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Massive parallel ultra deep sequencing of Influenza/Herpes antiviral resistance with the Solexa Genome Analyzer

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Massive parallel ultra deep sequencing of Influenza/Herpes antiviral resistance with the Solexa Genome Analyzer

Rechten: Alle rechten voorbehouden

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Summary
At the department of Virology of the Erasmus MC, sequencing of viruses is currently performed using the Sanger sequencing method. Mutations that are present at frequencies below 20% are not detectable however with Sanger sequencing methods. Next generation sequencing methods are capable of sequencing minority variants in a viral quasi species. It has previously been shown that both 454 and Solexa next generation sequencers are capable of detecting minority species up to 0,005%. [1]
As part of a bachelor study the ability to detect minority species in viral quasi species was investigated. In the Solexa system Influenza H3N2 Oseltamivir resistance mutations E119V, R292K and K308Q were used as model system. And for the 454 CTL epitopes in UL 1, UL 48 and UL 53 of HSV were used as model system.
Initially (RT-) PCR's were developed that would enable sequencing of these regions with Solexa and 454.
For Influenza E119V a nested RT-PCR was developed with a sensitivity of 105 viral particles per ml. For Influenza R292K and K308Q a nested RT-PCR was developed with a sensitivity of 5x105 viral particles per ml.
To test the sensitivity of the different methods for minority species, a mixture of mutant and wildtype H3N2 E119V, R292K and K308Q was made. Using quantification by real time RT-PCR a mixture was made with 25% mutant in the total population. Sanger sequencing was not able to detect minority species in the mixture. With real time RT-PCR's specific for either mutant or wildtype at positions 119 and 292 the mutant species in the mixture could be detected. Using the Solexa system mutant species for position 119 and 308 could be analyzed and proved to be 27%. The real time RT-PCR's and the Solexa system therefore gave comparable results, it was therefore decided to test lower percentages: 0,1% and 1% mutant in a total population. Results are pending.
PCR's for CTL epitope regions in the UL 1 and UL 53 genes were developed and sensitivity is proved to be approximately 500-1000 viral particles per ml for HSV-1. The sensitivity of the UL 1 PCR for HSV-2 was approximately one million viral particles per ml and the UL 53 PCR had a sensitivity on HSV-2 of approximately 10 000 viral particles per ml. The PCR for CTL epitope region for the UL 48 gene did not amplify correctly.
For Herpes Simplex Virus, test samples were also made to detect if the Solexa could detect a 0,1% and a 1% HSV-2 in HSV-1 for every epitope. Results are pending.
(RT)-PCR methods for Influenza A H3N2 Oseltamivir resistance mutations and HSV-1/2 CTL epitopes in UL 1 and UL 53 were developed to test the sensitivity of the Solexa and 454 Next Generation Sequencers for minority mutant species. Further analyses will be part of the subsequent study.

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OrganisatieAvans Hogeschool
AfdelingATGM Academie voor de technologie van Gezondheid en Milieu
PartnersErasmus MC-Rotterdam
Datum2010-06
TypeBachelorscriptie
TaalEngels

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