title: 16S metagenomic sequencing of homo sapiens saliva microbiota:  Subject S76, Visit 3, 41.5666666666667 months old, Healthy Caries Free, Candida Positive
design: At each study visit, whole, stimulated, saliva samples were obtained through a disposable saliva ejector attached to a 50 ml sterile centrifuge tube, which in turn was attached to a vacuum pump. Approximately 2 ml of saliva was collected for each sample. The saliva sample was kept on ice and then transferred to a cyrotube and frozen at -80C. Samples selected for sequencing were thawed and 50ul was transferred to a new sterile microfuge tube and frozen at -80C.  Total genomic DNA was extracted from the nasal samples using a modification of the ZymoBIOMICSTM DNA Miniprep Kit (Zymo Research, Irvine, CA) and FastPrep mechanical lysis (MPBio, Solon, OH).  16S ribosomal DNA (rRNA) was amplified with Phusion High-Fidelity polymerase (Thermo Scientific, Waltham, MA) and dual indexed primers specific to the V1-V3 (8F: 5 AGAGTTTGATCCTGGCTCAG 3;  534R: 3 ATTACCGCGGCTGCTGG 5) hypervariable regions. Amplicons were pooled and paired-end sequenced on an Illumina MiSeq (Illumina, San Diego, CA) in the University of Rochester Genomics Research Center.