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2015 ; 421
(ä): 112-121
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Mycobiome: Approaches to analysis of intestinal fungi
#MMPMID25891793
Tang J
; Iliev ID
; Brown J
; Underhill DM
; Funari VA
J Immunol Methods
2015[Jun]; 421
(ä): 112-121
PMID25891793
show ga
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.