Computational protocol: Multiple Reassortment between Pandemic (H1N1) 2009 and Endemic Influenza Viruses in Pigs, United States

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Protocol publication

[…] Specimens were sequenced by using an Illumina Genome Analyzer (Illumina, Inc., San Diego, CA, USA). An RT-PCR was performed on RNA templates by using Uni-12 and Uni-13 primers to amplify all 8 segments in 1 reaction with Invitrogen SuperScript III One-Step Reverse Transcriptase and Platinum Taq HiFi (Invitrogen, Carlsbad, CA, USA). Polymerase gene primers were added to optimize the sequencing reaction (). The obtained double-stranded DNA was sonicated in a Covaris AFA (Covaris, Woburn, MA, USA) until a broad peak at 200 bp appeared. The 3′ overhangs were removed from the sheared DNA by end repair, a Poly-A tail was added, and adapters were then ligated to the DNA fragments by using New England Biolabs (NEB) kits E6050L, E6053L, and E6056L (NEB, Ipswich, MA, USA). The ligation products were purified by gel electrophoresis by using E-Gel SizeSelect 2% agarose precast gels (Invitrogen). Index sequences were added to the DNA samples by Phusion DNA polymerase (NEB) before they were loaded on the illumina sequencer.For sequence analyses, samples were de-multiplexed and each genome was assembled by using CLC Genomics Workbench software (CLC bio, Germantown, MD, USA) by running a high stringency de novo assembly. Sequences were compared by using BioEdit () and ClustalW (). Phylogenetic analyses were performed by using MEGA version 4.0.2 (). The sequences of the 9 influenza viruses we studied were submitted to GenBank under accession nos. CY086877–CY086942. […]

Pipeline specifications

Software tools CLC Genomics Workbench, BioEdit, Clustal W, MEGA
Application Phylogenetics
Organisms Sus scrofa, Mustela putorius furo, Homo sapiens