Novel ITS1 Fungal Primers for Characterization of the Mycobiome

ABSTRACT Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features all...

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Autores principales:	Mykhaylo Usyk, Christine P. Zolnik, Hitesh Patel, Michael H. Levi, Robert D. Burk
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	ITS1 yeast fungi mycobiome oral primer design Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/aac9b4d591194366961ec53c12bed496
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spelling	oai:doaj.org-article:aac9b4d591194366961ec53c12bed4962021-11-15T15:21:52ZNovel ITS1 Fungal Primers for Characterization of the Mycobiome10.1128/mSphere.00488-172379-5042https://doaj.org/article/aac9b4d591194366961ec53c12bed4962017-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00488-17https://doaj.org/toc/2379-5042ABSTRACT Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states.Mykhaylo UsykChristine P. ZolnikHitesh PatelMichael H. LeviRobert D. BurkAmerican Society for MicrobiologyarticleITS1yeastfungimycobiomeoralprimer designMicrobiologyQR1-502ENmSphere, Vol 2, Iss 6 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	ITS1 yeast fungi mycobiome oral primer design Microbiology QR1-502
spellingShingle	ITS1 yeast fungi mycobiome oral primer design Microbiology QR1-502 Mykhaylo Usyk Christine P. Zolnik Hitesh Patel Michael H. Levi Robert D. Burk Novel ITS1 Fungal Primers for Characterization of the Mycobiome
description	ABSTRACT Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states.
format	article
author	Mykhaylo Usyk Christine P. Zolnik Hitesh Patel Michael H. Levi Robert D. Burk
author_facet	Mykhaylo Usyk Christine P. Zolnik Hitesh Patel Michael H. Levi Robert D. Burk
author_sort	Mykhaylo Usyk
title	Novel ITS1 Fungal Primers for Characterization of the Mycobiome
title_short	Novel ITS1 Fungal Primers for Characterization of the Mycobiome
title_full	Novel ITS1 Fungal Primers for Characterization of the Mycobiome
title_fullStr	Novel ITS1 Fungal Primers for Characterization of the Mycobiome
title_full_unstemmed	Novel ITS1 Fungal Primers for Characterization of the Mycobiome
title_sort	novel its1 fungal primers for characterization of the mycobiome
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/aac9b4d591194366961ec53c12bed496
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Novel ITS1 Fungal Primers for Characterization of the Mycobiome

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