RNA-combine: a toolkit for comprehensive analyses on transcriptome data from different sequencing platforms

Background Understanding the transcriptome has become an essential step towards the full interpretation of the biological function of a cell, a tissue or even an organ. Many tools are available for either processing, analysing transcriptome data, or visualizing analysis results. However, most existing tools are limited to data from a single sequencing platform and only several of them could handle more than one analysis module, which are far from enough to meet the requirements of users, especially those without advanced programming skills. Hence, we still lack an open-source toolkit that enables both bioinformatician and non-bioinformatician users to process and analyze the large transcriptome data from different sequencing platforms and visualize the results. Results We present a Linux-based toolkit, RNA-combine, to automatically perform the quality assessment, downstream analysis of the transcriptome data generated from different sequencing platforms, including bulk RNA-seq (Illumina platform), single cell RNA-seq (10x Genomics) and Iso-Seq (PacBio) and visualization of the results. Besides, this toolkit is implemented with at least 10 analysis modules more than other toolkits examined in this study. Source codes of RNA-combine are available on GitHub: https://github.com/dongxuemin666/RNA-combine. Conclusion Our results suggest that RNA-combine is a reliable tool for transcriptome data processing and result interpretation for both bioinformaticians and non-bioinformaticians.

Large-sample assessment of spatial scaling effects of the distributed wflow_sbm hydrological model shows that finer spatial resolution does not necessarily lead to better streamflow estimates

Distributed hydrological modelling moves into the realm of hyper-resolution modelling. This results in a plethora of scaling related challenges that remain unsolved. In light of model result interpretation, finer resolution output might implicate to the user an increase in understanding of the complex interplay of heterogeneity within the hydrological system. Here we investigate spatial scaling in the realm of hyper-resolution by evaluating the streamflow estimates of the distributed wflow_sbm hydrological model based on 454 basins from the large-sample CAMELS data set. Model instances were derived at 3 spatial resolutions, namely 3 km, 1 km, and 200 m. The results show that a finer spatial resolution does not necessarily lead to better streamflow estimates at the basin outlet. Statistical testing of the objective function distributions (KGE score) of the 3 model instances show only a statistical difference between the 3 km and 200 m streamflow estimates. However, results indicate strong locality in scaling behaviour between model instances expressed by differences in KGE scores of on average 0.22. This demonstrates the presence of scaling behavior throughout the domain and indicates where locality in results is strong. The results of this study open up research paths that can investigate the changes in flux and state partitioning due to spatial scaling. This will help further understand the challenges that need to be resolved for hyper resolution hydrological modelling.

Serologic titers to Leptospira in vaccinated pigs and interpretation for surveillance

Diagnosis and surveillance of pathogenic Leptospira is difficult as organisms may be intermittently shed and in small numbers. Therefore, serologic testing by the microscopic agglutination test (MAT) is the primary screening method for leptospirosis. While a MAT titer ≥1:100 is considered to be a positive result, interpretation is complicated by the use of commercial vaccines in pigs. Most guidelines for interpretation of MAT titers in pigs were published in the 1970’s and 1980’s, prior to the development of the current multivalent vaccines. We evaluated MAT titers in routinely vaccinated healthy research pigs compared to their unvaccinated cohorts. Our study confirmed previous reports that the Pomona serovar elicits minimal antibody response even after a second booster 6 months after initial vaccination. However, MAT titers of ≥1:3,200 were detected as early as 4 weeks post initial vaccination for serovars Bratislava and Icterohaemorrhagiae and remained as high as ≥1:1,600 prior to booster at 24 weeks post vaccination. Our study determined that high levels of MAT titers can occur from vaccination alone and high titers are not necessarily indicative of infection. Therefore, the interpretation of MAT titers as indicators of Leptospira infection should be readdressed.

148. Single-amplicon, Multiplex Real-time RT-PCR with Tiled Probes to Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern

Background Detection and surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is of great public health importance. Broadly accessible and inexpensive assays are needed to enhance variant surveillance and detection globally. We developed and validated a single-reaction multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations associated with variants of concern (VOC). Methods A single primer pair was designed to amplify a 348 bp region of spike. Probes were initially designed with locked nucleic acids (LNAs) to increase probe melting temperature, shorten probe length, and specifically detect 417K, E484K, and N501Y (Figure). The assay was optimized and evaluated using characterized variant sample pools. Clinical evaluation was performed on a convenience set of residual nasopharyngeal swabs, and variant calls were confirmed by SARS-CoV-2 genomic sequencing in a subset of samples. Following the initial evaluation, unmodified probes (without LNAs) were designed to detect L452R, L452Q, and E484Q. Figure. Spike SNP distinguishes mutations occurring in different lineages (A-C). Representative results of variant detection a single Spike SNP run are shown for mutations in the codons for 4177K (A) and mutations that encode 484K (B) and 501Y (C). Curves show dilutions of the following variants: blue, BEI 52286 (wild type); pink B.1.1.7; purple, B1.525; and green, P.1. Variant pools were used for B.1.17, B.1.525, and P.1 strains. Curves are displayed for a given dilution in each channel and result interpretation is shown (D). Results The lower limit of 95% detection was 2.46 to 2.48 log10 GE/mL for the three targets (~1-2 GE/reaction). Among 253 nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%), including all samples with Ct values < 30 (220/220) for the N2 target and 18/33 samples with N2 Ct values ≥ 30. Results were confirmed by SARS-CoV-2 genomic sequencing in 50/50 samples (100%). Subsequent addition of the 452R probe did not affect performance for the original targets, and probes for 452Q and 484Q performed similarly to LNA-modified probes. Conclusion The Spike SNP assay provides fast, inexpensive and sensitive detection of specific mutations associated with SARS-CoV-2 VOCs, and the assay can be quickly modified to detect new mutations in the receptor binding domain. Similar analytical performance of LNA-modified and unmodified probes presents options for future assay customization that balance the shorter probe length (LNAs) and increased accessibility (unmodified). The Spike SNP assay, if implemented across laboratories offering SARS-CoV-2 testing, could greatly increase capacity for variant detection and surveillance globally. Disclosures Colleen S. Kraft, MD, MSc, Rebiotix (Individual(s) Involved: Self): Advisor or Review Panel member

Isolation of Live Leukocytes from Human Inflammatory Muscles

In inflammatory myopathies, the self-reactive immune cells involved in muscle aggression have been studied mostly using histological assessment of muscle biopsy sections; this methodology provides the advantage of visualizing and identifying cells within the tissue, but it does not allow further investigation. To gain access to live and isolated cells, many studies utilized blood samples; however, in the absence of biological tools to discriminate the leukocytes associated with the autoimmune process from those that emerged from responses against pathogens, the information observed on circulating immune cells often lacks in specificity, and thus result interpretation may prove difficult. In order to selectively retrieve self-reactive immune cells, we developed a protocol to isolate live leukocytes from human muscle biopsies, which allows for further analysis using a large range of methodologies. The protocol uses enzymatic digestion to release live leukocytes from freshly collected skeletal muscle samples, followed by filtration and separation of the leukocytes from the myocytes by density gradient centrifugation. The isolated cells can be submitted immediately to various analysis strategies to characterize ex vivo the specific cellular and molecular mechanisms responsible for self-directed immune muscle aggression or may be placed in culture for expansion.

Czekanowski’s Diagram and Spatial Data Cluster Analysis for Planning Sustainable Development of Rural Areas

Defects in the spatial structure of agricultural land resulting from the common phenomenon of land fragmentation constitute one of the most important factors that contribute to the lack of rational land management. Reconstruction of the spatial structure of rural areas is essential for their sustainable development. The process of land consolidation is a tool that can arrange space and lead to the desired structural changes. It is reasonable to select objects for land consolidation in such a way as to obtain the best possible effect. This article presents an algorithm for grouping areas with the concentration of the external land ownership patchwork with the use of Czekanowski’s method of cluster analysis. The clusters determined this way can be treated as the whole objects subjected to land consolidation, for which the process will bring the greatest benefits in terms of the elimination of the external land ownership patchwork. The described algorithm is relatively simple to use and the graphical final form is easy for the result interpretation. It allows for multi-variant examination of the analyzed phenomenon and can be applied wherever there is access to reliable information from land registry and cadastral and GIS databases that are used to obtain a complete picture of the spatial and ownership structure of the analyzed areas.

Evaluation of the acid-neutralizing capacity and other properties of antacids marketed in Morocco

Aim. The aim of this study was to evaluate the acid-neutralizing capacity (ANC) and other properties of antacid drugs marketed in Morocco. Methods. Samples of 12 antacids were collected from pharmacies and were subjected to the test described in the US Pharmacopoeia in order to measure their ANC. Other properties such as price and sodium content were also studied. Results. All the tested brands met the minimal requirement of 5 mEq. However, Aluminum hydroxide/Magnesium hydroxide combinations showed a superior acid-neutralizing capacity over other products and oral suspensions showed better results compared to other pharmaceutical forms. Regarding the cost of antacids, Aluminum hydroxide/Magnesium hydroxide combinations and calcium carbonate/magnesium carbonate combinations showed the most favorable ANC/price ratio. Some of the antacids studied contain a high amount of sodium. Conclusion. All the antacids marketed in Morocco meet the USP requirement regarding their ANC. However, the ANC value should be included in the antacids’ labels so that both patients and physicians can choose the most appropriate product. The ANC value should be evaluated according to the dose of the active substance instead of the minimum labeled dosage in order to allow a better result interpretation.

Assessment of Partial Discharges in the Air by Application of Corona Camera

This paper reports the results of the analysis of measurements involving partial discharges (PD) occurring in the air using a corona camera (UV camera). The measurements were carried out in laboratory conditions and applied two electrode systems: needle–needle and needle–plate, in order to obtain various electric field distributions. The measurements of PDs, including a variety of alternatives, were carried out using a portable UV camera, taking into account the impact of the camera gain parameter and its distance from the PD sources. As a result, some important regularities and characteristics were identified that could significantly affect the ability to assess PDs by application of UV camera measurements. In addition, the results obtained can be employed for non-invasive diagnostic measurements performed on working power equipment and may be useful in further work on standardizing the result interpretation method obtained from measurements using a UV camera.

Validation study of canine urine cortisol measurement with the Immulite 2000 Xpi cortisol immunoassay

We report here validation of the Immulite 2000 Xpi cortisol immunoassay (Siemens; with kit lot numbers <550) for measurement of urine cortisol in dogs, with characterization of the precision (CV), accuracy (spiking-recovery [SR] bias), and observed total error (TEo = bias + 2CV) across the reportable range. Linearity assessed by simple linear regression was excellent. Imprecision, SR bias, and TEo increased markedly with decreasing urine cortisol concentration. Interlaboratory comparison studies determined range-based (RB) bias and average bias (AB). The 3 biases (SR, RB, and AB) and resulting TEo differed markedly. At 38.6 and 552 nmol/L (1.4 and 20 μg/dL), between-run CVs were 10% and 4.5%, respectively, and TEoRB were ~30% and 20%, respectively, similar to observations in serum in another validation study. These analytical performance parameters should be considered for urine cortisol:creatinine ratio (UCCR) result interpretation, given that, for any hypothetical errorless urine creatinine measurement, the error % on UCCR mirrors the error % on urine cortisol. Importantly, there is no commonly used interpretation threshold for UCCR, given that UCCR varies greatly depending on measurement methods and threshold computation. To date, there is no manufacturer-provided quality control material (QCM) with target values for urine cortisol with an Immulite; for Liquicheck QCM (Bio-Rad), between-run imprecision was ~5% for both QCM levels. Acceptable QC rules are heavily dependent on the desired total allowable error (TEa) for the QCM system, itself limited by the desired clinical TEa.