Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts.
Journal
Environmental microbiology
ISSN: 1462-2920
Titre abrégé: Environ Microbiol
Pays: England
ID NLM: 100883692
Informations de publication
Date de publication:
06 2020
06 2020
Historique:
received:
16
09
2019
accepted:
08
04
2020
pubmed:
15
4
2020
medline:
22
1
2021
entrez:
15
4
2020
Statut:
ppublish
Résumé
Reverse-transcriptase-quantitative PCR (RT-Q-PCR) and RT-PCR amplicon sequencing, provide a convenient, target-specific, high-sensitivity approach for gene expression studies and are widely used in environmental microbiology. Yet, the effectiveness and reproducibility of the reverse transcription step has not been evaluated. Therefore, we tested a combination of four commercial reverse transcriptases with two priming techniques to faithfully transcribe 16S rRNA and amoA transcripts from marine sediments. Both enzyme and priming strategy greatly affected quantification of the exact same target with differences of up to 600-fold. Furthermore, the choice of RT system significantly changed the communities recovered. For 16S rRNA, both enzyme and priming had a significant effect with enzyme having a stronger impact than priming. Inversely, for amoA only the change in priming strategy resulted in significant differences between the same samples. Specifically, more OTUs and better coverage of amoA transcripts diversity were obtained with GS priming indicating this approach was better at recovering the diversity of amoA transcripts. Moreover, sequencing of RNA mock communities revealed that, even though transcript α diversities (i.e., OTU counts within a sample) can be biased by the RT, the comparison of β diversities (i.e., differences in OTU counts between samples) is reliable as those biases are reproducible between environments.
Identifiants
pubmed: 32285609
doi: 10.1111/1462-2920.15017
doi:
Substances chimiques
Bacterial Proteins
0
RNA, Ribosomal, 16S
0
Oxidoreductases
EC 1.-
ammonia monooxygenase
EC 1.7.3.-
RNA-Directed DNA Polymerase
EC 2.7.7.49
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2383-2402Subventions
Organisme : Royal Academy of Engineering-Scottish Water Research Chair
ID : RCSRF1718643
Pays : International
Organisme : NERC IRF
ID : NE/L011956/1
Pays : International
Informations de copyright
© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.
Références
Botes, M., De Kwaadsteniet, M., and Cloete, T.E. (2013) Application of quantitative PCR for the detection of microorganisms in water. Anal Bioanal Chem 405: 91-108.
Browning, D.F., and Busby, S.J.W. (2016) Local and global regulation of transcription initiation in bacteria. Nat Rev Microbiol 14: 638-650.
Bustin, S., and Nolan, T. (2017) Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research. Eur J Clin Invest 47: 756-774.
Bustin, S.A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): Trends and problems. J Mol Endocrinol 29: 23-39.
Bustin, S.A., and Nolan, T. (2004) Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 15: 155-166.
Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., et al. (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335-336.
Cholet, F., Ijaz, U.Z., and Smith, C.J. (2019) Differential ratio amplicons (R amp) for the evaluation of RNA integrity extracted from complex environmental samples. Environ Microbiol 21: 827-844.
Christiansen, N.A., Fryirs, K.A., Green, T.J., and Hose, G.C. (2019) The impact of urbanisation on community structure, gene abundance and transcription rates of microbes in upland swamps of Eastern Australia. PLoS One 14: 1-20.
D'Amore, R., Ijaz, U.Z., Schirmer, M., Kenny, J.G., Gregory, R., Darby, A.C., et al. (2016) A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling. BMC Genomics 17: 55.
Damashek, J., Smith, J.M., Mosier, A.C., and Francis, C.A. (2015) Benthic ammonia oxidizers differ in community structure and biogeochemical potential across a riverine delta. Front Microbiol 6: 1-18.
Duff, A.M., Zhang, L.M., and Smith, C.J. (2017) Small-scale variation of ammonia oxidisers within intertidal sediments dominated by ammonia-oxidising bacteria Nitrosomonas sp. amoA genes and transcripts. Sci Rep 7: 1-13.
Feng, G., Sun, W., Zhang, F., Orlić, S., and Li, Z. (2018) Functional transcripts indicate phylogenetically diverse active ammonia-scavenging microbiota in sympatric sponges. Marine Biotechnol 20: 131-143.
Forootan, A., Sjöback, R., Björkman, J., Sjögreen, B., Linz, L., and Kubista, M. (2017) Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR). Biomol Detect Quantif 12: 1-6.
Gadkar, V.J., and Filion, M. (2013) Quantitative real-time polymerase chain reaction for tracking microbial gene expression in complex environmental matrices. Curr Issues Mol Biol 15: 45-58.
Gonçalves, J., Gutiérrez-Aguirre, I., Balasubramanian, M.N., Zagorščak, M., Ravnikar, M., and Turk, V. (2018) Surveillance of human enteric viruses in coastal waters using concentration with methacrylate monolithic supports prior to detection by RT-qPCR. Mar Pollut Bull 128: 307-317.
Graham, J.E., Wantland, N.B., Campbell, M., and Klotz, M.G. (2011) Characterizing bacterial gene expression in nitrogen cycle metabolism with RT-qPCR. Methods Enzymol. 496: 345-372. https://doi.org/10.1016/B978-0-12-386489-5.00014-2.
Griffiths, R.I., Whiteley, A.S., O'Donnell, A.G., and Bailey, M.J. (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl Environ Microbiol 66: 5488-5491.
Gutleben, J., Chaib De Mares, M., van Elsas, J.D., Smidt, H., Overmann, J., and Sipkema, D. (2018) The multi-omics promise in context: from sequence to microbial isolate. Crit Rev Microbiol 44: 212-229.
Hata, A., Katayama, H., and Furumai, H. (2015) Organic substances interfere with reverse transcription-quantitative PCR-based virus detection in water samples. Appl Environ Microbiol 81: 1585-1593.
Hoshino, T., and Inagaki, F. (2013) A comparative study of microbial diversity and community structure in marine sediments using poly(A) tailing and reverse transcription-PCR. Front Microbiol 4: 1-8.
Joshi, N.A. and Sickle, J.N.F. (2011) A sliding-window, adaptive, quality-based trimming tool for FastQ files: 1-9.
Kapoor, V., Pitkänen, T., Ryu, H., Elk, M., Wendell, D., and Santo Domingo, J.W. (2015) Distribution of human-specific Bacteroidales and fecal indicator bacteria in an urban watershed impacted by sewage pollution, determined using RNA- and DNA-based quantitative PCR assays. Appl Environ Microbiol 81: 91-99.
Katoh, K., Asimenos, G., and Toh, H. (2009) Multiple alignment of DNA sequences with MAFFT. Bioinformatics for DNA Sequence Analysis, Methods in Molecular Biology 537: 39-64.
Leininger, S., Urich, T., Schloter, M., Schwark, L., Qi, J., Nicol, G.W., et al. (2006) Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442: 806-809.
Lekanne Deprez, R.H., Fijnvandraat, A.C., Ruijter, J.M., and Moorman, A.F.M. (2002) Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 307: 63-69.
Levesque-Sergerie, J.-P., Duquette, M., Thibault, C., Delbecchi, L., and Bissonnette, N. (2007) Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR. BMC Mol Biol 8: 93.
Li, Y., Jia, Z., Sun, Q., Cheng, J., Yang, Y., Zhan, J., and Wang, D. (2017) Plant-mediated changes in soil n-cycling genes during revegetation of copper mine tailings. Front Environ Sci 5: 1-11.
Liu, S., Meng, C., Xu, G., Jian, H., and Wang, F. (2018) Validation of reference genes for reverse transcription real-time quantitative PCR analysis in the deep-sea bacterium Shewanella psychrophila WP2. FEMS Microbiol Lett 365: 1-5.
López-Maury, L., Marguerat, S., and Bähler, J. (2008) Tuning gene expression to changing environments: From rapid responses to evolutionary adaptation. Nat Rev Genet 9: 583-593.
Marotz, C., Sharma, A., Humphrey, G., Gotte, N., Daum, C., Gilbert, J., Eloe-Fadrosh, E., and Knight R. (2019). Triplicate PCRreactions for 16S rRNA ge.ne amplicon sequencing are unnecessary. Biotechniques 67: 6-9.
Mäki, A., and Tiirola, M. (2018) Directional high-throughput sequencing of RNAs without gene-specifi c primers. Biotechniques 65: 219-223.
Miranda, J.A., and Steward, G.F. (2017) Variables influencing the efficiency and interpretation of reverse transcription quantitative PCR (RT-qPCR): An empirical study using Bacteriophage MS2. J Virol Methods 241: 1-10.
Okello, J.B.A., Rodriguez, L., Poinar, D., Bos, K., Okwi, A.L., Bimenya, G.S., et al. (2010) Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA. PLoS One: 5: e13931.
Pelissari, C., Guivernau, M., Viñas, M., de Souza, S.S., García, J., Sezerino, P.H., and Ávila, C. (2017) Unraveling the active microbial populations involved in nitrogen utilization in a vertical subsurface flow constructed wetland treating urban wastewater. Sci Total Environ 584-585: 642-650.
Pelissari, C., Guivernau, M., Viñas, M., García, J., Velasco-Galilea, M., Souza, S.S., et al. (2018) Effects of partially saturated conditions on the metabolically active microbiome and on nitrogen removal in vertical subsurface flow constructed wetlands. Water Res 141: 185-195.
Pierre, S., Hewson, I., Sparks, J.P., Litton, C.M., Giardina, C., Groffman, P.M., and Fahey, T.J. (2017) Ammonia oxidizer populations vary with nitrogen cycling across a tropical montane mean annual temperature gradient. Ecology 98: 1896-1907.
Polumuri, S.K., Ruknudin, A., and Schulze, D.H. (2002) RNase H and its effects on PCR. Biotechniques 32: 1224-1225.
Posman, K.M., DeRito, C.M., and Madsen, E.L. (2017) Benzene degradation by a Variovorax species within a coal tar-contaminated groundwater microbial community. Appl Environ Microbiol 83: 1-13.
Price, M.N., Dehal, P.S., and Arkin, A.P. (2010) FastTree 2-Approximately maximum-likelihood trees for large alignments. PLoS One 5: e9490.
R Core Team (2013). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. http://www.R-project.org/
Saleh-Lakha, S., Shannon, K.E., Goyer, C., and Trevors, J.T. (2011) Challenges in quantifying microbial gene expression in soil using quantitative reverse transcription real-time PCR. J Microbiol Methods 85: 239-243.
Sanders, R., Mason, D.J., Foy, C.A., and Huggett, J.F. (2014) Considerations for accurate gene expression measurement by reverse transcription quantitative PCR when analysing clinical samples. Anal Bioanal Chem 406: 6471-6483.
Santoro, A.E., Casciotti, K.L., and Francis, C.A. (2010) Activity, abundance and diversity of nitrifying archaea and bacteria in the central California Current. Environ Microbiol 12: 1989-2006.
Santos, J.P., Mendes, D., Monteiro, M., Ribeiro, H., Baptista, M.S., Borges, M.T., and Magalhães, C. (2018) Salinity impact on ammonia oxidizers activity and amoA expression in estuarine sediments. Estuar Coast Shelf Sci 211: 177-187.
Schirmer, M., Ijaz, U.Z., D'Amore, R., Hall, N., Sloan, W.T., and Quince, C. (2015) Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform. Nucleic Acids Res 43: e37.
Segall-Shapiro, T.H., Meyer, A.J., Ellington, A.D., Sontag, E.D., and Voigt, C.A. (2014) A “resource allocator” for transcription based on a highly fragmented T7 RNA polymerase. Mol Syst Biol 10: 742-742.
Shakya, M., Quince, C., Campbell, J.H., Yang, Z.K., Schadt, C.W., and Podar, M. (2013) Comparative metagenomic and rRNA microbial diversity characterization using archaeal and bacterial synthetic communities. Environ Microbiol 15: 1882-1899.
Sieber, M.W., Recknagel, P., Glaser, F., Witte, O.W., Bauer, M., Claus, R.A., and Frahm, C. (2010) Substantial performance discrepancies among commercially available kits for reverse transcription quantitative polymerase chain reaction: A systematic comparative investigator-driven approach. Anal Biochem 401: 303-311.
Smith, C.J., Nedwell, D.B., Dong, L.F., and Osborn, A.M. (2006) Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples. Environ Microbiol 8: 804-815.
Smith, C.J., Nedwell, D.B., Dong, L.F., and Osborn, A.M. (2007) Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments. Appl Environ Microbiol 73: 3612-3622.
Smith, C.J., and Osborn, A.M. (2009) Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol 67: 6-20.
Smith, C.J., Dong, L.F., Wilson, J., Stott A., Osborn, A.M., and Nedwell, D.B. (2015). Seasonal variation in denitrification and dissimilatory nitrate reduction to ammonia process rates and corresponding key functional genes along an estuarine nitrate gradient. Front Microbiol 6: 1-11.
Ståhlberg, A., Håkansson, J., Xian, X., Semb, H., and Kubista, M. (2004a) Properties of the reverse transcription reaction in mRNA quantification. Clin Chem 50: 509-515.
Ståhlberg, A., Kubista, M., and Pfaffl, M. (2004b) Comparison of reverse transcriptases in gene expression analysis. Clin Chem 50: 1678-1680.
Stangegaard, M., Dufva, I.H., and Dufva, M. (2006) Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA. Biotechniques 40: 649-657.
Tolar, B.B., Ross, M.J., Wallsgrove, N.J., Liu, Q., Aluwihare, L.I., Popp, B.N., and Hollibaugh, J.T. (2016) Contribution of ammonia oxidation to chemoautotrophy in Antarctic coastal waters. ISME J 10: 2605-2619.
Wang, Y., Nagaoka, K., Hayatsu, M., Sakai, Y., Tago, K., Asakawa, S., and Fujii, T. (2012) A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil. Appl Microbiol Biotechnol 96: 793-802.
Wang, Z.B., Ni, S.Q., Zhang, J., Zhu, T., Ma, Y.G., Liu, X.L., et al. (2016) Gene expression and biomarker discovery of anammox bacteria in different reactors. Biochem Eng J 115: 108-114.
Werbrouck, H., Botteldoorn, N., Uyttendaele, M., Herman, L., and Van Coillie, E. (2007) Quantification of gene expression of Listeria monocytogenes by real-time reverse transcription PCR: Optimization, evaluation and pitfalls. J Microbiol Methods 69: 306-314.
Winter, D.J. (2017) rentrez: An R package for the NCBI eUtils API. R J 9: 520-526.
Yan, Y.W., Zou, B., Zhu, T., Hozzein, W.N., and Quan, Z.X. (2017) Modified RNA-seq method for microbial community and diversity analysis using rRNA in different types of environmental samples. PLoS One 12: 1-20.
Yergeau, E., Arbour, M., Brousseau, R., Juck, D., Lawrence, J.R., Masson, L., et al. (2009) Microarray and real-time PCR analyses of the responses of high-arctic soil bacteria to hydrocarbon pollution and bioremediation treatments. Appl Environ Microbiol 75: 6258-6267.
Zhang, L., Duff, A., and Smith, C. (2018) Community and functional shifts in ammonia oxidizers across terrestrial and marine (soil/sediment) boundaries in two coastal Bay ecosystems. Environ Microbiol 20: 2834-2853.
Zheng, Y., Hou, L., Liu, M., Lu, M., Zhao, H., Yin, G., and Zhou, J. (2013) Diversity, abundance, and activity of ammonia-oxidizing bacteria and archaea in Chongming eastern intertidal sediments. Appl Microbiol Biotechnol 97: 8351-8363.