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DNA suspension arrays: silencing discrete artifacts for high-sensitivity applications.

ABSTRACT: Detection of low frequency single nucleotide polymorphisms (SNPs) has important implications in early screening for tumorgenesis, genetic disorders and pathogen drug resistance. Nucleic acid arrays are a powerful tool for genome-scale SNP analysis, but detection of low-frequency SNPs in a mixed population on an array is problematic. We demonstrate a model assay for HIV-1 drug resistance mutations, wherein ligase discrimination products are collected on a suspension array. In developing this system, we discovered that signal from multiple polymorphisms was obscured by two discrete hybridization artifacts. Specifically: 1) tethering of unligated probes on the template DNA elicited false signal and 2) unpredictable probe secondary structures impaired probe capture and suppressed legitimate signal from the array. Two sets of oligonucleotides were used to disrupt these structures; one to displace unligated reporter labels from the bead-bound species and another to occupy sequences which interfered with array hybridization. This artifact silencing system resulted in a mean 21-fold increased sensitivity for 29 minority variants of 17 codons in our model assay for mutations most commonly associated with HIV-1 drug resistance. Furthermore, since the artifacts we characterized are not unique to our system, their specific inhibition might improve the quality of data from solid-state microarrays as well as from the growing number of multiple analyte suspension arrays relying on sequence-specific nucleic acid target capture.

SUBMITTER: Lalonde MS

PROVIDER: S-EPMC2975679 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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DNA suspension arrays: silencing discrete artifacts for high-sensitivity applications.

Lalonde Matthew S MS Arts Eric J EJ

PloS one 20101108 11

Detection of low frequency single nucleotide polymorphisms (SNPs) has important implications in early screening for tumorgenesis, genetic disorders and pathogen drug resistance. Nucleic acid arrays are a powerful tool for genome-scale SNP analysis, but detection of low-frequency SNPs in a mixed population on an array is problematic. We demonstrate a model assay for HIV-1 drug resistance mutations, wherein ligase discrimination products are collected on a suspension array. In developing this syst ...[more]

PMID: 21079742

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Project description:BackgroundWhole exome sequencing is increasingly used for the clinical evaluation of genetic disease, yet the variation of coverage and sensitivity over medically relevant parts of the genome remains poorly understood. Several sequencing-based assays continue to provide coverage that is inadequate for clinical assessment.MethodsUsing sequence data obtained from the NA12878 reference sample and pre-defined lists of medically-relevant protein-coding and noncoding sequences, we compared the breadth and depth of coverage obtained among four commercial exome capture platforms and whole genome sequencing. In addition, we evaluated the performance of an augmented exome strategy, ACE, that extends coverage in medically relevant regions and enhances coverage in areas that are challenging to sequence. Leveraging reference call-sets, we also examined the effects of improved coverage on variant detection sensitivity.ResultsWe observed coverage shortfalls with each of the conventional exome-capture and whole-genome platforms across several medically interpretable genes. These gaps included areas of the genome required for reporting recently established secondary findings (ACMG) and known disease-associated loci. The augmented exome strategy recovered many of these gaps, resulting in improved coverage in these areas. At clinically-relevant coverage levels (100 % bases covered at ≥20×), ACE improved coverage among genes in the medically interpretable genome (>90 % covered relative to 10-78 % with other platforms), the set of ACMG secondary finding genes (91 % covered relative to 4-75 % with other platforms) and a subset of variants known to be associated with human disease (99 % covered relative to 52-95 % with other platforms). Improved coverage translated into improvements in sensitivity, with ACE variant detection sensitivities (>97.5 % SNVs, >92.5 % InDels) exceeding that observed with conventional whole-exome and whole-genome platforms.ConclusionsClinicians should consider analytical performance when making clinical assessments, given that even a few missed variants can lead to reporting false negative results. An augmented exome strategy provides a level of coverage not achievable with other platforms, thus addressing concerns regarding the lack of sensitivity in clinically important regions. In clinical applications where comprehensive coverage of medically interpretable areas of the genome requires higher localized sequencing depth, an augmented exome approach offers both cost and performance advantages over other sequencing-based tests.

Project description:High-density oligonucleotide (oligo) arrays are a powerful tool for transcript profiling. Arrays based on GeneChip technology are amongst the most widely used, although GeneChip arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip array is available, using a GeneChip array designed for Arabidopsis thaliana (L.) Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P) stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.

Dataset Information

DNA suspension arrays: silencing discrete artifacts for high-sensitivity applications.

Publications

DNA suspension arrays: silencing discrete artifacts for high-sensitivity applications.

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