Computational protocol: Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus

Similar protocols

Protocol publication

[…] Total RNA was isolated as described in the previous section. The NP and polymerase genes were amplified using a nested PCR approach. First, a multisegment RT–PCR comprising 25 cycles was performed for each sample. Next, nested PCR reactions comprised of 28 cycles were performed to generate two overlapping amplicons for each polymerase segment and a single amplicon for the NP segment. All primer sequences are provided in . Nested amplicons were then combined into an amplicon pool, which was purified by using AMPure Beads (Beckman Coulter). The purified amplicon pool was processed with the Ion Xpress Plus Fragment Library Kit (Life Technologies) to generate sheared, barcoded and size-selected Ion Torrent PGM 200-base libraries, and then amplified in eight additional PCR cycles to increase the number of barcoded library fragments. Final pools for sequence analysis were constructed by combining equimolar amounts of the individually barcoded libraries (which correspond to a single isolate). The pools were then sequenced on the Ion Torrent PGM with 314 chips. Basecalling was performed by using TraceTuner, and the resulting sequences were quality-trimmed and then assembled using the merger utility from the EMBOSS software suite. All processing steps were integrated into a reproducible Galaxy workflow and are available upon request. The resulting assembled sequences were analysed by using the Galaxy bioinformatics suite and the Integrative Genomics Viewer. […]

Pipeline specifications

Software tools TraceTuner, EMBOSS, IGV
Application Sanger sequencing
Organisms Mus musculus, Homo sapiens
Diseases Bird Diseases, Influenza in Birds, Influenza, Human
Chemicals Amino Acids