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Pipeline publication

[…] al density at 600 nm of 0.1, and a 10-fold dilution series was plated on LB agar with or without spectinomycin (50 µg/ml; Millipore Sigma). Empty vector EPI300 was used as a comparator for false-positive enumeration, but no false-positive colonies were observed after 24 h of growth at 37°C. For each library, 12 spectinomycin-resistant cosmids were extracted (EZ-10 Plasmid DNA Miniprep kit; BioBasic) and analyzed for uniqueness by EcoRI/BamHI restriction digestion (New England Biolabs) (not shown). DNA from three unique cosmids from both HS31 and PM13 libraries was prepared for Illumina MiSeq PE250 sequencing (Génome Québec). Reads (~175,000/cosmid) and the Illumina adaptor were trimmed with Trimmomatic 0.36 (criteria: phred33, LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36; Reads surviving = >98%) (), assembled with SPAdes 3.10.1 (), and annotated with PROKKA () by using default settings. The pCC1FOS sequence (GenBank accession no. EU140751.1) was manually subtracted from resultant contigs in Geneious 8.1.9. The smallest cosmid insert was 28,221 bp, and the largest was 38,394 bp. The three contigs/cosmid inserts obtained for each strain were assembled to produce finalized sequences 48,228 and 30,236 bp in length for HS31 and PM13, respectively., The HS31 and PM13 sequences shared 87.9% pairwise identity (MUSCLE alignment) and shared synteny with the integrative and conjugative element (ICE) MGE of M. haemolytica strain M42548 (GenBank accession no. CP005383) (), which was isolated from a BRD case in Pennsylvania (). Annotation identified a 789-bp open reading frame designated adenylyltransferase (ANT1), which was further identified as a streptomycin-3″-adenylyltransferase via a c […]

Pipeline specifications

Software tools Trimmomatic, SPAdes, Prokka, Geneious