Κατεύθυνση Κλινική Χημεία
Library of the School of Science

2016-03-02

2016

Αναστασοπούλου Μαρίτα

Δημήτριος Παρασκευής Επίκ. Καθηγητής (Επιβλέπων), Ευρύκλεια Λιανίδου Καθηγήτρια, Χρήστος Κρούπης Επίκ. Καθηγητής

Σύγκριση των Μεθόδων Αλληλούχισης Sanger και NGS με pyrosequencing (454 GS Junior) ως προς τη Μελέτη Γενετικών Πολυμορφισμών του Ιου HIV-1

Greek

Comparison of Sanger sequencing with Ultra-Deep pyrosequencing (454 GS Junior) for the study of HIV-1 Genetic Diversity"

Human immunodeciency virus (HIV) exhibits remarkable diversity in its genomic
makeup and exists in any given individual as a complex distribution of closely
related but nonidentical genomes called viral quasispecies related by a similar
mutation or mutations, while these are subject to genetic variation,
competition, and selection. This viral diversity clinically manifests as a
selection of mutant variants based on viral tness in treatment-naive
individuals and based on drug-selective pressure in those on antiretroviral
therapy (ART). Although HIV patients have benefited from an increased life
span, many experience several episodes of virological failure after developing
resistance to a specific drug or drug combination, because of viral
heterogeneity through variant strains. The viral ratio of mutant variant to
wild type often decreases dramatically in the absence of selective pressure.
Recent data indicate that low abundance HIV variants can become clinically
relevant in cases where drug selection pressure results in rapid proliferation
and subsequent treatment failure. The FDA recommends variant identification in
clinical trials of new HIV drugs.
To optimize therapy, clinicians rely on viral genotypic information obtained
from conventional population sequencing–based assays (Sanger method), which
cannot reliably detect viral variants that constitute <20% of the circulating
viral quasispecies. These low-frequency variants can be detected by highly
sensitive genotyping methods collectively grouped under the moniker of deep
sequencing. With Ultra Deep 454 Sequencing, heterogeneity and subtype variation
within HIV protease and reverse transcriptase genes can be accurately and
quantitatively assessed, whereas interrelationships among individual
polymorphisms may eventually be elucidated. Additional uses of 454 Sequencing
method include correlation of the identified variants with functionally
translated biochemical changes in reverse transcriptase and protease function
as well as interactions with coreceptors and other host molecules. Given the
low limits of detection Roche 454 Sequencing technology provides the requisite
sensitivity for detection of minor variants to comprehensively evaluate
molecular HIV sample profiles.
The purpose of the present postgraduate work is to compare the conventional
method of Sanger and Roche 454 Sequencing method on the detection of low
frequency HIV viral strains. It is being demonstrated the high sensitivity of
Roche 454 Sequencing method as may reliably detect mutations that are not
detected by the method of Sanger, for an extended area of gene RT and PR. In
parallel, has taken place an analysis that focused only on the detection of
targeted mutations associated with resistance to therapy in the areas of the RT
and PR. Both methods responded equally, yielding qualitativily the same
variants.

HIV quasispecies, Low-frequency variants, Drug resistance, Sanger method, Roche 454 sequencing

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https://pergamos.lib.uoa.gr/uoa/dl/object/1319921