Critical issues in mycobiota analysis

Frontiers in Microbiology

Volumn 8, Issue FEB, 2017, Pages

  Halwachs, Bettina ^a,b   Madhusudhan, Nandhitha ^a   Krause, Robert ^a   Nilsson, R H ^c   Moissl Eichinger, Christine ^a,b   Högenauer, Christoph ^a,b   Thallinger, Gerhard G ^b,d   Gorkiewicz, Gregor ^a,b  

^a MEDICAL UNIVERSITY OF GRAZ   (Austria)

^b BIOTECHMED GRAZ   (Austria)

^c UNIVERSITY OF GOTHENBURG   (Sweden)

^d GRAZ UNIVERSITY OF TECHNOLOGY   (Austria)

Author keywords

16S rRNA gene; DNA isolation; Formalin fixed paraffin embedded tissue (FFPE); Internal transcribed spacer (ITS); Microbiota; Multiple sequence alignment (MSA); Mycobiota; OTU picking

Indexed keywords

INTERNAL TRANSCRIBED SPACER; INTERNAL TRANSCRIBED SPACER 1; INTERNAL TRANSCRIBED SPACER 2; RNA 16S;

AMPLICON; ARTICLE; BIOINFORMATICS; COMPUTER MODEL; DNA BARCODING; DNA EXTRACTION; FUNGAL COMMUNITY; FUNGAL FLORA; HUMAN; MICROBIAL COMMUNITY; NEXT GENERATION SEQUENCING; NONHUMAN; OPERON; PHYLOGENETIC TREE; PHYLOGENY; SEQUENCE ALIGNMENT; TAXONOMY;

EID: 85014580673     PISSN: None     EISSN: 1664302X     Source Type: Journal    
DOI: 10.3389/fmicb.2017.00180     Document Type: Article

References (84)

1. Albanese, D., Fontana, P., De Filippo, C., Cavalieri, D., and Donati, C. (2015). MICCA: a complete and accurate software for taxonomic profiling of metagenomic data. Sci. Rep. 5:9743. doi: 10.1038/srep09743.
2. Bazzicalupo, A. L., Bálint, M., and Schmitt, I. (2013). Comparison of ITS1 and ITS2 rDNA in 454 sequencing of hyperdiverse fungal communities. Fungal Ecol. 6, 102-109. doi: 10.1016/j.funeco.2012.09.003.
3. Belkaid, Y., and Naik, S. (2013). Compartmentalized and systemic control of tissue immunity by commensals. Nat. Immunol. 14, 646-653. doi: 10.1038/ni.2604.
4. Bellemain, E., Carlsen, T., Brochmann, C., Coissac, E., Taberlet, P., and Kauserud, H. (2010). ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. BMC Microbiol. 10:189. doi: 10.1186/1471-2180-10-189.
5. Bengtsson-Palme, J., Ryberg, M., Hartmann, M., Branco, S., Wang, Z., Godhe, A., et al. (2013). Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data. Methods Ecol. Evol. 4, 914-919. doi: 10.1111/2041-210x.12073.
6. Blaalid, R., Kumar, S., Nilsson, R. H., Abarenkov, K., Kirk, P. M., and Kauserud, H. (2013). ITS1 versus ITS2 as DNA metabarcodes for fungi. Mol. Ecol. Resour. 13, 218-224. doi: 10.1111/1755-0998.12065.
7. Blackwell, M. (2011). The fungi: 1, 2, 3.5.1 million species? Am. J. Bot. 98, 426-438. doi: 10.3732/ajb.1000298.
8. Bokulich, N. A., Subramanian, S., Faith, J. J., Gevers, D., Gordon, J. I., Knight, R., et al. (2013). Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat. Methods 10, 57-59. doi: 10.1038/nmeth.2276.
9. Bonin, S., and Stanta, G. (2013). Nucleic acid extraction methods from fixed and paraffin-embedded tissues in cancer diagnostics. Expert Rev. Mol. Diagn. 13, 271-282. doi: 10.1586/erm.13.14.
10. Brown, G. D., Denning, D. W., Gow, N. A., Levitz, S. M., Netea, M. G., and White, T. C. (2012). Hidden killers: human fungal infections. Sci. Transl. Med. 4:165rv13. doi: 10.1126/scitranslmed.3004404.
11. 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. doi: 10.1038/nmeth.f.303.
12. Charlson, E. S., Diamond, J. M., Bittinger, K., Fitzgerald, A. S., Yadav, A., Haas, A. R., et al. (2012). Lung-enriched organisms and aberrant bacterial and fungal respiratory microbiota after lung transplant. Am. J. Respir. Crit. Care Med. 186, 536-545. doi: 10.1164/rccm.201204-0693OC.
13. Coura, R., Prolla, J. C., Meurer, L., and Ashton-Prolla, P. (2005). An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue. J. Clin. Pathol. 58, 894-895. doi: 10.1136/jcp.2004.021352.
14. Cui, L., Morris, A., and Ghedin, E. (2013). The human mycobiome in health and disease. Genome Med. 5:63. doi: 10.1186/gm467.
15. de Boer, R., Peters, R., Gierveld, S., Schuurman, T., Kooistra-Smid, M., and Savelkoul, P. (2010). Improved detection of microbial DNA after bead-beating before DNA isolation. J. Microbiol. Methods 80, 209-211. doi: 10.1016/j.mimet.2009.11.009.
16. Diezmann, S., Cox, C. J., Schonian, G., Vilgalys, R. J., and Mitchell, T. G. (2004). Phylogeny and evolution of medical species of Candida and related taxa: a multigenic analysis. J. Clin. Microbiol. 42, 5624-5635. doi: 10.1128/JCM.42.12.5624-5635.2004.
17. Edgar, R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460-2461. doi: 10.1093/bioinformatics/btq461.
18. Edgar, R. C. (2013). UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996-998. doi: 10.1038/nmeth.2604.
19. Findley, K., Oh, J., Yang, J., Conlan, S., Deming, C., Meyer, J. A., et al. (2013). Topographic diversity of fungal and bacterial communities in human skin. Nature 498, 367-370. doi: 10.1038/nature12171.
20. Flury, B., Weisser, M., Prince, S. S., Bubendorf, L., Battegay, M., Frei, R., et al. (2014). Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? BMC Infect. Dis. 14:692. doi: 10.1186/s12879-014-0692-z.
21. Flynn, J. M., Brown, E. A., Chain, F. J., MacIsaac, H. J., and Cristescu, M. E. (2015). Toward accurate molecular identification of species in complex environmental samples: testing the performance of sequence filtering and clustering methods. Ecol. Evol. 5, 2252-2266. doi: 10.1002/ece3.1497.
22. Gardes, M., and Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol. Ecol. 2, 113-118. doi: 10.1111/j.1365-294X.1993.tb00005.x.
23. Ghannoum, M. A., Jurevic, R. J., Mukherjee, P. K., Cui, F., Sikaroodi, M., Naqvi, A., et al. (2010). Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog. 6:e1000713. doi: 10.1371/journal.ppat.1000713.
24. Goldschmidt, P., Degorge, S., Merabet, L., and Chaumeil, C. (2014). Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious agents. PLoS ONE 9:e94886. doi: 10.1371/journal.pone.0094886.
25. Gorkiewicz, G., Thallinger, G. G., Trajanoski, S., Lackner, S., Stocker, G., Hinterleitner, T., et al. (2013). Alterations in the colonic microbiota in response to osmotic diarrhea. PLoS ONE 8:e55817. doi: 10.1371/journal.pone.0055817.
26. Gosiewski, T., Salamon, D., Szopa, M., Sroka, A., Malecki, M. T., and Bulanda, M. (2014). Quantitative evaluation of fungi of the genus Candida in the feces of adult patients with type 1 and 2 diabetes-a pilot study. Gut Pathog. 6:43. doi: 10.1186/s13099-014-0043-z.
27. Gouba, N., and Drancourt, M. (2015). Digestive tract mycobiota: a source of infection. Med. Mal. Infect. 45, 9-16. doi: 10.1016/j.medmal.2015.01.007.
28. Hallen-Adams, H. E., Kachman, S. D., Kim, J., Legge, R. M., and Martínez, I. (2015). Fungi inhabiting the healthy human gastrointestinal tract: a diverse and dynamic community. Fungal Ecol. 15, 9-17. doi: 10.1016/j.funeco.2015.01.006.
29. Hartmann, M., Howes, C. G., Abarenkov, K., Mohn, W. W., and Nilsson, R. H. (2010). V-Xtractor: an open-source, high-throughput software tool to identify and extract hypervariable regions of small subunit (16S/18S) ribosomal RNA gene sequences. J. Microbiol. Methods 83, 250-253. doi: 10.1016/j.mimet.2010.08.008.
30. Hawksworth, D. (1991). The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol. Res. 95, 641-655. doi: 10.1016/S0953-7562(09)80810-1.
31. Hoarau, G., Mukherjee, P. K., Gower-Rousseau, C., Hager, C., Chandra, J., Retuerto, M. A., et al. (2016). Bacteriome and mycobiome interactions underscore microbial dysbiosis in familial Crohn's disease. MBio 7, e01250-e01216. doi: 10.1128/mBio.01250-16.
32. Hunt, J., Boddy, L., Randerson, P. F., and Rogers, H. J. (2004). An evaluation of 18S rDNA approaches for the study of fungal diversity in grassland soils. Microb. Ecol. 47, 385-395. doi: 10.1007/s00248-003-2018-3.
33. Ihrmark, K., Bödeker, I. T., Cruz-Martinez, K., Friberg, H., Kubartova, A., Schenck, J., et al. (2012). New primers to amplify the fungal ITS2 region-evaluation by 454-sequencing of artificial and natural communities. FEMS Microbiol. Ecol. 82, 666-677. doi: 10.1111/j.1574-6941.2012.01437.x.
34. Iliev, I. D., Funari, V. A., Taylor, K. D., Nguyen, Q., Reyes, C. N., Strom, S. P., et al. (2012). Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science 336, 1314-1317. doi: 10.1126/science.1221789.
35. Irinyi, L., Serena, C., Garcia-Hermoso, D., Arabatzis, M., Desnos-Ollivier, M., Vu, D., et al. (2015). International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database-the quality controlled standard tool for routine identification of human and animal pathogenic fungi. Med. Mycol. 53, 313-337. doi: 10.1093/mmy/myv008.
36. Kappe, R., Fauser, C., Okeke, C. N., and Maiwald, M. (1996). Universal fungus-specific primer systems and group-specific hybridization oligonucleotides for 18S rDNA. Mycoses 39, 25-30. doi: 10.1111/j.1439-0507.1996.tb00079.x.
37. Kim, S. H., Clark, S. T., Surendra, A., Copeland, J. K., Wang, P. W., Ammar, R., et al. (2015). Global analysis of the fungal microbiome in cystic fibrosis patients reveals loss of function of the transcriptional repressor nrg1 as a mechanism of pathogen adaptation. PLoS Pathog. 11:e1005308. doi: 10.1371/journal.ppat.1005308.
38. Kocjan, B. J., Hosnjak, L., and Poljak, M. (2015). Commercially available kits for manual and automatic extraction of nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues. Acta Dermatovenerol. Alp. Pannonica. Adriat. 24, 47-53. doi: 10.15570/actaapa.2015.12.
39. Kõljalg, U., Nilsson, R. H., Abarenkov, K., Tedersoo, L., Taylor, A. F. S., Bahram, M., et al. (2013). Towards a unified paradigm for sequence-based identification of fungi. Mol. Ecol. 22, 5271-5277. doi: 10.1111/mec.12481.
40. Kopylova, E., Navas-Molina, J. A., Mercier, C. ã., Xu, Z. Z., Mahé, F., He, Y., et al. (2016). Open-source sequence clustering methods improve the state of the art. mSystems 1, e00003-e00015. doi: 10.1128/mSystems.00003-15.
41. Krause, R., Halwachs, B., Thallinger, G. G., Klymiuk, I., Gorkiewics, G., Hoenigl, M., et al. (2016). Characterisation of Candida within the mycobiome/microbiome of the lower respiratory tract of ICU patients. PLoS ONE 11:e0155033. doi: 10.1371/journal.pone.0155033.
42. Kuczynski, J., Lauber, C. L., Walters, W. A., Parfrey, L. W., Clemente, J. C., Gevers, D., et al. (2012). Experimental and analytical tools for studying the human microbiome. Nat. Rev. Genet. 13, 47-58. doi: 10.1038/nrg3129.
43. Lindahl, B. D., Nilsson, R. H., Tedersoo, L., Abarenkov, K., Carlsen, T., Kjoller, R., et al. (2013). Fungal community analysis by high-throughput sequencing of amplified markers-a user's guide. New Phytol. 199, 288-299. doi: 10.1111/nph.12243.
44. Liu, K. L., Porras-Alfaro, A., Kuske, C. R., Eichorst, S. A., and Xie, G. (2012). Accurate, rapid taxonomic classification of fungal large-subunit rRNA genes. Appl. Environ. Microbiol. 78, 1523-1533. doi: 10.1128/AEM.06826-11.
45. Lozupone, C., Lladser, M. E., Knights, D., Stombaugh, J., and Knight, R. (2011). UniFrac: an effective distance metric for microbial community comparison. ISME J. 5, 169-172. doi: 10.1038/ismej.2010.133.
46. Mahé, F., Rognes, T., Quince, C., de Vargas, C., and Dunthorn, M. (2014). Swarm: robust and fast clustering method for amplicon-based studies. PeerJ 2:e593. doi: 10.7717/peerj.593.
47. Mahé, S., Duhamel, M., Le Calvez, T., Guillot, L., Sarbu, L., Bretaudeau, A., et al. (2012). PHYMYCO-DB: a curated database for analyses of fungal diversity and evolution. PLoS ONE 7:e43117. doi: 10.1371/journal.pone.0043117.
48. Martin, K. J., and Rygiewicz, P. T. (2005). Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol. 5:28. doi: 10.1186/1471-2180-5-28.
49. McLaughlin, D. J., Hibbett, D. S., Lutzoni, F., Spatafora, J. W., and Vilgalys, R. (2009). The search for the fungal tree of life. Trends Microbiol. 17, 488-497. doi: 10.1016/j.tim.2009.08.001.
50. Mello, A., Napoli, C., Murat, C., Morin, E., Marceddu, G., and Bonfante, P. (2011). ITS-1 versus ITS-2 pyrosequencing: a comparison of fungal populations in truffle grounds. Mycologia 103, 1184-1193. doi: 10.3852/11-027.
51. Muñoz-Cadavid, C., Rudd, S., Zaki, S. R., Patel, M., Moser, S. A., Brandt, M. E., et al. (2010). Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR. J. Clin. Microbiol. 48, 2147-2153. doi: 10.1128/JCM.00459-10.
52. Nilsson, R. H., Hyde, K., Pawlowska, J., Ryberg, M., Tedersoo, L., Aas, A. B., et al. (2014). Improving ITS sequence data for identification of plant pathogenic fungi. Fungal Divers. 67, 11-19. doi: 10.1007/s13225-014-0291-8.
53. O'Donnell, K., Sutton, D. A., Rinaldi, M. G., Sarver, B. A., Balajee, S. A., Schroers, H. J., et al. (2010). Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. J. Clin. Microbiol. 48, 3708-3718. doi: 10.1128/JCM.00989-10.
54. Oever, J. T., and Netea, M. G. (2014). The bacteriome-mycobiome interaction and antifungal host defense. Eur. J. Immunol. 44, 3182-3191. doi: 10.1002/eji.201344405.
55. Oh, J., Byrd, A. L., Deming, C., Conlan, S., Kong, H. H., and Segre, J. A. (2014). Biogeography and individuality shape function in the human skin metagenome. Nature 514, 59-64. doi: 10.1038/nature13786.
56. Oh, J., Freeman, A. F., Park, M., Sokolic, R., Candotti, F., Holland, S. M., et al. (2013). The altered landscape of the human skin microbiome in patients with primary immunodeficiencies. Genome Res. 23, 2103-2114. doi: 10.1101/gr.159467.113.
57. Ott, S. J., Kühbacher, T., Musfeldt, M., Rosenstiel, P., Hellmig, S., Rehman, A., et al. (2008). Fungi and inflammatory bowel diseases: alterations of composition and diversity. Scand. J. Gastroenterol. 43, 831-841. doi: 10.1080/00365520801935434.
58. Paulino, L. C., Tseng, C. H., Strober, B. E., and Blaser, M. J. (2006). Molecular analysis of fungal microbiota in samples from healthy human skin and psoriatic lesions. J. Clin. Microbiol. 44, 2933-2941. doi: 10.1128/JCM.00785-06.
59. Porras-Alfaro, A., Liu, K. L., Kuske, C. R., and Xie, G. (2014). From genus to phylum: large-subunit and internal transcribed spacer rRNA operon regions show similar classification accuracies influenced by database composition. Appl. Environ. Microbiol. 80, 829-840. doi: 10.1128/AEM.02894-13.
60. Reck, M., Tomasch, J., Deng, Z., Jarek, M., Husemann, P., and Wagner-Dobler, I. (2015). Stool metatranscriptomics: a technical guideline for mRNA stabilisation and isolation. BMC Genomics 16:494. doi: 10.1186/s12864-015-1694-y.
61. Rideout, J. R., He, Y., Navas-Molina, J. A., Walters, W. A., Ursell, L. K., Gibbons, S. M., et al. (2014). Subsampled open-reference clustering creates consistent, comprehensive OTU definitions and scales to billions of sequences. PeerJ 2:e545. doi: 10.7717/peerj.545.
62. Robinson, M. D., McCarthy, D. J., and Smyth, G. K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140. doi: 10.1093/bioinformatics/btp616.
63. Sangoi, A. R., Rogers, W. M., Longacre, T. A., Montoya, J. G., Baron, E. J., and Banaei, N. (2009). Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: a ten-year retrospective review at a single institution. Am. J. Clin. Pathol. 131, 364-375. doi: 10.1309/AJCP99OOOZSNISCZ.
64. 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. doi: 10.1093/nar/gku1341.
65. Schloss, P. D., Gevers, D., and Westcott, S. L. (2011). Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS ONE 6:e27310. doi: 10.1371/journal.pone.0027310.
66. Schloss, P. D., Westcott, S. L., Ryabin, T., Hall, J. R., Hartmann, M., Hollister, E. B., et al. (2009). Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537-7541. doi: 10.1128/AEM.01541-09.
67. Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., et al. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Natl. Acad. Sci. U.S.A. 109, 6241-6246. doi: 10.1073/pnas.1117018109.
68. Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W. S., et al. (2011). Metagenomic biomarker discovery and explanation. Genome Biol. 12:R60. doi: 10.1186/gb-2011-12-6-r60.
69. Simon, L., Lalonde, M., and Bruns, T. D. (1992). Specific amplification of 18S fungal ribosomal genes from vesicular-arbuscular endomycorrhizal fungi colonizing roots. Appl. Environ. Microbiol. 58, 291-295.
70. Smit, E., Leeflang, P., Glandorf, B., van Elsas, J. D., and Wernars, K. (1999). Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis. Appl. Environ. Microbiol. 65, 2614-2621.
71. Sokol, H., Leducq, V., Aschard, H., Pham, H. P., Jegou, S., Landman, C., et al. (2016). Fungal microbiota dysbiosis in IBD. Gut. doi: 10.1136/gutjnl-2015-310746. [Epub ahead of print].
72. Stackebrandt, E., and Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Evol. Microbiol. 44, 846-849. doi: 10.1099/00207713-44-4-846.
73. Tanabe, Y., Watanabe, M. M., and Sugiyama, J. (2002). Are Microsporidia really related to Fungi? a reappraisal based on additional gene sequences from basal fungi. Mycol. Res. 106, 1380-1391. doi: 10.1017/S095375620200686X.
74. Tang, J., Iliev, I. D., Brown, J., Underhill, D. M., and Funari, V. A. (2015). Mycobiome: approaches to analysis of intestinal fungi. J. Immunol. Methods 421, 112-121. doi: 10.1016/j.jim.2015.04.004.
75. Taylor, D. L., Hollingsworth, T. N., McFarland, J. W., Lennon, N. J., Nusbaum, C., and Ruess, R. W. (2014). A first comprehensive census of fungi in soil reveals both hyperdiversity and fine-scale niche partitioning. Ecol. Monogr. 84, 3-20. doi: 10.1890/12-1693.1.
76. Tedersoo, L., Bahram, M., Polme, S., Koljalg, U., Yorou, N. S., Wijesundera, R., et al. (2014). Fungal biogeography. Global diversity and geography of soil fungi. Science 346:1256688. doi: 10.1126/science.1256688.
77. Toju, H., Tanabe, A. S., Yamamoto, S., and Sato, H. (2012). High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. PLoS ONE 7:e40863. doi: 10.1371/journal.pone.0040863.
78. Underhill, D. M., and Iliev, I. D. (2014). The mycobiota: interactions between commensal fungi and the host immune system. Nat. Rev. Immunol. 14, 405-416. doi: 10.1038/nri3684.
79. van Burik, J. A., Schreckhise, R. W., White, T. C., Bowden, R. A., and Myerson, D. (1998). Comparison of six extraction techniques for isolation of DNA from filamentous fungi. Med. Mycol. 36, 299-303. doi: 10.1080/02681219880000471.
80. Wang, Q., Garrity, G. M., Tiedje, J. M., and Cole, J. R. (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261-5267. doi: 10.1128/AEM.00062-07.
81. Westcott, S. L., and Schloss, P. D. (2015). De novo clustering methods outperform reference-based methods for assigning 16S rRNA gene sequences to operational taxonomic units. PeerJ 3:e1487. doi: 10.7717/peerj.1487.
82. Wheeler, M. L., Limon, J. J., Bar, A. S., Leal, C. A., Gargus, M., Tang, J., et al. (2016). Immunological consequences of intestinal fungal dysbiosis. Cell Host Microbe 19, 865-873. doi: 10.1016/j.chom.2016.05.003.
83. White, T., Bruns, T., Lee, S., and Taylor, J. (1990). "Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics," in PCR Protocols: A Guide to Methods and Applications, eds M. Innis, D. Gelfand, J. Sninsky, and T. White (Orlando, FL: Academic Press), 315-322.
84. Yuille, M., van Ommen, G. J., Brechot, C., Cambon-Thomsen, A., Dagher, G., Landegren, U., et al. (2008). Biobanking for Europe. Brief. Bioinform. 9, 14-24. doi: 10.1093/bib/bbm050.