The insufficient standardization of diagnostic next-generation sequencing (NGS) still limits its implementation in clinical practice, with the correct detection of mutations at low variant allele ...frequencies (VAF) facing particular challenges. We address here the standardization of sequencing coverage depth in order to minimize the probability of false positive and false negative results, the latter being underestimated in clinical NGS. There is currently no consensus on the minimum coverage depth, and so each laboratory has to set its own parameters. To assist laboratories with the determination of the minimum coverage parameters, we provide here a user-friendly coverage calculator. Using the sequencing error only, we recommend a minimum depth of coverage of 1,650 together with a threshold of at least 30 mutated reads for a targeted NGS mutation analysis of ≥3% VAF, based on the binomial probability distribution. Moreover, our calculator also allows adding assay-specific errors occurring during DNA processing and library preparation, thus calculating with an overall error of a specific NGS assay. The estimation of correct coverage depth is recommended as a starting point when assessing thresholds of NGS assay. Our study also points to the need for guidance regarding the minimum technical requirements, which based on our experience should include the limit of detection (LOD), overall NGS assay error, input, source and quality of DNA, coverage depth, number of variant supporting reads, and total number of target reads covering variant region. Further studies are needed to define the minimum technical requirements and its reporting in diagnostic NGS.
Abstract
Extramedullary disease (EMM) represents a rare, aggressive and mostly resistant phenotype of multiple myeloma (MM). EMM is frequently associated with high-risk cytogenetics, but their ...complex genomic architecture is largely unexplored. We used whole-genome optical mapping (Saphyr, Bionano Genomics) to analyse the genomic architecture of CD138+ cells isolated from bone-marrow aspirates from an unselected cohort of newly diagnosed patients with EMM (n = 4) and intramedullary MM (n = 7). Large intrachromosomal rearrangements (> 5 Mbp) within chromosome 1 were detected in all EMM samples. These rearrangements, predominantly deletions with/without inversions, encompassed hundreds of genes and led to changes in the gene copy number on large regions of chromosome 1. Compared with intramedullary MM, EMM was characterised by more deletions (size range of 500 bp–50 kbp) and fewer interchromosomal translocations, and two EMM samples had copy number loss in the 17p13 region. Widespread genomic heterogeneity and novel aberrations in the high-risk
IGH/IGK/IGL
, 8q24 and 13q14 regions were detected in individual patients but were not specific to EMM/MM. Our pilot study revealed an association of chromosome 1 abnormalities in bone marrow myeloma cells with extramedullary progression. Optical mapping showed the potential for refining the complex genomic architecture in MM and its phenotypes.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Motivation
Optical mapping is a complementary technology to traditional DNA sequencing technologies, such as next-generation sequencing (NGS). It provides genome-wide, high-resolution ...restriction maps from single, stained molecules of DNA. It can be used to detect large and small structural variants, copy number variations and complex rearrangements. Optical mapping is affected by different kinds of errors in comparison with traditional DNA sequencing technologies. It is important to understand the source of these errors and how they affect the obtained data. This article proposes a novel approach to modeling errors in the data obtained from the Bionano Genomics Inc. Saphyr system with Direct Label and Stain (DLS) chemistry. Some studies have already addressed this issue for older instruments with nicking enzymes, but we are unaware of a study that addresses this new system.
Results
The main result is a framework for studying errors in the data obtained from the Saphyr instrument with DLS chemistry. The framework’s main component is a simulation that computes how major sources of errors for this instrument (a false site, a missing site and resolution errors) affect the distribution of fragment lengths in optical maps. The simulation is parametrized by variables describing these errors and we are using a differential evolution algorithm to evaluate parameters that best fit the data from the instrument. Results of the experiments manifest that this approach can be used to study errors in the optical mapping data analysis.
Availability and implementation
Source codes supporting the presented results are available at: https://github.com/mvasinek/olgen-om-error-prediction. The data underlying this article are available on the Bionano Genomics Inc. website, at: https://bionanogenomics.com/library/datasets/.
Supplementary information
Supplementary data are available at Bioinformatics online.
A combined experimental and modelling study of the structure of a laminar premixed ultra–lean (ϕ=0.33) dimethyl ether/air flame at atmospheric pressure and an elevated temperature was carried out. ...The work aimed to apply tunable diode laser absorption spectroscopy to the identification of various flame regimes that are relevant to the oxidation of dimethyl ether. One-dimensional calculations employing burner-stabilized flame assumptions confirmed the significance of low-temperature combustion chemistry. A stable double-flame structure was predicted using state-of-the-art chemical kinetic schemes and was revealed by the experimental observations. The feasibility of the novel experimental strategy based on the preheated flat-flame burner and scanned wavelength modulation spectroscopy was investigated in this context. The absorption features of hot water and the hydroxyl radical near 1572 nm were selected as appropriate targets for distinguishing the transition from the cool flame regime to the hot flame regime. Discrepancies in the water line position and intensities were found within the 1509 nm region.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
ABSTRACT
A global uncertainty analysis is performed for three current mechanisms describing the low‐temperature oxidation of dimethyl ether (Aramco Mech 1.3, Metcalfe et al., Int J Chem Kinet 2013, ...45, 638–675; Zheng et al., Proc Combust Inst 2005, 30, 1101–1109; Liu et al., Combust Flame 2013, 160, 2654–2668) with application to simulations of species concentrations (CH2O, H2O2, CH3OCHO) corresponding to existing data from an atmospheric pressure flow reactor and high‐pressure ignition delays. When incorporating uncertainties in reaction rates within a global sampling approach, the distributions of predicted targets can span several orders of magnitude. The experimental profiles, however, fall within the predictive uncertainty limits. A variance‐based sensitivity analysis is then undertaken using high dimensional model representations. The main contributions to predictive uncertainties come from the CH3OCH2 + O2 system, with isomerization, propagation, chain‐branching, secondary OH formation, and peroxy–peroxy reactions all playing a role. The response surface describing the relationship between sampled reaction rates and predicted outputs is complex in all cases. Higher order interactions between parameters contribute significantly to output variance, and no single reaction channel dominates for any of the conditions studied. Sensitivity scatter plots illustrate that many different parameter combinations could lead to good agreement with specific sets of experimental data. The Aramco scheme is then updated based on data from a recent study by Eskola et al. (J Phys Chem A, in press), which presents quite different temperature and pressure dependencies for the rates of CH3OCH2O2 → CH2OCH2O2H and CH2OCH2O2H → OH+2CH2O compared with currently used values and includes well skipping channels. The updates from Eskola worsen the agreement with experiments when used in isolation. However, if the rate of the CH2OCH2O2H + O2 channel is subsequently reduced, very good agreement can be achieved. Owing to the complex nature of the response surface, the tuning of this channel remains speculative. Further detailed studies of the temperature and pressure dependence of the CH3OCH2O2 + O2, CH2OCH2O2H + O2 system are recommended to reduce uncertainties within current dimethyl ether mechanisms for low‐temperature conditions.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
The change of zero order entropy is studied over different strategies of grammar production rule selection. The two major rules are distinguished: transformations leaving the message size intact and ...substitution functions changing the message size. Relations for zero order entropy changes were derived for both cases and conditions under which the entropy decreases were described. In this article, several different greedy strategies reducing zero order entropy, as well as message sizes are summarized, and the new strategy MinEnt is proposed. The resulting evolution of the zero order entropy is compared with a strategy of selecting the most frequent digram used in the Re-Pair algorithm.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
This communication reports technical notes on the development and application of an automated line-shape fitting procedure for wavelength modulation spectroscopy (WMS). Near-infrared transitions of ...carbon dioxide (CO
) around 1573 nm were measured in vertical cold (nonreacting) flow of CO
at atmospheric pressure using WMS with demodulation at second harmonic frequency. Semi-empirical model based on the set of so-called Gabor functions was developed and parameters of Lorentzian line-shape profile and its asymmetry resulting from simultaneous frequency and amplitude response of the current-modulated semiconductor laser were determined. Nonlinear least-square fitting procedure employing differential evolution algorithm was successfully utilized for performing this task. Line-shape fitting procedure enabling efficient signal de-noising and background subtraction of wavelength modulation spectra was implemented into an open-source code.
The availability of reliable modeling tools and input data required for the prediction of surface removal rate from the lithium fluoride targets irradiated by the intense photon beams is essential ...for many practical aspects. This study is motivated by the practical implementation of soft X-ray (SXR) or extreme ultraviolet (XUV) lasers for the pulsed ablation and thin film deposition. Specifically, it is focused on quantitative description of XUV laser-induced desorption/ablation from lithium fluoride, which is a reference large band-gap dielectric material with ionic crystalline structure. Computational framework was proposed and employed here for the reconstruction of plume expansion dynamics induced by the irradiation of lithium fluoride targets. The morphology of experimentally observed desorption/ablation craters were reproduced using idealized representation (two-zone approximation) of the laser fluence profile. The calculation of desorption/ablation rate was performed using one-dimensional thermomechanic model (XUV-ABLATOR code) taking into account laser heating and surface evaporation of the lithium fluoride target occurring on a nanosecond timescale. This step was followed by the application of two-dimensional hydrodynamic solver for description of laser-produced plasma plume expansion dynamics. The calculated plume lengths determined by numerical simulations were compared with a simple adiabatic expansion (blast-wave) model.
The current development in the Next-Generation Sequencing (NGS) technologies and the gradual growth of its use leads to the production of a huge amount of sequencing data. There is a need to ...efficiently transfer and store these data. This article introduces a novel lossy transformation algorithm for quality scores in sequencing data. Asymptotically, the algorithm preserves the likelihood of occurrence of particular quality score in individual positions of quality sequences. Such a model may be advantageous for sequencing data with very high coverage, such as targeted amplicon sequencing data. In experimental results, we show the comparison of characteristics of this algorithm with other algorithms performing lossy compression of the quality sequences. The proposed algorithm can be easily integrated into current sequencing pipelines. In this work we apply the algorithm to SAM files, which are then compressed into BAM files. The goal of the algorithm is to modify the data so that the subsequent Deflate algorithm application achieves a better compression ratio while minimizing the negative effects in the subsequent variant calling.
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