RNA Extraction and Sequencing for Viral Mutations

RNA was isolated from egg allantoic fluid using the E.Z.N.A. viral RNA extraction kit (Omega Bio-Tek) according to the manufacturer’s instructions and then underwent next-generation sequencing. Sequences were assembled using a pipeline designed at the Icahn School of Medicine at Mount Sinai as described previously (42 (link)). To identify point mutations, full-length coding sequences were compared to the sequenced wild-type A/Netherlands/602/2009 (H1N1)pdm09 strain used for escape mutagenesis. All reported mutations were numbered from the methionine of each protein.

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Kirkpatrick Roubidoux E., Sano K., McMahon M., Carreño J.M., Capuano C., Jiang K., Simon V., van Bakel H., Wilson P, & Krammer F. (2022). Novel Epitopes of the Influenza Virus N1 Neuraminidase Targeted by Human Monoclonal Antibodies. Journal of Virology, 96(9), e00332-22.

Publication 2022

E.Z.N.A. viral RNA extraction kit

Allantoic Coding sequences Methionine Mutagenesis Mutations Point mutations Protein Strain Viral rna

Corresponding Organization : Icahn School of Medicine at Mount Sinai

Other organizations : University of Chicago

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Variable analysis

independent variables

RNA isolation using the E.Z.N.A. viral RNA extraction kit (Omega Bio-Tek)

dependent variables

Point mutations in the full-length coding sequences compared to the sequenced wild-type A/Netherlands/602/2009 (H1N1)pdm09 strain

control variables

Manufacturer's instructions for the E.Z.N.A. viral RNA extraction kit
Sequenced wild-type A/Netherlands/602/2009 (H1N1)pdm09 strain used for escape mutagenesis

controls

Sequenced wild-type A/Netherlands/602/2009 (H1N1)pdm09 strain used as a control for comparison

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