The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot

Holman, Devin B.; Timsit, Edouard; Amat, Samat; Abbott, D. Wade; Buret, Andre G.; Alexander, Trevor W.

The 16S rRNA gene sequences were processed and analyzed within the QIIME (quantitative insights into microbial ecology) software package v. 1.9.1 [15] . Pairedend reads were joined using fastq-join with a percent allowed maximum difference of 15% and a minimum overlap of 35 bp [16] . Joined sequences were quality filtered with sequences being truncated following three consecutive base calls of a Phred score of less than 25. Sequences were retained only when 75% or more of the original sequence remained after truncation. Chimeric sequences were removed using the UCHIME algorithm [17] implemented in USEARCH v. 6.1544 [18] . Sequences were then assigned to operational taxonomic units (OTUs) at 97% similarity using an open-reference OTU picking method and the SILVA database v. 123 [19] . In this method, sequences that were less than 97% similar to those in the SILVA database were clustered into OTUs using the de novo approach and USEARCH. Taxonomy was assigned using the UCLUST consensus taxonomy assigner with a minimum similarity of 0.8 and max accepts of three [18] . Sequences were then aligned using PyNAST [20] and a phylogenetic tree was created using FastTree [21] . OTUs containing fewer than ten sequences were removed prior to analysis, as were sequences classified as mitochondria or chloroplasts.