA observed with removed, actually was that rRNA of amount the reflect accurately not did fraction rRNA postdepletion but appear the to observed effective, be very ( experiments both in removal greatest in the resulted treatments combined although below), (see ences between differ composition efficiencies community to due was experiments two removal the of order rank in difference The because degradation proportionbecause increasesthe of rRNA fragments i. 2b Fig. c ′ a -monophosphate ligated poorly to archaeal archaeal to poorly ligated -monophosphate Percentage removal of not did methods depletion rRNA applied the glance, first At Run 4 (Hyb 2 ii) Run 1 (Hyb 1 ii) 100,000 100,000 10,000 10,000 1,000 1,000 100 100 10 10 ). For each community member we also calculated calculated also we member community each For ). , 10 10 d . e upc ta sm o te rcse 5 processed the of some that suspect We ). r r Overall correlation: rRNAs =0.997 =0.950 rRNAs by exonuclease, however, was unexpected unexpected was however, exonuclease, by rRNAs Fig. 2 Fig. 2 Fig. 100 100 Run 3(Hyb2i) Run 1(Hybi) and 2 d 4 d ). The most poorly performing treatment for . This indicates that it will be difficult to to difficult be will it that indicates This . 1,000 1,000 ). Rather, we noticed that the the that noticed Rather,we ). Catenulispora Lactobacillus Halorhabdus 4 Spirochaeta Kangiella Streptomyces ). ). Halorhabdus utahensis Halorhabdus 10,000 10,000 G+C 43% 46% 63% 65% 70% 100,000 100,000 sp. str. LCC rRNA was mark Fig. Fig. 2 2.126 Slope 0.639 0.773 1.407 1.406 Table Table Fig. Fig. aeuipr acidiphila Catenulispora 2 c 5 (RIN of 5.5; Fig. 2 Fig. 2 and ). 1 b d ). Low removal of of removal Low ). Run 4 (Hyb 2 ii) Run 2 (Exo 1 ii) 100,000 100,000 Supplementary Supplementary 10,000 10,000 1,000 d 1,000 100 exhibited the the exhibited 100 10 and and 10 Streptomyces 10 10 Sulfolobus Sulfolobus Table r Overall correlation: r Supple­ =0.992 =0.998 ′ ends ends 100 100 1 Run 1(Exoi) ). Run 3(Hyb2i) - - -

1,000 1,000 Kangiella Lactobacillus Halorhabdus Spirochaeta Catenulispora ing in lower mRNA enrichment. mRNA lower in ing rRNAs of the and number reduced Hyb rRNA result sites, target that using exonuclease as the first treatment produced fragmented tion sensitivity than Exo followed by Hyb ( in mRNA improvement by detec gave Exo Hyb greater followed ( results different statistically produced kits removal of the combination of treatments, the order of applying the rRNA ( rounds for two Hyb to 172% for one-round 159% from improvement sensitivity detection the treatments. removal rRNA of cation (50% increase in mRNA detection sensitivity) justifying the appli removal) resulted observed in more use efficient of data sequence sensitivity ( detection mRNA in increase 50–230% a calculated we Methods), Online size; dataset for normalization (after gene control. corresponding the mRNAs in detected of number the to relative treatment given a in mRNAs detected of number the in increase percentage the as calculated sensitivity detection mRNA in improvement be may metric tive informa more a treatments, removal rRNA of effect the express way is to an intuitive mRNA enrichment fold Although removal. rRNA observed the with consistent largely 5.7-fold, to 1.9- from ( treatment each control and the between reads mRNA mapped total of proportion the comparing by mRNAs in enrichment fold the Wecalculated I rRNA removal efficiency for these methods. 3 Fig. rRNA removal and RIN values for most treatments ( ( treatments the of some organismin rRNAlower this removalfor rity of Fig. 2 Fig. Fig. 3 mprovement in m in mprovement Two rounds of subtractive hybridization marginally increased increased marginally hybridization Two subtractive of rounds per reads mapped 30 as threshold detection the Defining 10,000 10,000 ) suggesting that RIN can be used as an indicator of likely of indicator an as used be can RIN that suggesting ) Desulfovibrio vulgaris d b ). Indeed, we observed a high positive correlation between ). ). This shows that even a modest depletion of rRNA (3.6% G+C 43% 46% 63% 65% 70% 100,000 100,000 1.004 Slope 0.971 1.067 0.921 0.979 n a ture methods RN Fig. 3 Fig. targeted by exonuclease skewing the mRNA: mRNAs degraded partially be can because of the Exo treatment on explain the apparently reduced effectiveness also may integrity RNA Lower organism. this in sites target rRNA the of presence effec the despite treatment Hyb the reduced of tiveness the explains likely This sites target Hyb conserved the without points for each organism are indicated in is shown. Slopes from linear regression of data all data points regardless of source organism product moment correlation coefficient, for G+C content by organism ( and Hyb between runs 3 and 4 after normalizing runs 3 and 4 ( ( replicates (i and ii) of Hyb in Illumina run 1 two technical replicates. Analysis of technical abundance of an individual mRNA transcript in the correlation plots, each point indicates the Figure removal (Online Methods). Lower integ Lower Methods). (Online removal rRNA actual calculate to used ratio rRNA A detection sensitivity A detection a ), Exo between runs 1 and 2 ( RNA (RIN 7.4) may similarly explain a 1 ), which produced results ranging ranging results produced which ), P |

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© 2010 Nature America, Inc. All rights reserved. sn te ae elto methods depletion communities same natural the of using sequence- analyses published for metatranscriptomic calculated based range 5–58% the within hypothesis. this to of sequences Illumina mouse transcripts in reported been previously lower-quality has bias content G+C A with ­templates. content G+C high quality, of run underrepresentation greater having Illumina runs to linked directly is bias this that We speculate range. organisms content G+C reference broad a with multiple from data without missed have been would that comparison. bias content G+C interrun consistent one a of discovered We exception notable the with ible technical replicates of Illumina sequencing were highly reproduc samples environmental and isolate terial bac a on (Roche) pyrosequencing FLX Sequencer Genome and on sequencing a using Illumina reproducibility bacterial technical high has RNA-seq that showed studies two Recently, DIS applied. were kits the which in order the of independent and reproducible also was yet and alone has kit each that effect the of addition simple a than fidelity mRNA on effect complex This suggests that the the between interaction two kits 66.6 has a more = similarity (BC alone treatment Hyb and 1.8%) 67.2 = similarity (BC alone treatment Exo to correlation lower = 90.6 2 similarity (BC experiment in combinations tested two the by introduced was skew mRNA ( applied were they than twofold change from control) regardlessgreater ofshowing the order transcripts in which detectable of (>50% fidelity mRNA different removal treatments varied greatly in greatly preserving varied treatments removal ­different Observed rRNA removal for the synthetic communities was was communities synthetic the for removal rRNA Observed in skew greatest the produced together applied Exo and Hyb a C 60 USSION 70 Simila 80 ri ty 90 Exo (1ii Exo (1i) Hyb (1ii) Hyb (1i) Control (1ii) Control (1i) Exo (1 Hyb + 10 0 Fig. Fig. ) ) 4 ). Somewhat unexpectedly, a consistent consistent a unexpectedly, Somewhat ). Hyb + Exo (1) Exo (1) Hyb (1) 10 10 10 10 10 10 10 10 10 3 5 3 5 3 5 10 10 10 3.2 51% 29.5 Control (1 Control (1 Control (1 ± 10 10 10 2.0% ± 0.7% ± 3 3 3 1.1%) despite both having a having both despite 1.1%) ) 10 10 10 ) ) 14,17,21–23 5 5 5 2 7 2 , , lending some support 6 . We, too, found that that found We,too, . b Exo + Hyb (2) Hyb + Exo (2) Exo (2) 2Hyb (2) Hyb (2) 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 . However, the the However, . 3 5 3 5 3 5 3 5 3 5 10 10 10 10 10 2.5 51.7 5.2 53.0 2.2 Control (2 Control (2 Control (2 Control (2 Control (2 ± ± ±

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© 2010 Nature America, Inc. All rights reserved. 3. 2. 1. com/reprintsandpermissions/. R Published online at http://www.nature.com/naturemethods/. The authors declare no competing financial interests. C the data analysis and S.H. and P.H. wrote the manuscript. S.Y. executed the experiments, S.H., O.W., J.L.F., Z.W., R.S. and P.H. performed S.H., K.S., S.G.T., F.C., E.A.L. and P.H. planned the experiments, S.H., K.S. and A contract DE-AC02-06NA25396. under contract DE-AC52-07NA27344 and Los Alamos National Laboratory under under contract DE-AC02-05CH11231, Lawrence Livermore National Laboratory and by the University of California, Lawrence Berkeley National Laboratory Energy’s Office of Science, Biological and Environmental Research Program, The work was performed in part under the auspices of the US Department of for Systems Biology of the Human Cell and an Azrieli Foundation Fellowship. the Yeda-Sela Center for basic research. O.W. was supported by the Kahn Center (1615/09), the EMBO Young Investigator program, the Minerva Foundation and Foundation Focal Initiatives in Research in Science and Technology program Energy Biosciences Institute. R.S. was supported, in part, by the Israel Science for sequencing support. S.H. and P.H. were supported by a grant from the of research and development and production teams at the Joint Genome Institute biomass, L. Pennacchio for supporting and facilitating this project, and members T. Zhang and F. Warnecke (Joint Genome Institute) for providing microbial feedback, H.-P. Klenk (German Collection of Microorganisms and Cell Cultures), We thank M. Allgaier, T. Zhan, M. Hess and E. DeLong for helpful discussions and A Note: information Supplementary is available on the at http://www.nature.com/naturemethods/. paper the of version and are Methods references any in available the online associated M atmay be play.many more variables which in communities mRNAfor enrich to how on decisions informed making in researchers guide help the potential for G+C content bias. These recommendations should samples between Illumina runs should be of viewed cautiously given comparisons Finally, treatment. Hyb the of effectiveness tial poten the maximize to 7.0 > RIN with RNAs sequencing only RNAs of should include a the determination RIN, and we suggest logical interest in studying full-length transcripts. Workup of total bio is a specific there unless used not be should alone treatment should be avoided as they severely impact mRNA fidelity, and Exo not fidelity. should treatments compromise Combined transcript capture generic to beads approach alternative or complement ising prom a are strategies hybridization subtractive sample-specific  OM eprints and permissions information is available online at http://npg.nature. UTHOR c

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© 2010 Nature America, Inc. All rights reserved. genomic sample prep kit was used for Illumina library prepara library Illumina for used was kit prep sample genomic sequencing. and construction library Illumina . by nick synthesized strand second the and hexamers random by primed strand first the with (Invitrogen), Kit Synthesis cDNA Double-Stranded SuperScript using RNAs 5 min. Double-stranded cDNAs were synthesized from fragmented with incubation 1× solution (Ambion) fragmentation at 70 °C for ments (~5–60 ng) were chemically fragmented to ~150–250 bp by RNAs from controls synthesis. (100 ng) and mRNA-enriched cDNA RNAs by and treat fragmentation RNA ( electropherograms RNA Bioanalyzer 2100 Agilent by assessed visually was treatment each after rRNA removal ment evaluated. total RNA pooled was as for used material the each starting treat ~8.5 1. For experiments, for both Hyb + in Exo experiment replicate (following RNAtechnical was except included, pooling) a treatment, each For instructions. manufacturers’ to according in mRNA-ONLY applied were the various combinations to 1 communities synthetic and 2 ( (Epicentre) and Kit Isolation mRNA (Ambion) Prokaryotic Kit Enrichment mRNA treatments. removal rRNA technical ( replicates corresponding their as well as (rRNA procedures), treatments removal various (total and control removal) the rRNA for without RNA material starting the form to aliquoted in described as organisms, five of sets two the from RNAs total of amounts pool equimolar by ing constructed then were Two metatranscriptomes electropherograms. synthetic Bioanalyzer 2100 Agilent evalu was using RNA of ated quality and quantity The residues pellet. salt cell high the in the with cope to buffer RNAwiz of volume that grows by only in salt) the recommended high applying twice modification RNA from A extracting when (bp)). made was pairs this base by (<200 size excluded their on largely based are kit tRNAs and rRNAs 5S that with instructions. Note manufacturer’s (Ambion) the Kit following Bacteria digestion, I DNase RiboPure the using isolate bial tion. construc metatranscriptome synthetic and extraction RNA ( available is contigs 5,197 Streptomyces of exception the with genomes reference complete had isolates ( conditions and media ommended rec DSMZ using grown were isolates microbial Other °C. 30 at grown and broth extract malt yeast into transferred were plates Cultures (DSMZ) medium 84) at 30 °C, and from colonies axenic on of oatmeal agar Collection Microorganisms (German and Cell at 30 °C. flasks shake ml 50 in overnight incubated were both and 416, medium brevis glucose, 0.5% with supplemented (Difco) medium in study ( used this Microbial isolates. ONLINE doi:10.1038/nmeth.1507 fragmented Briefly, instructions. manufacturer’s following tion, Supplementary Fig. 1 Fig. Supplementary Total RNA was extracted independently from each micro each from independently Totalextracted RNA was was grown in American Type Culture Collection (ATCC) Collection Type Culture American in grown was

METHODS Table sp. str. LCC for which a draft genome comprising comprising genome draft a which for LCC str. sp. Streptomyces 2 ). Table One archaeal and seven bacterial isolates were ). 1 h ). ). t sp. str. LCC was initially maintained sp. maintained str. was initially LCC Table Lactococcus lactis Lactococcus t p The MICROBExpress Bacterial Bacterial MICROBExpress The : / / i m 1 g . The pooled total RNA was was RNA total pooled The . . j h g t Halorhabdus i t . p d : o / e / w . g was grown in M17 The pooled total total pooled The w o v w / . e d r The Illumina Illumina The s ). Lactobacillus Lactobacillus (a halophile halophile (a m z . d Table e / ). All All ). μ g g of 2 ------) ­

3.0 bp. This indicates that the vast majority of sequences were were sequences of majority vast the that indicates This bp. 3.0 age length of the alignment for samples in experiment 2 was 72.5 a required of length minimal 42 bp up allowing alignment to six mismatches. The aver The 11. of genomes word-size a reference with blastn the using to evaluation. aligned were abundance reads transcript Quality-filtered relative and Alignment (flowcells) ( runs Illumina four between and within distributed were replicates Technical data. paired-end was which (1-ii), Exo sample of exception the with reads bp 76 single-end generate to II platform Analyzer Genome Illumina the using performed was sequencing All fragments. cDNA adaptor-modified of amounts fragments (A+T)-rich of to was applied comparable adaptor primers PCR with A 12-cycle denaturation minimize to °C, 55 recommended the of instead °C, 37 at dissolved was gel except the that instructions, Extraction manufacturer’s to Gel according MiniElute (Qiagen) Kit the using elec gel purification by conducted trophoresis was products ligation of selection size bp 200–500 A adaptors. Illumina of ligation subsequent the ing 3 the to added was adenine An ends. blunt generate to repaired end was cDNA rdc-oet orlto cefcet ( Pearson’s coefficient the correlation using product-moment evaluated was reproducibility Technical content G+C shown). not data testing; based on empirical simulations (corrected for multiple hypothesis ( expression differential for test statistical Chi-square a to pass needed of reads number minimum the to be found it was mRNA a was given for abundance) transcript (relative count read size–normalized each experiment. An mRNA was defined as in ‘detected’ controls the if the in library reads mapped total of number average the by multiplying then and lane Illumina each in reads mapped total of number the by transcript per count read raw the dividing by performed was normalization This sample. each for generated reads of numbers different for account to size library by the ized genic regions; reads of tRNA rRNA, inter mRNA, and (including number gene to each mapped The origin. of genome their to mapped fully fit that minimizes the sum of the difference between between difference the of sum the minimizes that fit best the by determined B, and A runs between factor correction gene in run A; %G+C is the G+C content of that gene; and count and genes, individual of content below. G+C described the by also abundance tran the we script normalized which available, gene), (by be strategy not second a in tried may real in organism datasets mRNAs of source metatranscriptomic classification its taxonomic from the As count run. each read mRNA total the by mRNAs individual of abundances normaliz transcript by relative the ing performed was strategies. organism) (by normalization strategy first simple The two tried we observed, content G+C was with associated variation interrun As replicates. technical two between mRNAs individual of abundances script for all mRNAs with technical replicates in runs A and B. and A runs in replicates technical mRNAs with all for B) run in replicate technical corresponding its from count (read Table For each gene, gene, each For 2 ). N A ′ – is G+C content–normalized read is count G+C for content–normalized a given Supplementary TableSupplementary 2 ′ end of the blunt-ended cDNA fragments, allow fragments, cDNA blunt-ended the of end associated interrun variation normalization. variation interrun associated N A ≥ ′ 30. This detection threshold was selected as selected was threshold detection This 30. = = N A × (%G+C) × ) ) was counted and normal a , in which which in , r o rltv tran relative of ) n N a ture methods A is raw read read raw is N A P ′ and and < 0.05) 0.05) < a is the N 2 8 ± B ------.

© 2010 Nature America, Inc. All rights reserved. Obse mapped reads in the control and treatment respectively; and and respectively; treatment and control the in reads mapped were as: calculated sensitivity detection mRNA in improvement percentage tion. calcula increase sensitivity detection and mRNA enrichment 2. experiment in 4 and 3 runs from comparisons intrarun available all and 1 experiment s.d. in comparisons and treatment-control available all average from derived was The 4. and 3 runs between bias content observed G+C the of because 2 experiment in run same the to pairs below), described we as restricted comparisons of treatment-control fidelity, and improvement sensitivity detection enrichment, respectively. treatment, and control the in reads as: calculated was removal percentage rRNA rRNA calculation. removal percentage n these transcripts may appear to be skewed because of statisti of cally insignificant read because counts in the control. skewed Therefore, we used a be to appear may transcripts these treatment, enrichment mRNA an after detectable become may transcripts low-abundance As control. treat corresponding and a ment between abundance transcript relative of preservation evaluation. fidelity mRNA respectively. treatment, and trol in by size) the con (30 reads per gene library after normalization and Ac r m Pe mRNA − = a 0 To accurately assess the rRNA (mRNAdepletion efficiency fold ture methods and and tual 0 (( rc and and M M M rved The fold enrichment of total mRNA abundance and the the and abundance mRNA total of enrichment fold The entage 1 rR 0 1 are the number of mRNAs above the detection threshold threshold of are mRNAs number the detection the above r fold 1 m NA are the percentages of rRNA reads among all mapped mapped all among reads rRNA of percentages the are rR 1 are the percentage of total mRNA reads among all all among reads mRNA total of percentage the are improvemen NA enrichment percentage / ) percentage M 0 ) × 100 re = ti m nm m m remova ooval mRNA fidelity is a measure of the the of measure a is fidelity mRNA 0 1 RN / − = Ad l 1 − = etecti r r / ( 1 0 r r 0 1 The observed and The actual observed oon ( ) sensitivit − × ) ( 1 1 r r 1 0 y ( / − M )) 0 - - - ­

mined by performing a chi-square test of the GEI of all mRNAs all of GEI the of test chi-square a performing by mined in the sample. Differential expression between samples was deter reads mRNA of number total the to according normalization by dividing the number of reads in the mRNA followed per kilobase, by calculated was mRNAs all for (GEI) index expression gene A identification. category functional mRNA Underrepresented control. corresponding the mRNAs in detected total of portion and corresponding control was counted and represented as a pro treatment a between difference twofold than greater exhibiting mRNAs of number The treatment. each in counts read malized nor their against plotted were control the in individual detected mRNAs for counts read normalized addition, In algorithm. clustering UPGMA the using performed was samples of tering vidual mRNAs included. clushierarchical A BC similarity-based indi for abundance transcript relative of comparisons pairwise on based matrices similarity generate to Ltd) (PRIMER-E 6.1.9) 6 (version Primer using were calculated samples between larities simi BC analysis. the in included mRNAs all from counts read total by normalized further were counts read mRNA Individual fidelity. mRNA assess to controls the of majority a in threshold ­filter to only include mRNAs that are above the detection 30-read 28. RIN. and removal percent ( The Pearson’scoefficient correlation variables. product-moment ( significance statistical obtain analyses. statistical Other testing. a by multiple-hypothesis for correction followed Bonferonni test exact Fisher’s single-sided a with calculated was categories functional COG different in mRNAs represented ( US from records (NCBI) Information downloaded Biotechnology for Center was National genes of classifications functional multiple-hypothesis testing with a threshold of for correction Bonferonni a by followed conditions, different in h r ) was used to determine the correlation between actual rRNA rRNA actual between correlation the determine to used was ) t

t p sequencing system. sequencing Quail, M.A. Quail, : / / w w w . n et al. et c b i . n A large genome center’s improvements to the Illumina the to improvements center’s genomelarge A l m Nat. Methods Nat. . n i h . g o Student’s v / C

5 O , 1005–1010 (2008). 1005–1010 , P value) when comparing two two comparing when value) G / ), and enrichment for under t -tests were performed to to performed were -tests doi:10.1038/nmeth.1507 P < 0.05. Available ------