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Reply to ‘Reconciling disparate estimates of viral genetic diversity during human influenza infections’

Nature Genetics volume 51, pages 1301–1303 (2019)Cite this article

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Poon et al. reply — In their Correspondence, Xue and Bloom1 identify an important discrepancy in the sequence data that we provided in our 2016 paper2 on the transmission of influenza virus in Hong Kong during the first wave of the H1N1 pandemic in 2009. The goal of the study was to provide a quantitative approach to characterizing the within-host genetic diversity of influenza virus and how this genomic information might be used in tracking transmission. We have determined that the potential source of this discrepancy was due to the technical limitations of the methods used at the time, which inadvertently led to the reporting of substantially higher levels of within-host diversity than were likely to be present.

The primary swabs and cell culture samples were collected in 2009 and sequenced in 2010. Extracted RNA was used as input for multi-segment RT–PCR (M-RTPCR)3 for amplification of influenza segments, followed by sequence-independent single-primer amplification (SISPA)4 for the creation of randomly primed amplicons with nucleotide barcodes (index). The amplicons were then pooled for sequencing on the Illumina platform. We have determined that the primary source of the inconsistency in the data is probably due to an experimental issue that may be inherent to the SISPA method, in which single-stranded DNA present after amplification of the genomic library could lead to the annealing of single-stranded DNA from mismatched samples during the pooling process and extension by the Taq polymerase (Supplementary Fig. 1), thus resulting in the creation of chimeric molecules. In our case, there appeared to be an unusually high percentage of chimeras created. With today’s standards, these chimeras would have been identified bioinformatically and eliminated from the downstream analyses. However, at the time (2010), most of the sequencing reactions for Illumina were unidirectional, and the demultiplexing pipeline did not use the paired read information. Consequently, all reads were processed as single-end data, and each read was assigned to its individual sample according to the SISPA barcode information.

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Fig. 1: Within-host viral genetic diversity of human influenza virus samples for the Hong Kong 2009 study.

References

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Authors and Affiliations

  1. Division of Public Health Laboratory Sciences, School of Public Health, University of Hong Kong, Hong Kong, China

    Leo L. M. Poon & Joseph S. M. Peiris

  2. Memorial Sloan Kettering, New York, NY, USA

    Timothy Song

  3. Centers for Disease Control and Prevention, Atlanta, GA, USA

    David E. Wentworth

  4. Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Biological Sciences, University of Sydney, Sydney, New South Wales, Australia

    Edward C. Holmes

  5. Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia

    Edward C. Holmes

  6. Tisch Cancer Institute, Departments of Medicine, Hematology and Medical Oncology, and Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Benjamin D. Greenbaum

  7. Division of Epidemiology and Biostatistics, School of Public Health, University of Hong Kong, Hong Kong, China

    Benjamin J. Cowling

  8. Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA

    Elodie Ghedin

Authors
  1. Leo L. M. Poon
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Correspondence to Elodie Ghedin.

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Poon, L.L.M., Song, T., Wentworth, D.E. et al. Reply to ‘Reconciling disparate estimates of viral genetic diversity during human influenza infections’. Nat Genet 51, 1301–1303 (2019). https://doi.org/10.1038/s41588-019-0486-8

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