Determining the Optimal Primers for Studying Microeukaryotic Diversity of the Human Gut

T. A. Auchtung, A. K. Nash, M. C. Wong, N. J. Ajami, J. F. Petrosino

Author address: 

Baylor Coll. of Med., Houston, TX

Abstract: 

Microeukaryotes are an understudied component of the human gut microbiome — in healthy adults, fungi and protists compose typically 0.01 - 1% of the genetic material. They have been shown to be present at abnormal levels in some diseases. The extent of this diversity is infrequently examined, and it is often unclear whether the detected microeukaryotes are active components that have colonized the gastrointestinal tract, or are simply passing through with consumed food or saliva.To gain insight into the microeukaryotic diversity of the gut, we are amplifying, sequencing, and analyzing ribosomal internal transcribed spacer (ITS) and 18S rRNA genes that are useful for identifying fungi, and other microeukaryotes, respectively. As an initial step, it was necessary to determine which of the many published ITS primer sets was best for use with MiSeq 2x300 sequencing of fecal samples. The most important criteria were which primers had the best amplification efficiency, and which the broadest and least biased taxonomic coverage. We tested those commonly used to target ITS1 (ITS1F and ITS2R) and ITS2 (ITS3F and ITS4R), the published winner of another fungal primer set comparison, ITS2-targeting ITS86F and ITS4R, and as a control, the broad fungal 18S rRNA gene-targeting 775F and 1121R.DNA extracted from five children's fecal samples, five adult fecal samples, and a mock community were used as template in PCR with each the of four primer sets. When the 11 PCRamplified samples were run on an agarose gel, there was a detectable band from 5/11 for ITS1FITS2R, 8/11 for ITS3F-ITS4R and ITS86F-ITS4R, and 9/11 for 775F-1121R. No primer set yielded consistently greater alpha diversity. ITS3F-ITS4R and ITS86F-ITS4R amplicons had no major taxonomic differences, but neither detected Rhodotorula (Sporidiobolales). ITS1F-ITS2R missed Malassezia, and mock community members Cryptococcus neosporans and Aspergillus fumigatus completely.Based on the above results, ITS3F-ITS4R was chosen for use in our large scale fungal analysis projects. This ITS primer set, in concert with 18S rRNA gene primers, are now yielding insights into the diversity, activity, and functions of the microeukaryotic portion of the human gut.
2016

abstract No: 

FRIDAY-666

Full conference title: 

ASM Microbe 2016
    • ASM microbe 1st (2016)