"Total nucleic acids were extracted from a frozen specimen without a buffer using the DNeasy UltraClean DNA Extraction Kit (Qiagen) and quantified using the Qubit™ assay (Invitrogen) from one of the four aliquots prepared from the original sample. The 16S rRNA gene V4–V5 region was amplified using a modified 515F–806R primer pair.20 Sequencing of amplicons was completed using the Illumina MiSeq platform to generate 2 × 250 paired-end reads."