Fungal DNA from feces from 20 year old American human female, normoweight
Fungal DNA was selectively PCR-amplified using the fungal-specific forward primer ITS 1F (Gardes and Bruns, 1993) and the eukaryotic reverse primer TW13 (White et al., 1990). Due to the low abundance of fungal DNA (compared with bacterial DNA), a nested PCR reaction was then performed using ITS 1F and the eukaryotic ITS4 (White et al., 1990) primer, which amplifies the internal transcribed spacer (ITS) 1, 5.8S, and ITS2 regions of the ribosomal RNA genes. The nested primers were modified following the Roche-454 Titanium protocol, and the A or B Titanium sequencing adapters were added (shown in italics). In the forward primer, the adapter was followed immediately by a unique 8-base barcode sequence (BBBBBBBB), and finally the ITS1F primer, as shown:   5’ - CCATCTCATCCCTGCGTGTCTCCGACTCAGBBBBBBBBCTTGGTCATTTAGAGGAAGTAA-3’. The reverse primer was composed of the Titanium B sequencing adapter followed by ITS4: 5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGTCCTCCGCTTATTGATATGC-3’. Individual samples were amplified with primers containing unique barcodes, which allowed multiplexing. PCR amplification was performed on a Mastercycler Pro (Eppendorf) using Ex-Taq polymerase (TaKaRa Bio, USA) following the procedure of Martínez and colleagues (2010).  Gardes, M. Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol. Ecol. 2, 113-118.  Martínez, I., Kim, J., Duffy, P.R., Schlegel, V.L., Walter, J., 2010. Resistant starches types 2 and 4 have differential effects on the composition of the fecal microbiota in human subjects. PLoS One 5(11), e15046.  White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols: a guide to methods and applications. Academic Press, San Diego, pp. 315-322.