Mycobiome: Approaches to analysis of intestinal fungi.

Massively parallel sequencing (MPSS) of bacterial 16S rDNA has been widely used to characterize the microbial makeup of the human and mouse gastrointestinal tract. However, techniques for fungal microbiota (mycobiota) profiling remain relatively under-developed. Compared to 16S profiling, the size and sequence context of the fungal Internal Transcribed Spacer 1 (ITS1), the most common target for mycobiota profiling, are highly variable. Using representative gastrointestinal tract fungi to build a known "mock" library, we examine how this sequence variability affects data quality derived from Illumina Miseq and Ion Torrent PGM sequencing pipelines. Also, while analysis of bacterial 16S profiles is facilitated by the presence of high-quality well-accepted databases of bacterial 16S sequences, such an accepted database has not yet emerged to facilitate fungal ITS sequence characterization, and we observe that redundant and inconsistent ITS1 sequence representation in publically available fungal reference databases affect quantitation and annotation of species in the gut. To address this problem, we have constructed a manually curated reference database optimized for annotation of gastrointestinal fungi. This targeted host-associated fungi (THF) database contains 1817 ITS1 sequences representing sequence diversity in genera previously identified in human and mouse gut. We observe that this database consistently outperforms three common ITS database alternatives on comprehensiveness, taxonomy assignment accuracy and computational efficiency in analyzing sequencing data from the mouse gastrointestinal tract.

Tang J ，Iliev ID ，Brown J ，Underhill DM ，Funari VA ... -  《-》

Age-Related Variation of Bacterial and Fungal Communities in Different Body Habitats across the Young, Elderly, and Centenarians in Sardinia.

Human body microbes interact with the host, forming microbial communities that are in continual flux during the aging process. Previous studies have mostly focused on surveying a single body habitat to determine the age-related variation in the bacterial and fungal communities. A more comprehensive understanding of the variation in the human microbiota and mycobiota across multiple body habitats related to aging is still unclear. To obtain an integrated view of the spatial distribution of microbes in a specific Mediterranean population across a wide age range, we surveyed the bacterial and fungal communities in the skin, oral cavity, and gut in the young, elderly, and centenarians in Sardinia using 16S rRNA gene and internal transcribed spacer 1 (ITS1) sequencing. We found that the distribution and correlation of bacterial and fungal communities in Sardinians were largely determined by body site. In each age group, the bacterial and fungal communities found in the skin were significantly different in structure. In the oral cavity, age had a marginal impact on the structures of the bacterial and fungal communities. Furthermore, the gut bacterial communities in centenarians clustered separately from those of the young and elderly, while the fungal communities in the gut habitat could not be separated by host age.IMPORTANCE Site-specific microbial communities are recognized as important factors in host health and disease. To better understand how the human microbiota potentially affects and is affected by its host during the aging process, the fundamental issue to address is the distribution of microbiota related to age. Here, we show an integrated view of the spatial distribution of microbes in a specific Mediterranean population (Sardinians) across a wide age range. Our study indicates that age plays a critical role in shaping the human microbiota in a habitat-dependent manner. The dynamic age-related microbiota changes we observed across multiple body sites may provide possibilities for modulating microbe communities to maintain or improve health during aging.

Wu L ，Zeng T ，Deligios M ，Milanesi L ，Langille MGI ，Zinellu A ，Rubino S ，Carru C ，Kelvin DJ ... -  《mSphere》

Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity.

Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies.IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

De Filippis F ，Laiola M ，Blaiotta G ，Ercolini D ... -  《-》

Early gut mycobiota and mother-offspring transfer.

Schei K ，Avershina E ，Øien T ，Rudi K ，Follestad T ，Salamati S ，Ødegård RA ... -  《Microbiome》

Fungal ITS1 Deep-Sequencing Strategies to Reconstruct the Composition of a 26-Species Community and Evaluation of the Gut Mycobiota of Healthy Japanese Individuals.

Motooka D ，Fujimoto K ，Tanaka R ，Yaguchi T ，Gotoh K ，Maeda Y ，Furuta Y ，Kurakawa T ，Goto N ，Yasunaga T ，Narazaki M ，Kumanogoh A ，Horii T ，Iida T ，Takeda K ，Nakamura S ... -  《-》