“…Genomic DNA was prepared for metagenomic 16S rRNA gene amplicon sequencing of the V3-V4 hypervariable region using a modified phenol-chloroform protocol recently described for complex samples, such as parasitic roundworms ( 6 , 7 ), fastidious anaerobic rumen bacteria ( 8 – 10 ), and spore-forming psychrotolerant Clostridium sp. isolated from spoiled meat ( 11 , 12 ). A DNA library was prepared using the 16S V3-V4 rRNA library preparation method (Illumina, Inc., San Diego, CA) according to the manufacturer’s instructions ( 13 ) and sequenced on the Illumina MiSeq platform with the 2 × 250-bp paired-end (PE) reagent kit v2, producing a total of 5,208,027 PE raw reads.…”