Evaluation of Ribosomal RNA Removal Protocols for Salmonella RNA-Seq Projects
Affiliation(s)
Environmental Microbial & Food Safety Laboratory, Beltsville, USA. Diet Genomics and Immunology Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, Beltsville, USA.
Environmental Microbial & Food Safety Laboratory, Beltsville, USA. Diet Genomics and Immunology Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, Beltsville, USA.
ABSTRACT
Next generation sequencing is a powerful technology whose application in sequencing entire RNA populations (RNA-Seq) of food-borne pathogens will provide valuable insights. A problem unique to prokaryotic RNA-Seq is removal of ribosomal RNA. Unlike eukaryotic messenger RNA (mRNA), bacterial mRNA species are devoid of polyadenylation at the 3’-end and thus the approach of affinity enrichment of mRNA using oligo-dT probes is not an option. Among several approaches to enriching mRNA molecules, removal of ribosomal RNA (rRNA) by subtractive hybridization has been widely used. This approach is a single-step procedure for which several rRNA-depletion kits are commercially available. We evaluated three commercially available rRNA-depletion kits to determine their respective efficiencies of rRNA removal from Salmonella enterica serovar Typhimurium strain SL1344. The three protocols achieved varying degrees of rRNA depletion and resulted in 8 to 1000-fold enrichment of mRNA. rRNA removal probes from two of the three kits were unable to titrate out 23S rRNA species while removal of 16S rRNA was less efficient. The Ribo-Zero kit was most efficient in eliminating 16S, 23S and 5S ribosomal RNA species from the transcriptome of S. enterica serovar Typhimurium strain SL1344.
Cite this paper
A. Bhagwat, Z. Ying and A. Smith, "Evaluation of Ribosomal RNA Removal Protocols for Salmonella RNA-Seq Projects," Advances in Microbiology, Vol. 4 No. 1, 2014, pp. 25-32. doi: 10.4236/aim.2014.41006.
A. Bhagwat, Z. Ying and A. Smith, "Evaluation of Ribosomal RNA Removal Protocols for Salmonella RNA-Seq Projects," Advances in Microbiology, Vol. 4 No. 1, 2014, pp. 25-32. doi: 10.4236/aim.2014.41006.
References
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http://dx.doi.org/10.1016/S0167-7012(03)00175-1 [17] S. H. Liming and A. A. Bhagwat, “Application of a Molecular Beacon—Real-Time PCR Technology to Detect Salmonella Species Contaminating Fruits and Vegetables,” International Journal of Food Microbiology, Vol. 95, No. 2, 2004, pp. 177-187.
http://dx.doi.org/10.1016/j.ijfoodmicro.2004.02.013 [18] M. H. Queiroz, C. Madrid, S. Paytubi, C. Balsalobre and A. Juárez, “Integration Host Factor Alleviates H-NS Silencing of the Salmonella enterica Serovar Typhimurium Master Regulator of SPI1, hilA,” Microbiology, Vol. 157, No. 9, 2011, pp. 2504-2514.
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http://dx.doi.org/10.1099/mic.0.056499-0
[1] J. A. Kimbrel, Y. Di, J. S. Cumbie and J. H. Chang, “RNA-Seq for Plant Pathogenic Bacteria,” Gene, Vol. 2, 2011, pp. 689-705. [2] K. D. Passalacqua, A. Varadarajan, B. D. Ondov, D. T. Okou, M. E. Zwick and N. H. Bergman, “Structure and Complexity of a Bacterial Transcriptome,” Journal of Bacteriology, Vol. 191, No. 10, 2009, pp. 3203-3211.
http://dx.doi.org/10.1128/JB.00122-09 [3] M. Guell, E. Yus, M. Lluch-Senar and L. Serrano, “Bacterial Transcriptomics: What Is beyond the RNA Horizome?” Nature Reviews Microbiology, Vol. 9, 2011, pp. 658-669.
http://dx.doi.org/10.1038/nrmicro2620 [4] A. H. M. van Vilet, “Next Generation Sequencing of Microbial Transcriptomes: Challenges and Opportunities,” FEMS Microbiology Letters, Vol. 302, No. 1, 2010, pp. 1-7.
http://dx.doi.org/10.1111/j.1574-6968.2009.01767.x [5] M. J. Filiatrault, “Progress in Prokaryotic Transcriptomics,” Current Opinion in Microbiology, Vol. 14, No. 5, 2011, pp. 579-586.
http://dx.doi.org/10.1016/j.mib.2011.07.023 [6] N. J. Croucher and N. R. Thomson, “Studying Bacterial Transcriptomes Using RNA-seq,” Current Opinion in Microbiology, Vol. 13, No. 5, 2010, pp. 619-624.
http://dx.doi.org/10.1016/j.mib.2010.09.009 [7] S. He, O. Wurtzel, K. Singh, J. L. Froula, S. Yilmaz, S. G. Tringe, Z. Wang, F. Chen, E. A. Lindquist, R. Sorek and P. Hungenholtz, “Validation of Two Ribosomal RNA Removal Methods for Microbial Metatranscriptomics,” Nature Methods, Vol. 7, 2010, pp. 807-812.
http://dx.doi.org/10.1038/nmeth.1507 [8] F. J. Stewart, E. Ottesen and E. F. DeLong, “Development and Quantitative Analyses of a Universal rRNASubtraction Protocol for Microbial Metatranscriptomics,” ISME Journal, Vol. 4, 2010, pp. 896-907.
http://dx.doi.org/10.1038/ismej.2010.18 [9] G. Giannoukos, D. Ciulla, K. Huang, B. Haas, J. Izard, J. Levin, J. Livny, A. Earl, D. Gevers, D. Ward, C. Nusbaum, B. Birren and A. Gnirke, “Efficient and Robust RNA-seq Process for Cultured Bacteria and Complex Community Transcriptomes,” Genome Biology, Vol. 13, 2012, p. r23.
http://dx.doi.org/10.1186/gb-2012-13-3-r23 [10] Z. Chen and X. Duan, “Ribosomal RNA Depletion for Massively Parallel Bacterial RNA-Sequencing Applications,” Methods in Molecular Biology, Vol. 733, 2012, pp. 93-103. [11] C. Peano, A. Pietrelli, C. Consolandi, E. Rossi, L. Petiti, L. Tagliabue, G. De Bellis and P. Landini, “An Efficient rRNA Removal Method for RNA Sequencing in GC-Rich Bacteria,” Microbial Informatics and Experimentation, Vol. 3, 2013, p. 1.
http://dx.doi.org/10.1186/2042-5783-3-1 [12] D. A. Braasch and D. R. Corey, “Locked Nucleic Acid (LNA): Fine-Tuning the Recognition of DNA and RNA,” Chemistry & Biology, Vol. 8, No. 1, 2001, pp. 1-7.
http://dx.doi.org/10.1016/S1074-5521(00)00058-2 [13] P. M. McTigue, R. J. Peterson and J. D. Kahn, “Sequence-Dependent Thermodynamic Parameters for Locked Nucleic Acid (LNA)-DNA Duplex Formation,” Biochemistry, Vol. 43, 2004. [14] A. A. Bhagwat, W. Jun, L. Liu, P. Kannan, M. Dharne, B. Pheh, B. D. Tall, M. H. Kothary, K. C. Gross, S. Angle, J. Meng and A. Smith, “Osmoregulated Periplasmic Glucans of Salmonella enterica Serovar Typhimurium Are Required for Optimal Virulence in Mice,” Microbiology, Vol. 155, No. 1, 2009, pp. 229-237.
http://dx.doi.org/10.1099/mic.0.023747-0 [15] A. A. Bhagwat and M. A. Bhagwat, “Comparative Analysis of Transcriptional Regulatory Elements of Glutamate-Dependent Acid-Resistance Systems of Shigella flexneri and Escherichia coli O157:H7,” FEMS Microbiology Letters, Vol. 234, No. 1, 2004, pp. 139-147.
http://dx.doi.org/10.1111/j.1574-6968.2004.tb09525.x [16] A. A. Bhagwat, R. P. Phadke, D. Wheeler, S. Kalantre, M. Ram and M. Bhagwat, “Computational Methods and Evaluation of RNA Stabilization Reagents for GenomeWide Expression Studies,” Journal of Microbiological Methods, Vol. 55, No. 2, 2003, pp. 399-409.
http://dx.doi.org/10.1016/S0167-7012(03)00175-1 [17] S. H. Liming and A. A. Bhagwat, “Application of a Molecular Beacon—Real-Time PCR Technology to Detect Salmonella Species Contaminating Fruits and Vegetables,” International Journal of Food Microbiology, Vol. 95, No. 2, 2004, pp. 177-187.
http://dx.doi.org/10.1016/j.ijfoodmicro.2004.02.013 [18] M. H. Queiroz, C. Madrid, S. Paytubi, C. Balsalobre and A. Juárez, “Integration Host Factor Alleviates H-NS Silencing of the Salmonella enterica Serovar Typhimurium Master Regulator of SPI1, hilA,” Microbiology, Vol. 157, No. 9, 2011, pp. 2504-2514.
http://dx.doi.org/10.1099/mic.0.049197-0 [19] T. A. Lehti, P. Bauchart, U. Dobrindt, T. K. Korhonen and B. Westerlund-Wikström, “The Fimbriae Activator MatA Switches off Motility in Escherichia coli by Repression of the Flagellar Master Operon flhDC,” Microbiology, Vol. 158, 2012, pp. 1444-1455.
http://dx.doi.org/10.1099/mic.0.056499-0