Recent Advancements in Intestinal Microbiota Analyses: A Review for Non-Microbiologists

Abstract

Microbial constituents naturally inhabiting the gastrointestinal tract may influence the homeostasis of the gut environment. The presence or overabundance of some bacterial taxa has been reported to be associated with complex diseases, and the metabolites of certain bacteria may contribute to diverse disorders by influencing signaling pathways. Therefore, the study of gut microbial population has emerged as a crucial field and a new potential area of clinical significance. Advances in the methods of microbiota analysis have shed light upon the details including species diversity, microfloral activities as well as the entire gut microbiota. Nevertheless, comprehensive reviews on this subject are still limited. For elucidating the appropriate selection strategy of the methods to address a particular research question, we comprehensively reviewed the continuously improving technologies, classical to newly developed, and dissected their relative advantages and drawbacks. In addition, aiming at the rapidly advancing next-generation sequencing, we enumerated the improvements in mainstream platforms and made the horizontal and vertical comparison among them. Additionally, we demonstrated the four main -omics methods, which may provide further mechanistic insights into the role of microbiota, to propel phylotyping analysis to functional analysis.

Keywords

gut microbiota / methods / analysis / advantages / drawbacks

References

[1] SteinhoffU. Who controls the crowd? New findings and old questions about the intestinal microflora. Immunol Lett, 2005, 99(1): 12-16

[2] Human Microbiome ProjectC.. A framework for human microbiome research. Nature, 2012, 486(7402): 215-221

[3] Human Microbiome Project C.. Structure, function and diversity of the healthy human microbiome. Nature, 2012, 486(7402): 207-214

[4] Keates-BaleeiroJ, MooreP, KoyamaT, et al.. Incidence and outcome of idiopathic pneumonia syndrome in pediatric stem cell transplant recipients. Bone Marrow Transplant, 2006, 38(4): 285-289

[5] SommerF, BackhedF. The gut microbiota—masters of host development and physiology. Nat Rev Microbiol, 2013, 11(4): 227-238

[6] ClementeJC, UrsellLK, ParfreyLW, et al.. The impact of the gut microbiota on human health: an integrative view. Cell, 2012, 148(6): 1258-1270

[7] KhannaS, ToshPK. A clinician’s primer on the role of the microbiome in human health and disease. Mayo Clin Proc, 2014, 89(1): 107-114

[8] GuarnerF, MalageladaJR. Gut flora in health and disease. Lancet, 2003, 361(9356): 512-519

[9] DinanTG, CryanJF. The impact of gut microbiota on brain and behaviour: implications for psychiatry. Curr Opin Clin Nutr Metab Care, 2015, 18(6): 552-558

[10] QinJ, LiY, CaiZ, et al.. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature, 2012, 490(7418): 55-60

[11] PerryRJ, PengL, BarryNA, et al.. Acetate mediates a microbiome-brain-beta-cell axis to promote metabolic syndrome. Nature, 2016, 534(7606): 213-217

[12] BoulangeCL, NevesAL, ChillouxJ, et al.. Impact of the gut microbiota on inflammation, obesity, and metabolic disease. Genome Med, 2016, 8(1): 42

[13] GerritsenJ, SmidtH, RijkersGT, et al.. Intestinal microbiota in human health and disease: the impact of probiotics. Genes Nutr, 2011, 6(3): 209-240

[14] HsiaoEY, McBrideSW, HsienS, et al.. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell, 2013, 155(7): 1451-1463

[15] HillJM, BhattacharjeeS, PogueAI, et al.. The gastrointestinal tract microbiome and potential link to Alzheimer’s disease. Front Neurol, 2014, 5: 43

[16] FraherMH, O’ToolePW, QuigleyEM. Techniques used to characterize the gut microbiota: a guide for the clinician. Nat Rev Gastroenterol Hepatol, 2012, 9(6): 312-322

[17] KuczynskiJ, LauberCL, WaltersWA, et al.. Experimental and analytical tools for studying the human microbiome. Nat Rev Genet, 2012, 13(1): 47-58

[18] MaronPA, RanjardL, MougelC, et al.. Metaproteomics: a new approach for studying functional microbial ecology. Microb Ecol, 2007, 53(3): 486-493

[19] TurnbaughPJ, HamadyM, YatsunenkoT, et al.. A core gut microbiome in obese and lean twins. Nature, 2009, 457(7228): 480-484

[20] FinegoldSM, AtteberyHR, SutterVL. Effect of diet on human fecal flora: comparison of Japanese and American diets. Am J Clin Nutr, 1974, 27(12): 1456-1469

[21] GosslingJ, SlackJM. Predominant gram-positive bacteria in human feces: numbers, variety, and persistence. Infect Immun, 1974, 9(4): 719-729

[22] MooreWE, HoldemanLV. Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbiol, 1974, 27(5): 961-979

[23] ZenglerK, ToledoG, RappeM, et al.. Cultivating the uncultured. Proc Natl Acad Sci USA, 2002, 99(24): 15681-15686

[24] ZenglerK, WalcherM, ClarkG, et al.. High-throughput cultivation of microorganisms using microcapsules. Methods Enzymol, 2005, 397: 124-130

[25] BollmannA, LewisK, EpsteinSS. Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl Environ Microbiol, 2007, 73(20): 6386-6390

[26] KaeberleinT, LewisK, EpsteinSS. Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science, 2002, 296(5570): 1127-1129

[27] InghamCJ, SprenkelsA, BomerJ, et al.. The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms. Proc Natl Acad Sci USA, 2007, 104(46): 18217-18222

[28] McDonaldJA, SchroeterK, FuentesS, et al.. Evaluation of microbial community reproducibility, stability and composition in a human distal gut chemostat model. J Microbiol Methods, 2013, 95(2): 167-174

[29] MaL, KimJ, HatzenpichlerR, et al.. Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project’s Most Wanted taxa. Proc Natl Acad Sci USA, 2014, 111(27): 9768-9773

[30] EckburgPB, BikEM, BernsteinCN, et al.. Diversity of the human intestinal microbial flora. Science, 2005, 308(5728): 1635-1638

[31] LagierJC, ArmougomF, MillionM, et al.. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect, 2012, 18(12): 1185-1193

[32] LauJT, WhelanFJ, HerathI, et al.. Capturing the diversity of the human gut microbiota through culture-enriched molecular profiling. Genome Med, 2016, 8(1): 72

[33] LagierJC, MillionM, HugonP, et al.. Human gut microbiota: repertoire and variations. Front Cell Infect Microbiol, 2012, 2: 136

[34] ClarridgeJ 3rd. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev, 2004, 17(4): 840-862

[35] KolbertCP, PersingDH. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr Opin Microbiol, 1999, 2(3): 299-305

[36] LuT, StrootPG, OertherDB. Reverse transcription of 16S rRNA to monitor ribosome-synthesizing bacterial populations in the environment. Appl Environ Microbiol, 2009, 75(13): 4589-4598

[37] OlsenGJ, LaneDJ, GiovannoniSJ, et al.. Microbial ecology and evolution: a ribosomal RNA approach. Annu Rev Microbiol, 1986, 40: 337-365

[38] von WintzingerodeF, GobelUB, StackebrandtE. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev, 1997, 21(3): 213-229

[39] KielerIN, MolbakL, HansenLL, et al.. Overweight and the feline gut microbiome -a pilot study. J Anim Physiol Anim Nutr (Berl), 2016, 100(3): 478-484

[40] Radilla-VazquezRB, Parra-RojasI, Martinez-HernandezNE, et al.. Gut Microbiota and Metabolic Endotoxemia in Young Obese Mexican Subjects. Obes Facts, 2016, 9(1): 1-11

[41] DubocH, RajcaS, RainteauD, et al.. Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases. Gut, 2013, 62(4): 531-539

[42] PonnusamyK, ChoiJN, KimJ, et al.. Microbial community and metabolomic comparison of irritable bowel syndrome faeces. J Med Microbiol, 2011, 60(6): 817-827

[43] ObermajerT, LipoglavsekL, TompaG, et al.. Colostrum of healthy Slovenian mothers: microbiota composition and bacteriocin gene prevalence. PLoS One, 2014, 10(4): e0123324

[44] RiddleMS, ConnorBA. The Traveling Microbiome. Curr Infect Dis Rep, 2016, 18(9): 29

[45] FischerSG, LermanLS. DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci USA, 1983, 80(6): 1579-1583

[46] RamnaniP, CostabileA, BustilloAG, et al.. Arandomised, double-blind, cross-over study investigating the prebiotic effect of agave fructans in healthy human subjects. J Nutr Sci, 2015, 4: e10

[47] FischerSG, LermanLS. Separation of random fragments of DNA according to properties of their sequences. Proc Natl Acad Sci USA, 1980, 77(8): 4420-4424

[48] LiuWT, MarshTL, ChengH, et al.. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol, 1997, 63(11): 4516-4522

[49] DaveM, JohnsonLA, WalkST, et al.. A randomised trial of sheathed versus standard forceps for obtaining uncontaminated biopsy specimens of microbiota from the terminal ileum. Gut, 2011, 60(8): 1043-1049

[50] BrooksJP, EdwardsDJ, HarwichM Jr, et al.. The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies. BMC Microbiol, 2015, 15: 66

[51] HayashiH, SakamotoM, KitaharaM, et al.. Molecular analysis of fecal microbiota in elderly individuals using 16S rDNA library and T-RFLP. Microbiol Immunol, 2003, 47(8): 557-570

[52] MatsumotoM, SakamotoM, HayashiH, et al.. Novel phylogenetic assignment database for terminalrestriction fragment length polymorphism analysis of human colonic microbiota. J Microbiol Methods, 2005, 61(3): 305-319

[53] Mark WelchJL, RossettiBJ, RiekenCW, et al.. Biogeography of a human oral microbiome at the micron scale. Proc Natl Acad Sci USA, 2016, 113(6): E791-800

[54] SmithPA. The tantalizing links between gut microbes and the brain. Nature, 2015, 526(7573): 312-314

[55] IngberDE. Reverse Engineering Human Pathophysiology with Organs-on-Chips. Cell, 2016, 164(6): 1105-1109

[56] DewulfEM, CaniPD, ClausSP, et al.. Insight into the prebiotic concept: lessons from an exploratory, double blind intervention study with inulin-type fructans in obese women. Gut, 2013, 62(8): 1112-1121

[57] MichailS, DurbinM, TurnerD, et al.. Alterations in the gut microbiome of children with severe ulcerative colitis. Inflamm Bowel Dis, 2012, 18(10): 1799-1808

[58] SangerF, NicklenS, CoulsonAR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA, 1977, 74(12): 5463-5467

[59] CollinsFS, MorganM, PatrinosA. The Human Genome Project: lessons from large-scale biology. Science, 2003, 300(5617): 286-290

[60] HallN. Advanced sequencing technologies and their wider impact in microbiology. J Exp Biol, 2007, 210(9): 1518-1525

[61] de MagalhaesJP, FinchCE, JanssensG. Next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solutions. Ageing Res Rev, 2010, 9(3): 315-323

[62] JefferyIB, O’ToolePW, OhmanL, et al.. An irritable bowel syndrome subtype defined by species-specific alterations in faecal microbiota. Gut, 2012, 61(7): 997-1006

[63] TangJ, IlievID, BrownJ, et al.. Mycobiome: Approaches to analysis of intestinal fungi. J Immunol Methods, 2015, 421: 112-121

[64] GoodwinS, McPhersonJD, McCombieWR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet, 2016, 17(6): 333-351

[65] YatsunenkoT, ReyFE, ManaryMJ, et al.. Human gut microbiome viewed across age and geography. Nature, 2012, 486(7402): 222-227

[66] JaeggiT, KortmanGA, MorettiD, et al.. Iron fortification adversely affects the gut microbiome, increases pathogen abundance and induces intestinal inflammation in Kenyan infants. Gut, 2015, 64(5): 731-742

[67] OlivaresM, NeefA, CastillejoG, et al.. The HLADQ2 genotype selects for early intestinal microbiota composition in infants at high risk of developing coeliac disease. Gut, 2015, 64(3): 406-417

[68] LarsenN, VogensenFK, van den BergFW, et al.. Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PLoS One, 2010, 5(2): e9085

[69] AnderssonAF, LindbergM, JakobssonH, et al.. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS One, 2008, 3(7): e2836

[70] TurnbaughPJ, QuinceC, FaithJJ, et al.. Organismal, genetic, and transcriptional variation in the deeply sequenced gut microbiomes of identical twins. Proc Natl Acad Sci USA, 2010, 107(16): 7503-7508

[71] POC, Aguirre de CarcerD, JonesM, et al.. The effects from DNA extraction methods on the evaluation of microbial diversity associated with human colonic tissue. Microb Ecol, 2011, 61(2): 353-362

[72] YuanS, CohenDB, RavelJ, et al.. Evaluation of methods for the extraction and purification of DNA from the human microbiome. PLoS One, 2012, 7(3): e33865

[73] GillesA, MegleczE, PechN, et al.. Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing. BMC Genomics, 2011, 12: 245

[74] LuoC, TsementziD, KyrpidesN, et al.. Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample. PLoS One, 2012, 7(2): e30087

[75] KonstantinidisKT, TiedjeJM. Prokaryotic taxonomy and phylogeny in the genomic era: advancements and challenges ahead. Curr Opin Microbiol, 2007, 10(5): 504-509

[76] Sanschagrin S, Yergeau E. Next-generation sequencing of 16S ribosomal RNA gene amplicons. J Vis Exp, 2014(90). doi:10.3791/51709

[77] LagierJC, HugonP, KhelaifiaS, et al.. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol Rev, 2015, 28(1): 237-264

[78] XiongW, AbrahamPE, LiZ, et al.. Microbial metaproteomics for characterizing the range of metabolic functions and activities of human gut microbiota. Proteomics, 2015, 15(20): 3424-3438

[79] ReigstadCS, KashyapPC. Beyond phylotyping: understanding the impact of gut microbiota on host biology. Neurogastroenterol Motil, 2013, 25(5): 358-372

[80] WangWL, XuSY, RenZG, et al.. Application of metagenomics in the human gut microbiome. World J Gastroenterol, 2015, 21(3): 803-814

[81] StadenR. A strategy of DNA sequencing employing computer programs. Nucleic Acids Res, 1979, 6(7): 2601-2610

[82] PoinarHN, SchwarzC, QiJ, et al.. Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Science, 2006, 311(5759): 392-394

[83] MetzkerML. Sequencing technologies -the next generation. Nat Rev Genet, 2010, 11(1): 31-46

[84] SekirovI, RussellSL, AntunesLC, et al.. Gut microbiota in health and disease. Physiol Rev, 2010, 90(3): 859-904

[85] RanjanR, RaniA, MetwallyA, et al.. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem Biophys Res Commun, 2016, 469(4): 967-977

[86] De VosWM. Mining the microbes—the human microbiome as model. Microb Biotechnol, 2009, 2(2): 153-154

[87] KarlssonFH, TremaroliV, NookaewI, et al.. Gut metagenome in European women with normal, impaired and diabetic glucose control. Nature, 2013, 498(7452): 99-103

[88] KarlssonFH, FakF, NookaewI, et al.. Symptomatic atherosclerosis is associated with an altered gut metagenome. Nat Commun, 2012, 3: 1245

[89] ZhangX, ZhangD, JiaH, et al.. The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment. Nat Med, 2015, 21(8): 895-905

[90] MaJ, PrinceA, AagaardKM. Use of whole genome shotgun metagenomics: a practical guide for the microbiome-minded physician scientist. Semin Reprod Med, 2014, 32(1): 5-13

[91] WangZ, GersteinM, SnyderM. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet, 2009, 10(1): 57-63

[92] WestermannAJ, GorskiSA, VogelJ. Dual RNA-seq of pathogen and host. Nat Rev Microbiol, 2012, 10(9): 618-630

[93] BashiardesS, Zilberman-SchapiraG, ElinavE. Use of Metatranscriptomics in Microbiome Research. Bioinform Biol Insights, 2016, 10: 19-25

[94] FranzosaEA, MorganXC, SegataN, et al.. Relating the metatranscriptome and metagenome of the human gut. Proc Natl Acad Sci USA, 2014, 111(22): E2329-2338

[95] GosalbesMJ, DurbanA, PignatelliM, et al.. Metatranscriptomic approach to analyze the functional human gut microbiota. PLoS One, 2011, 6(3): e17447

[96] PeanoC, PietrelliA, ConsolandiC, et al.. An efficient rRNA removal method for RNA sequencing in GC-rich bacteria. Microb Inform Exp, 2013, 3(1): 1

[97] SimonC, DanielR. Metagenomic analyses: past and future trends. Appl Environ Microbiol, 2011, 77(4): 1153-1161

[98] AlvesP, ArnoldRJ, NovotnyMV, et al.. Advancement in protein inference from shotgun proteomics using peptide detectability. Pac Symp Biocomput, 2007409-420

[99] HuL, YeM, JiangX, et al.. Advances in hyphenated analytical techniques for shotgun proteome and peptidome analysis—a review. Anal Chim Acta, 2007, 598(2): 193-204

[100] JordanKW, NordenstamJ, LauwersGY, et al.. Metabolomic characterization of human rectal adenocarcinoma with intact tissue magnetic resonance spectroscopy. Dis Colon Rectum, 2009, 52(3): 520-525

[101] AldridgeBB, RheeKY. Microbial metabolomics: innovation, application, insight. Curr Opin Microbiol, 2014, 19: 90-96

[102] VernocchiP, Del ChiericoF, PutignaniL. Gut Microbiota Profiling: Metabolomics Based Approach to Unravel Compounds Affecting Human Health. Front Microbiol, 2016, 7: 1144

[103] Aguiar-PulidoV, HuangW, Suarez-UlloaV, et al.. Metagenomics, Metatranscriptomics, and Metabolomics Approaches for Microbiome Analysis. Evol Bioinform Online, 2016, 12(1): 5-16