Targeted resequencing of HIV variants by microarray thermodynamics

Abstract

Within a single infected individual, a virus population can have a high genomic variability. In the case of HIV, several mutations can be present even in a small genomic window of 20–30 nucleotides. For diagnostics purposes, it is often needed to resequence genomic subsets where crucial mutations are known to occur. In this article, we address this issue using DNA microarrays and inputs from hybridization thermodynamics. Hybridization signals from multiple probes are analysed, including strong signals from perfectly matching (PM) probes and a large amount of weaker cross-hybridization signals from mismatching (MM) probes. The latter are crucial in the data analysis. Seven coded clinical samples (HIV-1) are analyzed, and the microarray results are in full concordance with Sanger sequencing data. Moreover, the thermodynamic analysis of microarray signals resolves inherent ambiguities in Sanger data of mixed samples and provides additional clinically relevant information. These results show the reliability and added value of DNA microarrays for point-of-care diagnostic purposes.

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

“…The current manuscript builds on previous work on DNA hybridization [ 6 – 8 , 12 , 15 , 16 ], the novelty of the presented work is in the use of DNA thermodynamics in the design of an assay capable to detect multiple and specific mutations with a well-defined limit of detection derived from physical principles and capable to perform this on clinical FFPE samples. The example of the FFPE samples illustrates that our method can deal with the limited quality and heterogeneity of the tissue material.…”

Genetic Variability of HIV-1 for Drug Resistance Assay Development

“…We examined a span of 27 nucleotides encompassing each drug-resistance position as well as 12 upstream and 12 downstream nucleotides. These flanking nucleotides are important for hybridization strategies relying on a terminal 3’ mismatch for either positive or negative-stranded cDNA and for those that rely on a central mismatch [ 11 , 17 , 18 ].…”

Microarray experiments and factors which affect their reliability

“…Microarrays can be also used to track differential expression patterns among various tissues and thus evaluate variability among individuals [ 1 – 3 ], they are used in SNP (single-nucleotide polymorphism) genotyping [ 4 – 7 ] and identification of transcription factor binding sites using the ChIP-chip (ChIP: chromatin immunoprecipitation) method [ 8 – 12 ]. Microarrays are also used to estimate genomic copy number using Comparative Genomic Hybridization (CGH) arrays [ 13 – 16 ] and in resequencing [ 17 – 22 ].…”