Development of a Novel Long-Range 16S rRNA Universal Primer Set for Metagenomic Analysis of Gastrointestinal Microbiota in Newborn Infants (original) (raw)

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Research article

Food Microbiology and Biotechnology

Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea

Received: March 12, 2014; Accepted: April 8, 2014

J. Microbiol. Biotechnol. 2014; 24(6): 812-822

Published June 28, 2014 https://doi.org/10.4014/jmb.1403.03032

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new longrange 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.

Keywords

16S rRNA universal primer, fecal DNA extraction, gastrointestinal microbiota, metagenomics, next-generation sequencing

References

1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.
   
2. Andersson AF, Lindberg M, Jakobsson H, Backhed F, Nyren P, Engstrand L. 2008. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS One 3: e2836.
   
3. Benson D A, Cavanaugh M , Clark K , Karsch-Mizrachi I , Lipman DJ, Ostell J, Sayers EW. 2013. GenBank. Nucleic Acids Res. 41: D36-D42.
   
4. Bezirtzoglou E. 1997. The intestinal microflora during the first weeks of life. Anaerobe 3: 173-177.
   
5. Bullen C, Tearle P, Willis A. 1976. Bifidobacteria in the intestinal tract of infants: an in-vivo study. J. Med. Microbiol. 9: 325-333.
   
6. Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. 2014. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42: D633D642.
7. Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, Knight R. 2010. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc. Natl. Acad. Sci. USA 107: 11971-11975.
   
8. Duncan S, Louis P, Flint H. 2007. Cultivable bacterial diversity from the human colon. Lett. Appl. Microbiol. 44:343-350.
   
9. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al. 2005. Diversity of the human intestinal microbial flora. Science 308: 1635-1638.
   
10. Fan W , Huo G , Li X, Yang L, Duan C, Wang T, Chen J. 2013. Diversity of the intestinal microbiota in different patterns of feeding infants by Illumina high-throughput sequencing. World J. Microbiol. Biotechnol. 29: 2365-2372.
    
11. Fleming DW, Cochi SL, MacDonald KL, Brondum J , Hayes PS, Plikaytis BD, et al. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407.
    
12. Gasson MJ. 1983. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplastinduced curing. J. Bacteriol. 154: 1-9.
13. Gill S R, Pop M, DeBoy R T, Eckburg PB, Turnbaugh P J, Samuel BS, et al. 2006. Metagenomic analysis of the human distal gut microbiome. Science 312: 1355-1359.
    
14. Grumbt B, Eck S, Hinrichsen T, Hirv K. 2013. D iagnostic applications of next generation sequencing in immunogenetics and molecular oncology. Transfus. Med. Hemother. 40: 196206.
    
15. Guarner F, Malagelada JR. 2003. Gut flora in health and disease. Lancet 361: 512-519.
    
16. Hoiseth SK, Stocker B. 1981. Aromatic-dependent Salmonella Typhimurium are non-virulent and effective as live vaccines. Nature 291: 238-239.
    
17. Holzapfel WH, Haberer P, Snel J, Schillinger U, Huis in't Veld JH. 1998. Overview of gut flora and probiotics. Int. J. Food Microbiol. 41: 85-101.
    
18. Hooper LV, Macpherson AJ. 2010. Immune adaptations that maintain homeostasis with the intestinal microbiota. Nat. Rev. Immunol. 10: 159-169.
    
19. Huse SM, Dethlefsen L, Huber JA, Welch DM, Relman DA, Sogin ML. 2008. Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing. PLoS Genetics 4: e1000255.
    
20. Koenig JE, Spor A, Scalfone N, Fricker AD, Stombaugh J, Knight R, et al. 2011. Succession of microbial consortia in the developing infant gut microbiome. Proc. Natl. Acad. Sci. USA 108: 4578-4585.
    
21. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948.
    
22. Lee J-H, O’Sullivan DJ. 2010. Genomic insights into bifidobacteria. Microbiol. Mol. Biol. Rev. 74: 378-416.
    
23. Matamoros S, Gras-Leguen C, Le Vacon F, Potel G, de La Cochetiere M-F. 2013. Development of intestinal microbiota in infants and its impact on health. Trends Microbiol. 21: 167173.
    
24. McOrist A L, Jackson M , B ird AR. 2 002. A comparison o f five methods for extraction of bacterial DNA from human faecal samples. J. Microbiol. Methods 50: 131-139.
    
25. Onmus-Leone F, Hang J, Clifford RJ, Yang Y, Riley MC, Kuschner RA, et al. 2013. Enhanced de novo assembly of high throughput pyrosequencing data using whole genome mapping. PloS One 8: e61762.
    
26. Palmer C, Bik EM, DiGiulio DB, Relman DA, and Brown PO. 2007. Development of the human infant intestinal microbiota. PLoS Biol. 5: e177.
    
27. Penders J , Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, et al. 2006. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 118: 511-521.
    
28. Qin J , Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, et al. 2010. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464: 59-65.
    
29. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning. Cold Spring Harbor Laboratory Press, New York.
30. Shokralla S, Spall JL, Gibson JF, Hajibabaei M. 2012. Nextgeneration sequencing technologies for environmental DNA research. Mol. Ecol. 21: 1794-1805.
    
31. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley R E, et al. 2009. A core gut microbiome in obese and lean twins. Nature 457: 480-484.
    
32. Turroni F , Peano C , Pass DA, Foroni E, Severgnini M, Claesson MJ, et al. 2012. Diversity of bifidobacteria within the infant gut microbiota. PLoS One 7: e36957.
    
33. Yoshioka H, Iseki K , Fujita K. 1 983. D evelopment a nd differences of intestinal flora in the neonatal period in breast-fed and bottle-fed infants. Pediatrics 72: 317-321.
34. Yu Z, Morrison M. 2004. Improved extraction of PCRquality community DNA from digesta and fecal samples. Biotechniques 36: 808-813.

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Article

Research article

Development of a Novel Long-Range 16S rRNA Universal Primer Set for Metagenomic Analysis of Gastrointestinal Microbiota in Newborn Infants

Hye-Jin Ku 1 and Ju-Hoon Lee 1*

Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea

Received: March 12, 2014; Accepted: April 8, 2014

Abstract

Metagenomic analysis of the human intestinal microbiota has extended our understanding of
the role of these bacteria in improving human intestinal health; however, a number of reports
have shown that current total fecal DNA extraction methods and 16S rRNA universal primer
sets could affect the species coverage and resolution of these analyses. Here, we improved the
extraction method for total DNA from human fecal samples by optimization of the lysis buffer,
boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new longrange
16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA
product length. This new 16S rRNA primer set was evaluated by comparison with two
previously developed 16S rRNA universal primer sets and showed high species coverage and
resolution. The optimized total fecal DNA extraction method and newly designed long-range
16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of
adult and infant intestinal microbiota with minimization of any bias.

Keywords: 16S rRNA universal primer, fecal DNA extraction, gastrointestinal microbiota, metagenomics, next-generation sequencing

• Abstract

References

1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.
   
2. Andersson AF, Lindberg M, Jakobsson H, Backhed F, Nyren P, Engstrand L. 2008. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS One 3: e2836.
   
3. Benson D A, Cavanaugh M , Clark K , Karsch-Mizrachi I , Lipman DJ, Ostell J, Sayers EW. 2013. GenBank. Nucleic Acids Res. 41: D36-D42.
   
4. Bezirtzoglou E. 1997. The intestinal microflora during the first weeks of life. Anaerobe 3: 173-177.
   
5. Bullen C, Tearle P, Willis A. 1976. Bifidobacteria in the intestinal tract of infants: an in-vivo study. J. Med. Microbiol. 9: 325-333.
   
6. Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. 2014. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42: D633D642.
7. Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, Knight R. 2010. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc. Natl. Acad. Sci. USA 107: 11971-11975.
   
8. Duncan S, Louis P, Flint H. 2007. Cultivable bacterial diversity from the human colon. Lett. Appl. Microbiol. 44:343-350.
   
9. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al. 2005. Diversity of the human intestinal microbial flora. Science 308: 1635-1638.
   
10. Fan W , Huo G , Li X, Yang L, Duan C, Wang T, Chen J. 2013. Diversity of the intestinal microbiota in different patterns of feeding infants by Illumina high-throughput sequencing. World J. Microbiol. Biotechnol. 29: 2365-2372.
    
11. Fleming DW, Cochi SL, MacDonald KL, Brondum J , Hayes PS, Plikaytis BD, et al. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407.
    
12. Gasson MJ. 1983. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplastinduced curing. J. Bacteriol. 154: 1-9.
13. Gill S R, Pop M, DeBoy R T, Eckburg PB, Turnbaugh P J, Samuel BS, et al. 2006. Metagenomic analysis of the human distal gut microbiome. Science 312: 1355-1359.
    
14. Grumbt B, Eck S, Hinrichsen T, Hirv K. 2013. D iagnostic applications of next generation sequencing in immunogenetics and molecular oncology. Transfus. Med. Hemother. 40: 196206.
    
15. Guarner F, Malagelada JR. 2003. Gut flora in health and disease. Lancet 361: 512-519.
    
16. Hoiseth SK, Stocker B. 1981. Aromatic-dependent Salmonella Typhimurium are non-virulent and effective as live vaccines. Nature 291: 238-239.
    
17. Holzapfel WH, Haberer P, Snel J, Schillinger U, Huis in't Veld JH. 1998. Overview of gut flora and probiotics. Int. J. Food Microbiol. 41: 85-101.
    
18. Hooper LV, Macpherson AJ. 2010. Immune adaptations that maintain homeostasis with the intestinal microbiota. Nat. Rev. Immunol. 10: 159-169.
    
19. Huse SM, Dethlefsen L, Huber JA, Welch DM, Relman DA, Sogin ML. 2008. Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing. PLoS Genetics 4: e1000255.
    
20. Koenig JE, Spor A, Scalfone N, Fricker AD, Stombaugh J, Knight R, et al. 2011. Succession of microbial consortia in the developing infant gut microbiome. Proc. Natl. Acad. Sci. USA 108: 4578-4585.
    
21. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948.
    
22. Lee J-H, O’Sullivan DJ. 2010. Genomic insights into bifidobacteria. Microbiol. Mol. Biol. Rev. 74: 378-416.
    
23. Matamoros S, Gras-Leguen C, Le Vacon F, Potel G, de La Cochetiere M-F. 2013. Development of intestinal microbiota in infants and its impact on health. Trends Microbiol. 21: 167173.
    
24. McOrist A L, Jackson M , B ird AR. 2 002. A comparison o f five methods for extraction of bacterial DNA from human faecal samples. J. Microbiol. Methods 50: 131-139.
    
25. Onmus-Leone F, Hang J, Clifford RJ, Yang Y, Riley MC, Kuschner RA, et al. 2013. Enhanced de novo assembly of high throughput pyrosequencing data using whole genome mapping. PloS One 8: e61762.
    
26. Palmer C, Bik EM, DiGiulio DB, Relman DA, and Brown PO. 2007. Development of the human infant intestinal microbiota. PLoS Biol. 5: e177.
    
27. Penders J , Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, et al. 2006. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 118: 511-521.
    
28. Qin J , Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, et al. 2010. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464: 59-65.
    
29. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning. Cold Spring Harbor Laboratory Press, New York.
30. Shokralla S, Spall JL, Gibson JF, Hajibabaei M. 2012. Nextgeneration sequencing technologies for environmental DNA research. Mol. Ecol. 21: 1794-1805.
    
31. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley R E, et al. 2009. A core gut microbiome in obese and lean twins. Nature 457: 480-484.
    
32. Turroni F , Peano C , Pass DA, Foroni E, Severgnini M, Claesson MJ, et al. 2012. Diversity of bifidobacteria within the infant gut microbiota. PLoS One 7: e36957.
    
33. Yoshioka H, Iseki K , Fujita K. 1 983. D evelopment a nd differences of intestinal flora in the neonatal period in breast-fed and bottle-fed infants. Pediatrics 72: 317-321.
34. Yu Z, Morrison M. 2004. Improved extraction of PCRquality community DNA from digesta and fecal samples. Biotechniques 36: 808-813.