In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5 sequences were used for analysis after processing for read length (150？bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis. 1. Introduction Molecular tools such as the recently introduced method of massively parallel sequencing (deep sequencing) [1, 2] greatly facilitate the study of complex bacterial communities and provide deep insights into their compositions [3–5]. Combined with the technique of barcoded PCR amplicons, deep sequencing methods are able to process many samples at a relatively low cost per sequence [6, 7]. Deep sequencing is, therefore, a promising tool for examining the influence of nutritional and other factors on intestinal microbial communities and functionalities. However, as with any new technology, pitfalls exist. For barcoded PCR amplicon sequencing studies, nucleic acids must be extracted and the resulting DNA extract should ideally represent the entire bacterial diversity in a given habitat. Furthermore, barcoding requires a PCR step, which depends on primers that should ideally cover the complete bacterial diversity. Finally, the evaluation of sequence reads is based on databases, most of which are not yet suited for massive sequence inputs [8] and sequence quality is often found to be suboptimal [9, 10]. In regard to DNA extraction from complex samples, a multitude of studies have reported that any given nucleic acid extraction method is biased towards certain bacterial groups [11–13]. Complex samples such as
References
[1]
S. R. Gill, M. Pop, R. T. DeBoy et al., “Metagenomic analysis of the human distal gut microbiome,” Science, vol. 312, no. 5778, pp. 1355–1359, 2006.
[2]
L. A. S. Snyder, N. Loman, M. J. Pallen, and C. W. Penn, “Next-generation sequencing—the promise and perils of charting the great microbial unknown,” Microbial Ecology, vol. 57, no. 1, pp. 1–3, 2009.
[3]
L. V. Hooper, T. Midwedt, and J. I. Gordon, “How host-microbial interactions shape the nutrient environment of the mammalian intestine,” Annual Review of Nutrition, vol. 22, pp. 283–307, 2002.
[4]
H. N. Shi and A. Walker, “Bacterial colonization and the development of intestinal defences,” Canadian Journal of Gastroenterology, vol. 18, no. 8, pp. 493–500, 2004.
[5]
C. Cenciarini-Borde, S. Courtois, and B. la Scola, “Nucleic acids as viability markers for bacteria detection using molecular tools,” Future Microbiology, vol. 4, no. 1, pp. 45–64, 2009.
[6]
F. Armougom and D. Raoult, “Use of pyrosequencing and DNA barcodes to monitor variations in Firmicutes and Bacteroidetes communities in the gut microbiota of obese humans,” BMC Genomics, vol. 9, article 576, 2008.
[7]
M. Hamady, J. J. Walker, J. K. Harris, N. J. Gold, and R. Knight, “Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex,” Nature Methods, vol. 5, no. 3, pp. 235–237, 2008.
[8]
M. Hamady and R. Knight, “Microbial community profiling for human microbiome projects: tools, techniques, and challenges,” Genome Research, vol. 19, no. 7, pp. 1141–1152, 2009.
[9]
P. D. Bridge, P. J. Roberts, B. M. Spooner, and G. Panchal, “On the unreliability of published DNA sequences,” New Phytologist, vol. 160, no. 1, pp. 43–48, 2003.
[10]
R. Christen, “Global sequencing: a review of current molecular data and new methods available to assess microbial diversity,” Microbes and Environments, vol. 23, no. 4, pp. 253–268, 2008.
[11]
H. Morita, T. Kuwahara, K. Ohshima, et al., “An improved DNA isolation method for metagenomic analysis of the microbial flora of the human intestine,” Microbes and Environments, vol. 22, no. 3, pp. 214–222, 2007.
[12]
J. M. Nechvatal, J. L. Ram, M. D. Basson et al., “Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces,” Journal of Microbiological Methods, vol. 72, no. 2, pp. 124–132, 2008.
[13]
A. Salonen, J. Nikkil？, J. Jalanka-Tuovinen et al., “Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis,” Journal of Microbiological Methods, vol. 81, no. 2, pp. 127–134, 2010.
[14]
C. C. Tebbe and W. Vahjen, “Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast,” Applied and Environmental Microbiology, vol. 59, no. 8, pp. 2657–2665, 1993.
[15]
R. Sipos, A. J. Székely, M. Palatinszky, S. Révész, K. Márialigeti, and M. Nikolausz, “Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis,” FEMS Microbiology Ecology, vol. 60, no. 2, pp. 341–350, 2007.
[16]
G. C. Baker, J. J. Smith, and D. A. Cowan, “Review and re-analysis of domain-specific 16S primers,” Journal of Microbiological Methods, vol. 55, no. 3, pp. 541–555, 2003.
[17]
A. Schmalenberger, F. Schwieger, and C. C. Tebbe, “Effect of primers hybridizing to different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling,” Applied and Environmental Microbiology, vol. 67, no. 8, pp. 3557–3563, 2001.
[18]
R. Ducluzeau and P. Raibaud, “Microbial ecology of the digestive system,” Agressologie, vol. 26, no. 2, pp. 161–163, 1985.
[19]
F. Meyer, D. Paarmann, M. D'Souza et al., “The metagenomics RAST server—a public resource for the automatic phylogenetic and functional analysis of metagenomes,” BMC Bioinformatics, vol. 9, article 386, 2008.
[20]
E. Pruesse, C. Quast, K. Knittel et al., “SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB,” Nucleic Acids Research, vol. 35, no. 21, pp. 7188–7196, 2007.
[21]
S. M. Huse, J. A. Huber, H. G. Morrison, M. L. Sogin, and D. M. Welch, “Accuracy and quality of massively parallel DNA pyrosequencing,” Genome Biology, vol. 8, no. 7, article R143, 2007.
[22]
V. Kunin, A. Engelbrektson, H. Ochman, and P. Hugenholtz, “Wrinkles in the rare biosphere: Pyrosequencing errors can lead to artificial inflation of diversity estimates,” Environmental Microbiology, vol. 12, no. 1, pp. 118–123, 2010.
[23]
A. F. Andersson, M. Lindberg, H. Jakobsson, F. B？ckhed, P. Nyrén, and L. Engstrand, “Comparative analysis of human gut microbiota by barcoded pyrosequencing,” PLoS ONE, vol. 3, no. 7, Article ID e2836, 2008.
[24]
S. E. Dowd, T. R. Callaway, R. D. Wolcott et al., “Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP),” BMC Microbiology, vol. 8, article 125, 2008.
[25]
J. D. Coolon, K. L. Jones, S. Narayanan, and S. M. Wisely, “Microbial ecological response of the intestinal flora of Peromyscus maniculatus and P. leucopus to heavy metal contamination,” Molecular Ecology, vol. 19, supplement 1, pp. 67–80, 2010.
[26]
C. Zhang, M. Zhang, S. Wang et al., “Interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice,” ISME Journal, vol. 4, no. 2, pp. 232–241, 2010.
[27]
S. E. Dowd, Y. Sun, R. D. Wolcott, A. Domingo, and J. A. Carroll, “Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) for microbiome studies: Bacterial diversity in the ileum of newly weaned Salmonella-infected pigs,” Foodborne Pathogens and Disease, vol. 5, no. 4, pp. 459–472, 2008.
[28]
M. J. Roossinck, P. Saha, G. B. Wiley et al., “Ecogenomics: using massively parallel pyrosequencing to understand virus ecology,” Molecular Ecology, vol. 19, supplement 1, pp. 81–88, 2010.
[29]
M. Buée, M. Reich, C. Murat et al., “454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity,” New Phytologist, vol. 184, no. 2, pp. 449–456, 2009.
[30]
R. T. Jones, M. S. Robeson, C. L. Lauber, M. Hamady, R. Knight, and N. Fierer, “A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses,” ISME Journal, vol. 3, no. 4, pp. 442–453, 2009.
[31]
C. L. Lauber, M. Hamady, R. Knight, and N. Fierer, “Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale,” Applied and Environmental Microbiology, vol. 75, no. 15, pp. 5111–5120, 2009.
[32]
S. R. Miller, A. L. Strong, K. L. Jones, and M. C. Ungerer, “Bar-coded pyrosequencing reveals shared bacterial community properties along the temperature gradients of two alkaline hot springs in Yellowstone National Park,” Applied and Environmental Microbiology, vol. 75, no. 13, pp. 4565–4572, 2009.
[33]
R. M. Bowers, C. L. Lauber, C. Wiedinmyer et al., “Characterization of airborne microbial communities at a high-elevation site and their potential to act as atmospheric ice nuclei,” Applied and Environmental Microbiology, vol. 75, no. 15, pp. 5121–5130, 2009.
[34]
S. W. Roh, K. Kim, Y. Nam, H. Chang, E. Park, and J. Bae, “Investigation of archaeal and bacterial diversity in fermented seafood using barcoded pyrosequencing,” ISME Journal, vol. 4, no. 1, pp. 1–16, 2010.
[35]
S. D. Boyd, E. L. Marshall, J. D. Merker et al., “Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing,” Science Translational Medicine, vol. 1, no. 12, article 12ra23, 2009.
[36]
C. Labarca and K. Paigen, “A simple, rapid, and sensitive DNA assay procedure,” Analytical Biochemistry, vol. 102, no. 2, pp. 344–352, 1980.
[37]
F. Vitzthum, G. Geiger, H. Bisswanger, H. Brunner, and J. Bernhagen, “A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR green I and a standard ultraviolet transilluminator gel imaging system,” Analytical Biochemistry, vol. 276, no. 1, pp. 59–64, 1999.
[38]
A. J. Merz and M. So, “Interactions of pathogenic Neisseriae with epithelial cell membranes,” Annual Review of Cell and Developmental Biology, vol. 16, pp. 423–457, 2000.
[39]
H. Wu and P. M. Fives-Taylor, “Molecular strategies for fimbrial expression and assembly,” Critical Reviews in Oral Biology and Medicine, vol. 12, no. 2, pp. 101–115, 2001.
[40]
Y. Shimoji, Y. Ogawa, M. Osaki et al., “Adhesive surface proteins of Erysipelothrix rhusiopathiae bind to polystyrene, fibronectin, and type I and IV collagens,” Journal of Bacteriology, vol. 185, no. 9, pp. 2739–2748, 2003.
[41]
J. Merritt, G. Niu, T. Okinaga, and F. Qi, “Autoaggregation response of Fusobacterium nucleatum,” Applied and Environmental Microbiology, vol. 75, no. 24, pp. 7725–7733, 2009.
[42]
Z. Yu and M. Morrison, “Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis,” Applied and Environmental Microbiology, vol. 70, no. 8, pp. 4800–4806, 2004.
[43]
S. Chakravorty, D. Helb, M. Burday, N. Connell, and D. Alland, “A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria,” Journal of Microbiological Methods, vol. 69, no. 2, pp. 330–339, 2007.
[44]
N. Youssef, C. S. Sheik, L. R. Krumholz, F. Z. Najar, B. A. Roe, and M. S. Elshahed, “Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys,” Applied and Environmental Microbiology, vol. 75, no. 16, pp. 5227–5236, 2009.
[45]
W. Vahjen, R. Pieper, and J. Zentek, “Bar-coded pyrosequencing of 16S rRNA gene amplicons reveals changes in ileal porcine bacterial communities due to high dietary Zinc intake,” Applied and Environmental Microbiology, vol. 76, no. 19, pp. 6689–6691, 2010.
