A flexible and fully integrated system for amplification, detection and genotyping of genomic DNA targets based on microfluidic oligonucleotide arrays

Summerer, Daniel; Hevroni, Dona; Jain, Amit; Oldenburger, Olga; Parker, Jefferson D.; Caruso, Anthony; Stähler, Cord; Stähler, Peer F.; Beier, Markus

doi:10.1016/j.nbt.2010.03.005

Cited by 12 publications

(11 citation statements)

References 29 publications

(24 reference statements)

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“…Four target enrichment and three NGS methods were preliminarily evaluated for multiplexed carrier testing. Preliminary experiments suggested that existing protocols for Agilent SureSelect hybrid capture (15) and RainDance microdroplet polymerase chain reaction (PCR) (16) but not Febit HybSelect microarray-based biochip capture (30) or Olink padlock probe ligation and PCR (31) yielded consistent target enrichment. Therefore, workflows and software pipelines were developed for comprehensive carrier testing by hybrid capture or microdroplet PCR, followed by NGS (Fig.…”

Section: Resultsmentioning

confidence: 99%

Carrier Testing for Severe Childhood Recessive Diseases by Next-Generation Sequencing

et al. 2011

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Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160× average target coverage, 93% of nucleotides had at least 20× coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.

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Section: Resultsmentioning

confidence: 99%

Carrier Testing for Severe Childhood Recessive Diseases by Next-Generation Sequencing

et al. 2011

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“…Given the need for highly accurate genotype detection, we developed a decision tree for identifying and genotyping mutations based on previous studies of genotypes identified in NGS genome sequen ces (Figure 5) [32,33,37,38]. SNPs in 26 samples were genotyped by both Infinium arrays and NGS.…”

Section: Sequence Analysis Strategymentioning

confidence: 99%

Adopting orphans: comprehensive genetic testing of Mendelian diseases of childhood by next-generation sequencing

Kingsmore

Dinwiddie

Miller

et al. 2011

Expert Review of Molecular Diagnostics

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Orphan diseases are individually uncommon but collectively contribute significantly to pediatric morbidity, mortality and healthcare costs. Current molecular testing for rare genetic disorders is often a lengthy and costly endeavor, and in many cases a molecular diagnosis is never achieved despite extensive testing. Diseases with locus heterogeneity or overlapping signs and symptoms are especially challenging owing to the number of potential targets. Consequently, there is immense need for scalable, economical, rapid, multiplexed diagnostic testing for rare Mendelian diseases. Recent advances in next-generation sequencing and bioinformatic technologies have the potential to change the standard of care for the diagnosis of rare genetic disorders. These advances will be reviewed in the setting of a recently developed test for 592 autosomal recessive and X-linked diseases.

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“…Furthermore, while traditional sample preparation, amplification, and microarray hybridization chemistries can be incorporated into a consumable via microfluidic transfer steps (9)(10)(11)(12)(13)(14)(15), the engineering, manufacturing, and quality control challenges of such consumables (and control instruments) scale with the complexity of the microfluidic architecture. An alternative approach for simplifying microarray technology for routine monitoring applications, then, is to minimize the biochemistry and processing steps that precede and interface with the microarray itself.…”

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confidence: 99%

Profiling In Situ Microbial Community Structure with an Amplification Microarray

Chandler

Knickerbocker

Bryant

et al. 2013

Appl Environ Microbiol

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The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO 3 ؊ ) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO 3 , but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications.

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A flexible and fully integrated system for amplification, detection and genotyping of genomic DNA targets based on microfluidic oligonucleotide arrays

Cited by 12 publications

References 29 publications

Carrier Testing for Severe Childhood Recessive Diseases by Next-Generation Sequencing

Carrier Testing for Severe Childhood Recessive Diseases by Next-Generation Sequencing

Adopting orphans: comprehensive genetic testing of Mendelian diseases of childhood by next-generation sequencing

Profiling In Situ Microbial Community Structure with an Amplification Microarray

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