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Large-scale evolutionary surveillance of the 2009 H1N1 influenza A virus using resequencing arrays.

Lee CW, Koh CW, Chan YS, Aw PP, Loh KH, Han BL, Thien PL, Nai GY, Hibberd ML, Wong CW, Sung WK - Nucleic Acids Res. (2010)

Bottom Line: The accompanying base-calling software (EvolSTAR) introduces novel methods that utilize neighbourhood hybridization intensity profiles and substitution bias of probes on the microarray for mutation confirmation and recovery of ambiguous base queries.Our results demonstrate that EvolSTAR is highly accurate and has a much improved call rate.The high throughput and short turn-around time from sample to sequence and analysis results (30 h for 24 samples) makes this kit an efficient large-scale evolutionary biosurveillance tool.

View Article: PubMed Central - PubMed

Affiliation: Genome Institute of Singapore, Genome, 60 Biopolis Street, Singapore.

ABSTRACT
In April 2009, a new influenza A (H1N1 2009) virus emerged that rapidly spread around the world. While current variants of this virus have caused widespread disease, particularly in vulnerable groups, there remains the possibility that future variants may cause increased virulence, drug resistance or vaccine escape. Early detection of these virus variants may offer the chance for increased containment and potentially prevention of the virus spread. We have developed and field-tested a resequencing kit that is capable of interrogating all eight segments of the 2009 influenza A(H1N1) virus genome and its variants, with added focus on critical regions such as drug-binding sites, structural components and mutation hotspots. The accompanying base-calling software (EvolSTAR) introduces novel methods that utilize neighbourhood hybridization intensity profiles and substitution bias of probes on the microarray for mutation confirmation and recovery of ambiguous base queries. Our results demonstrate that EvolSTAR is highly accurate and has a much improved call rate. The high throughput and short turn-around time from sample to sequence and analysis results (30 h for 24 samples) makes this kit an efficient large-scale evolutionary biosurveillance tool.

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Visualization map of EvolSTAR. Visualization map of all eight segments of the 2009 influenza A(H1N1) virus and the locations of known drug binding sites (marked with green lines) on the neuraminidase (NA) gene (segment 6). A heat map bar is used to represent the quality and coverage of its sequence calls. The locations of all mutation calls made by EvolSTAR are represented by red triangles beneath the heat map bar. Sequences with coverage <90% are automatically flagged as ‘low coverage’. Other details such as coverage: percentage of base calls successfully made, match: number of base calls that match the reference sequence i.e. non-mutation base calls, strong mismatch: number of high confidence base calls that do not match the reference sequence i.e. mutation base calls, weak mismatch: number of low-confidence base-calls that do not match the reference sequence i.e. mutation base calls and Ns: number of ‘N’ calls, for each sequence call are also shown on the visualization map.
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Figure 8: Visualization map of EvolSTAR. Visualization map of all eight segments of the 2009 influenza A(H1N1) virus and the locations of known drug binding sites (marked with green lines) on the neuraminidase (NA) gene (segment 6). A heat map bar is used to represent the quality and coverage of its sequence calls. The locations of all mutation calls made by EvolSTAR are represented by red triangles beneath the heat map bar. Sequences with coverage <90% are automatically flagged as ‘low coverage’. Other details such as coverage: percentage of base calls successfully made, match: number of base calls that match the reference sequence i.e. non-mutation base calls, strong mismatch: number of high confidence base calls that do not match the reference sequence i.e. mutation base calls, weak mismatch: number of low-confidence base-calls that do not match the reference sequence i.e. mutation base calls and Ns: number of ‘N’ calls, for each sequence call are also shown on the visualization map.

Mentions: Besides a FASTA output of the virus sequence, EvolSTAR generates a visualization map of the sequence calls using a heat map based on the percentage identity of the called sequence to the reference sequence measured at 50 bp windows (Figure 8). The map template consists of all eight segments of the 2009 influenza A(H1N1) virus and the locations of known drug binding sites (marked with green lines) on the NA gene. Locations of all mutation calls are denoted by red triangles beneath the heat map bar. Sequences that are of low coverage (<90%) are automatically flagged, and the overall PM/MM discrimination ratio for each segment is displayed. The heat map bar allows the technician to rapidly assess the quality of the sequence data obtained from the microarray and identify regions where PCR did not work well, or presence of potential recombination/reassortment events. Mutations, especially those in close proximity to drug binding sites, can be quickly visualized. Other details such as coverage, number of base calls successfully made, number of mutations and number of ‘N’ calls for each sequence call are also shown on the visualization map.Figure 8.


Large-scale evolutionary surveillance of the 2009 H1N1 influenza A virus using resequencing arrays.

Lee CW, Koh CW, Chan YS, Aw PP, Loh KH, Han BL, Thien PL, Nai GY, Hibberd ML, Wong CW, Sung WK - Nucleic Acids Res. (2010)

Visualization map of EvolSTAR. Visualization map of all eight segments of the 2009 influenza A(H1N1) virus and the locations of known drug binding sites (marked with green lines) on the neuraminidase (NA) gene (segment 6). A heat map bar is used to represent the quality and coverage of its sequence calls. The locations of all mutation calls made by EvolSTAR are represented by red triangles beneath the heat map bar. Sequences with coverage <90% are automatically flagged as ‘low coverage’. Other details such as coverage: percentage of base calls successfully made, match: number of base calls that match the reference sequence i.e. non-mutation base calls, strong mismatch: number of high confidence base calls that do not match the reference sequence i.e. mutation base calls, weak mismatch: number of low-confidence base-calls that do not match the reference sequence i.e. mutation base calls and Ns: number of ‘N’ calls, for each sequence call are also shown on the visualization map.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: Visualization map of EvolSTAR. Visualization map of all eight segments of the 2009 influenza A(H1N1) virus and the locations of known drug binding sites (marked with green lines) on the neuraminidase (NA) gene (segment 6). A heat map bar is used to represent the quality and coverage of its sequence calls. The locations of all mutation calls made by EvolSTAR are represented by red triangles beneath the heat map bar. Sequences with coverage <90% are automatically flagged as ‘low coverage’. Other details such as coverage: percentage of base calls successfully made, match: number of base calls that match the reference sequence i.e. non-mutation base calls, strong mismatch: number of high confidence base calls that do not match the reference sequence i.e. mutation base calls, weak mismatch: number of low-confidence base-calls that do not match the reference sequence i.e. mutation base calls and Ns: number of ‘N’ calls, for each sequence call are also shown on the visualization map.
Mentions: Besides a FASTA output of the virus sequence, EvolSTAR generates a visualization map of the sequence calls using a heat map based on the percentage identity of the called sequence to the reference sequence measured at 50 bp windows (Figure 8). The map template consists of all eight segments of the 2009 influenza A(H1N1) virus and the locations of known drug binding sites (marked with green lines) on the NA gene. Locations of all mutation calls are denoted by red triangles beneath the heat map bar. Sequences that are of low coverage (<90%) are automatically flagged, and the overall PM/MM discrimination ratio for each segment is displayed. The heat map bar allows the technician to rapidly assess the quality of the sequence data obtained from the microarray and identify regions where PCR did not work well, or presence of potential recombination/reassortment events. Mutations, especially those in close proximity to drug binding sites, can be quickly visualized. Other details such as coverage, number of base calls successfully made, number of mutations and number of ‘N’ calls for each sequence call are also shown on the visualization map.Figure 8.

Bottom Line: The accompanying base-calling software (EvolSTAR) introduces novel methods that utilize neighbourhood hybridization intensity profiles and substitution bias of probes on the microarray for mutation confirmation and recovery of ambiguous base queries.Our results demonstrate that EvolSTAR is highly accurate and has a much improved call rate.The high throughput and short turn-around time from sample to sequence and analysis results (30 h for 24 samples) makes this kit an efficient large-scale evolutionary biosurveillance tool.

View Article: PubMed Central - PubMed

Affiliation: Genome Institute of Singapore, Genome, 60 Biopolis Street, Singapore.

ABSTRACT
In April 2009, a new influenza A (H1N1 2009) virus emerged that rapidly spread around the world. While current variants of this virus have caused widespread disease, particularly in vulnerable groups, there remains the possibility that future variants may cause increased virulence, drug resistance or vaccine escape. Early detection of these virus variants may offer the chance for increased containment and potentially prevention of the virus spread. We have developed and field-tested a resequencing kit that is capable of interrogating all eight segments of the 2009 influenza A(H1N1) virus genome and its variants, with added focus on critical regions such as drug-binding sites, structural components and mutation hotspots. The accompanying base-calling software (EvolSTAR) introduces novel methods that utilize neighbourhood hybridization intensity profiles and substitution bias of probes on the microarray for mutation confirmation and recovery of ambiguous base queries. Our results demonstrate that EvolSTAR is highly accurate and has a much improved call rate. The high throughput and short turn-around time from sample to sequence and analysis results (30 h for 24 samples) makes this kit an efficient large-scale evolutionary biosurveillance tool.

Show MeSH
Related in: MedlinePlus