Implementation of non-invasive prenatal testing by semiconductor sequencing in a genetic laboratory

Objectives: To implement non-invasive prenatal testing (NIPT) for fetal aneuploidies with semiconductor sequencing in an academic cytogenomic laboratory and to evaluate the first 15-month experience on clinical samples. Methods: We validated a NIPT protocol for cell-free fetal DNA sequencing from maternal plasma for the detection of trisomy 13, 18 and 21 on a semiconductor sequencing instrument. Fetal DNA fraction calculation for all samples and several quality parameters were implemented in the workflow. One thousand eighty-one clinical NIPT samples were analysed, following the described protocol. Results: Non-invasive prenatal testing was successfully implemented and validated on 201 normal and 74 aneuploid samples. From 1081 clinical samples, 17 samples showed an abnormal result: 14 trisomy 21 samples, one trisomy 18 and one trisomy 16 were detected. Also a maternal copy number variation on chromosome 13 was observed, which could potentially lead to a false positive trisomy 13 result. One sex discordant result was reported, possibly attributable to a vanishing twin. Moreover, our combined fetal fraction calculation enabled a more reliable risk estimate for trisomy 13, 18 and 21. Conclusions: Non-invasive prenatal testing for trisomy 21, 18 and 13 has a very high specificity and sensitivity. Because of several biological phenomena, diagnostic invasive confirmation of abnormal results remains required

WisecondorX : improved copy number detection for routine shallow whole-genome sequencing

Shallow whole-genome sequencing to infer copy number alterations (CNAs) in the human genome is rapidly becoming the method par excellence for routine diagnostic use. Numerous tools exist to deduce aberrations from massive parallel sequencing data, yet most are optimized for research and often fail to redeem paramount needs in a clinical setting. Optimally, a read depth-based analytical software should be able to deal with single-end and low-coverage datathis to make sequencing costs feasible. Other important factors include runtime, applicability to a variety of analyses and overall performance. We compared the most important aspect, being normalization, across six different CNA tools, selected for their assumed ability to satisfy the latter needs. In conclusion, WISECONDOR, which uses a within-sample normalization technique, undoubtedly produced the best results concerning variance, distributional assumptions and basic ability to detect true variations. Nonetheless, as is the case with every tool, WISECONDOR has limitations, which arise through its exclusiveness for non-invasive prenatal testing. Therefore, this work presents WisecondorX in addition, an improved WISECONDOR that enables its use for varying types of applications. WisecondorX is freely available at https://github.com/CenterForMedicalGeneticsGhent/WisecondorX

Preface : in silico pipeline for accurate cell-free fetal DNA fraction prediction

Objective During routine noninvasive prenatal testing (NIPT), cell-free fetal DNA fraction is ideally derived from shallow-depth whole-genome sequencing data, preventing the need for additional experimental assays. The fraction of aligned reads to chromosome Y enables proper quantification for male fetuses, unlike for females, where advanced predictive procedures are required. This study introduces PREdict FetAl ComponEnt (PREFACE), a novel bioinformatics pipeline to establish fetal fraction in a gender-independent manner. Methods PREFACE combines the strengths of principal component analysis and neural networks to model copy number profiles. Results For sets of roughly 1100 male NIPT samples, a cross-validated Pearson correlation of 0.9 between predictions and fetal fractions according to Y chromosomal read counts was noted. PREFACE enables training with both male and unlabeled female fetuses. Using our complete cohort (n(female) = 2468, n(male) = 2723), the correlation metric reached 0.94. Conclusions Allowing individual institutions to generate optimized models sidelines between-laboratory bias, as PREFACE enables user-friendly training with a limited amount of retrospective data. In addition, our software provides the fetal fraction based on the copy number state of chromosome X. We show that these measures can predict mixed multiple pregnancies, sex chromosomal aneuploidies, and the source of observed aberrations

Robot-Assisted Epiretinal Membrane Peeling: A Prospective Assessment of Pre- and Intra-Operative Times and of Surgeons' Subjective Perceptions.

PURPOSE The Preceyes Surgical System (PSS) is a robotic assistive device that may enhance surgical precision. This study assessed pre- and intra-operative times and surgeons' perceptions of robot-assisted epiretinal membrane peeling (RA-MP). METHODS We analyzed the time requirement of three main tasks: the preparation of the PSS (I), patient preparation (II), and surgery (III). Following surgery, the surgeons were asked questions about their experience. RESULTS RA-MP was performed in nine eyes of nine patients. Task I required an average time of 12.3 min, initially taking 15 min but decreasing to 6 min in the last surgery. Task II showed a mean time of 47.2 (range of 36-65) min. Task III had a mean time of 72.4 (range of 57-100) min. A mean time of 27.9 (range of 9-46) min was necessary for RA-MP. The responses to the questionnaire revealed a trend towards increasing ease and reduced stress as familiarity with the PSS increased. CONCLUSIONS A substantial reduction in pre- and intra-operative times, decreasing to a total of 115 min, was demonstrated. RA-MP was positively anticipated by the surgeons and led to no hand or arm strain while being more complex than manual MP

String Field Theory Projectors for Fermions of Integral Weight

The interaction vertex for a fermionic first order system of weights (1,0) such as the twisted bc-system, the fermionic part of N=2 string field theory and the auxiliary \eta\xi system of N=1 strings is formulated in the Moyal basis. In this basis, the Neumann matrices are diagonal; as usual, the eigenvectors are labeled by \kappa\in\R. Oscillators constructed from these eigenvectors make up two Clifford algebras for each nonzero value of \kappa. Using a generalization of the Moyal-Weyl map to the fermionic case, we classify all projectors of the star-algebra which factorize into projectors for each \kappa-subspace. At least for the case of squeezed states we recover the full set of bosonic projectors with this property. Among the subclass of ghost number-homogeneous squeezed state projectors, we find a single class of BPZ-real states parametrized by one (nearly) arbitrary function of \kappa. This class is shown to contain the generalized butterfly states. Furthermore, we elaborate on sufficient and necessary conditions which have to be fulfilled by our projectors in order to constitute surface states. As a byproduct we find that the full star product of N=2 string field theory translates into a canonically normalized continuous tensor product of Moyal-Weyl products up to an overall normalization. The divergent factors arising from the translation to the continuous basis cancel between bosons and fermions in any even dimension.Comment: LaTeX, 1+23 pages, minor improvements, references adde

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies