Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Harrison, A. Brand; Binder, Hans; Buhot, Arnaud; Burden, Conrad J.; Carlon, Enrico; Gibas, Cynthia; Gamble, Lara J.; Halperin, A.; Hooyberghs, Jef; Kreil, David P.; Levicky, Rastislav; Noble, Peter A.; Ott, Albrecht; Pettitt, B. Montgomery; Tautz, Diethard; Pozhitkov, Alex

doi:10.1093/nar/gks1358

Cited by 51 publications

(44 citation statements)

References 89 publications

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“…This effect has been attributed to electrostatic repulsion that suppresses the ability of target molecules to approach the surface, inhibited physical access of the target to the probes, and modified probe conformation and probe− probe partial hybridization. 13,14 Crowding effects on the performance of various hybridization platforms have commonly been studied using some form of self-assembled surface layer. Thiolated oligonucleotide probes have been used extensively.…”

Section: ■ Introductionmentioning

confidence: 99%

Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes

Libera

2016

Langmuir

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Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes.

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Section: ■ Introductionmentioning

confidence: 99%

Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes

Libera

2016

Langmuir

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“…EHC variation is unsurprising since the process is driven by mostly unevaluated interactions between experimental parameters, target choice, and DNA sample characteristics (19, 20). Of these, the absolute and/or relative abundance of targets are likely particularly relevant, especially given the highly complex non-target DNA content typical of aDNA extracts.…”

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confidence: 99%

Quantitative PCR as a Predictor of Aligned Ancient DNA Read Counts Following Targeted Enrichment

2013

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Targeted DNA enrichment through hybridization capture (EHC) is rapidly replacing PCR as the method of choice for enrichment prior to genomic resequencing. This is especially true in the case of ancient DNA (aDNA) from long-dead organisms, where targets tend to be highly fragmented and outnumbered by contaminant DNA. However, the behavior of EHC using aDNA has been quite variable, making success difficult to predict and preventing efficient sample equilibration during multiplexed sequencing runs. Here, we evaluate whether quantitative PCR (qPCR) measurements of aDNA samples correlate with on-target read counts before and after EHC. Our data indicate that not only do simple target qPCRs correlate strongly with high-throughput sequencing (HTS) data but that certain sample characteristics, such as overall target abundance as well as experimental parameters (e.g., bait concentration and secondary structure propensity), consistently influenced enrichment of our diverse set of aDNA samples. Taken together, our results should help guide experimental design, screening strategies, and multiplexed sample equilibration, increasing yield and reducing the expected and actual cost of aDNA EHC high-throughput sequencing projects in the future.

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“…Although DNA microarrays are considered nowadays a mature technology and these devices are providing high quality and reproducibile data, their potentials have not been fully exploited. A better understanding of the underlying physico-chemical principles of hybridization is an issue of central interest and can lead to novel methods to improve the data analysis (34). …”

Section: Discussionmentioning

confidence: 99%

Targeted resequencing of HIV variants by microarray thermodynamics

Hadiwikarta¹,

Dorst²,

Hollanders³

et al. 2013

Nucleic Acids Research

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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.

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Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Cited by 51 publications

References 89 publications

Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes

Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes

Quantitative PCR as a Predictor of Aligned Ancient DNA Read Counts Following Targeted Enrichment

Targeted resequencing of HIV variants by microarray thermodynamics

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