Statistical considerations for digital approaches to non-invasive fetal genotyping

Chu, Tianjiao; Bunce, Kimberly; Hogge, W. Allen; Peters, David G.

doi:10.1093/bioinformatics/btq544

Cited by 5 publications

(5 citation statements)

References 21 publications

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“…As mentioned above for HIV viral load monitoring, an ideal system would be able to achieve 3-fold resolution for as low as 500 molecules/mL. To be able to correctly resolve two different concentrations, the risk of both false positives (Type I error) and false negatives (Type II error) need to be taken into account. ,,,, Samples must give results at the desired confidence level (1-α, measure of Type I error) and demonstrate this confidence level again and again (Power: 1-β, measure of Type II error). When comparing two results, the null hypothesis is that the results come from samples that have statistically the same concentration.…”

Section: Resultsmentioning

confidence: 99%

“…The development of simple stand-alone devices for quantitative nucleic acid diagnostics would further enable diagnosis and treatment in point-of-care settings. Precise, absolute quantification of nucleic acid levels, especially at low levels of detection, would have particular impact in applications such as viral load analysis (e.g., HIV, hepatitis, cytomegalovirus (CMV), enterovirus), − bacterial detection, and quantification in food or water sources without culturing, − multiplexed diagnostics, and minimal residual disease. , Real time PCR is often considered the gold standard for nucleic acid quantification − but has limited utility in the field because it requires data collection and analysis over the entire course of the reaction, careful control of conditions, and internal calibration standards and typically gives relative levels rather than absolute concentrations. ,− Digital PCR − provides a way to obtain absolute nucleic acid levels directly using only end point analysis with high resolution and sensitivity.…”

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confidence: 99%

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Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

et al. 2011

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This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for “digital” (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12,000 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices, and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically-relevant levels of HIV viral load at the point-of-care (in plasma, <500 molecules/mL to >1,000,000 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space and thus enables multiplexing by using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general, and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip, and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In the accompanying paper by Shen et al. (JACS 2011), this approach is used to design and test digital RT-PCT devices for quantifying RNA.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

et al. 2011

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“…Collection of tissue samples was approved by the University of Pittsburgh Institutional Review Board (PRO07070298). CV samples were obtained between gestational weeks 11 and 13 from the Magee Women's Hospital Cytogenetic Screening Laboratory and carefully dissected by experienced technicians to remove maternal contamination as previously . The CV samples were obtained from three male and three female fetuses.…”

Section: Methodsmentioning

confidence: 99%

High‐resolution analysis of the human placental DNA methylome in early gestation

et al. 2020

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Background/Objective We communicate high‐read‐depth bisulfite sequencing analysis of the chorionic villus (CV) DNA methylome from samples obtained between 11 and 13 weeks gestation and samples of gestationally age‐matched maternal blood cells (MBC). Methods This was achieved through solution‐phase targeted region capture (84 Mb) of bisulfite converted human DNA. Results We identified biphasic distribution of methylation in CV and MBC genomes. We found greater numbers of intermediate methylated sites (20%‐80% methylated) in CV and greater number of high methylation sites in MBC and investigated distributions of these in promoters, introns, exons, CpG islands, CpG islands shores, and enhancers. We identified differentially methylated sites distinguishing CV and MBC. These are less likely to occur in CpG islands (CGIs), particularly those that exist outside promoters, exons, and introns. We found that gene promoter and gene body methylation patterns are associated with mRNA transcriptional profiles in CV. Despite the relative hypomethylation of CV genomes, we found that these contain DM regions that are more likely to be hypermethylated in CV relative to MBC. Conclusions Our data provide novel insight into the structure and organization of the CV epigenome, which may inform future studies of placental biology and noninvasive prenatal phenotyping.

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“…It is not trivial to tell apart the informative and non-informative SNPs (Sparks et al, 2012). The main reason for this difficulty lays in the existence of the allelic bias of the sequencing technology (Chu et al, 2010b). There are several methods for identifying the informative SNPs.…”

Section: Theorymentioning

confidence: 99%

Uncertainty of fetal fraction determination in Non-Invasive Prenatal Screening by highly polymorphic SNPs

Grendár

Loderer

Laučeková

et al. 2019

Journal of Biotechnology

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Statistical considerations for digital approaches to non-invasive fetal genotyping

Cited by 5 publications

References 21 publications

Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

High‐resolution analysis of the human placental DNA methylome in early gestation

Uncertainty of fetal fraction determination in Non-Invasive Prenatal Screening by highly polymorphic SNPs

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