tetano
Editor, Senior Moderator
PLoS One. 2013 Jul 2;8(7):e67010. doi: 10.1371/journal.pone.0067010. Print 2013.
Using High-Throughput Sequencing to Leverage Surveillance of Genetic Diversity and Oseltamivir Resistance: A Pilot Study during the 2009 Influenza A(H1N1) Pandemic.
T?llez-Sosa J, Rodr?guez MH, G?mez-Barreto RE, Valdovinos-Torres H, Hidalgo AC, Cruz-Hervert P, Luna RS, Carrillo-Valenzo E, Ramos C, Garc?a-Garc?a L, Mart?nez-Barnetche J.
Source
Centro de Investigaciones sobre Enfermedades Infecciosas, Instituto Nacional de Salud P?blica, Cuernavaca, M?xico.
Abstract
BACKGROUND:
Influenza viruses display a high mutation rate and complex evolutionary patterns. Next-generation sequencing (NGS) has been widely used for qualitative and semi-quantitative assessment of genetic diversity in complex biological samples. The "deep sequencing" approach, enabled by the enormous throughput of current NGS platforms, allows the identification of rare genetic viral variants in targeted genetic regions, but is usually limited to a small number of samples.
METHODOLOGY AND PRINCIPAL FINDINGS:
We designed a proof-of-principle study to test whether redistributing sequencing throughput from a high depth-small sample number towards a low depth-large sample number approach is feasible and contributes to influenza epidemiological surveillance. Using 454-Roche sequencing, we sequenced at a rather low depth, a 307 bp amplicon of the neuraminidase gene of the Influenza A(H1N1) pandemic (A(H1N1)pdm) virus from cDNA amplicons pooled in 48 barcoded libraries obtained from nasal swab samples of infected patients (n = 299) taken from May to November, 2009 pandemic period in Mexico. This approach revealed that during the transition from the first (May-July) to second wave (September-November) of the pandemic, the initial genetic variants were replaced by the N248D mutation in the NA gene, and enabled the establishment of temporal and geographic associations with genetic diversity and the identification of mutations associated with oseltamivir resistance.
CONCLUSIONS:
NGS sequencing of a short amplicon from the NA gene at low sequencing depth allowed genetic screening of a large number of samples, providing insights to viral genetic diversity dynamics and the identification of genetic variants associated with oseltamivir resistance. Further research is needed to explain the observed replacement of the genetic variants seen during the second wave. As sequencing throughput rises and library multiplexing and automation improves, we foresee that the approach presented here can be scaled up for global genetic surveillance of influenza and other infectious diseases.
PMID:
23843978
[PubMed - in process]
Free full text
http://www.ncbi.nlm.nih.gov/pubmed/23843978
Using High-Throughput Sequencing to Leverage Surveillance of Genetic Diversity and Oseltamivir Resistance: A Pilot Study during the 2009 Influenza A(H1N1) Pandemic.
T?llez-Sosa J, Rodr?guez MH, G?mez-Barreto RE, Valdovinos-Torres H, Hidalgo AC, Cruz-Hervert P, Luna RS, Carrillo-Valenzo E, Ramos C, Garc?a-Garc?a L, Mart?nez-Barnetche J.
Source
Centro de Investigaciones sobre Enfermedades Infecciosas, Instituto Nacional de Salud P?blica, Cuernavaca, M?xico.
Abstract
BACKGROUND:
Influenza viruses display a high mutation rate and complex evolutionary patterns. Next-generation sequencing (NGS) has been widely used for qualitative and semi-quantitative assessment of genetic diversity in complex biological samples. The "deep sequencing" approach, enabled by the enormous throughput of current NGS platforms, allows the identification of rare genetic viral variants in targeted genetic regions, but is usually limited to a small number of samples.
METHODOLOGY AND PRINCIPAL FINDINGS:
We designed a proof-of-principle study to test whether redistributing sequencing throughput from a high depth-small sample number towards a low depth-large sample number approach is feasible and contributes to influenza epidemiological surveillance. Using 454-Roche sequencing, we sequenced at a rather low depth, a 307 bp amplicon of the neuraminidase gene of the Influenza A(H1N1) pandemic (A(H1N1)pdm) virus from cDNA amplicons pooled in 48 barcoded libraries obtained from nasal swab samples of infected patients (n = 299) taken from May to November, 2009 pandemic period in Mexico. This approach revealed that during the transition from the first (May-July) to second wave (September-November) of the pandemic, the initial genetic variants were replaced by the N248D mutation in the NA gene, and enabled the establishment of temporal and geographic associations with genetic diversity and the identification of mutations associated with oseltamivir resistance.
CONCLUSIONS:
NGS sequencing of a short amplicon from the NA gene at low sequencing depth allowed genetic screening of a large number of samples, providing insights to viral genetic diversity dynamics and the identification of genetic variants associated with oseltamivir resistance. Further research is needed to explain the observed replacement of the genetic variants seen during the second wave. As sequencing throughput rises and library multiplexing and automation improves, we foresee that the approach presented here can be scaled up for global genetic surveillance of influenza and other infectious diseases.
PMID:
23843978
[PubMed - in process]
Free full text
http://www.ncbi.nlm.nih.gov/pubmed/23843978