SARS-CoV-2 McGill Nextera Flex sequencing protocol_Lunascript_ARTIC.V3_5uLRT v1

How the Nextera DNA Flex Assay Works The Nextera DNA Flex library prep kit uses a bead-based transposome complex to tagment genomic DNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in one step. After it is saturated with input DNA, the bead-based transposome complex fragments a set number of DNA molecules. This fragmentation provides flexibility to use a wide DNA input range to generate normalized libraries of consistent tight fragment size distribution. Following tagmentation, a limited-cycle PCR adds Nextera DNA Flex-specific index adapter sequences to the ends of a DNA fragment. This step enables capability across all Illumina sequencing platforms. A subsequent Sample Purification Beads (SPB) cleanup step then purifies libraries for use on an Illumina sequencer. The double-stranded DNAlibrary is denatured before hybridization of the biotin probe oligonucleotide pool. PCR Amplicons for Nextera Flex When starting with PCR amplicons, the PCR amplicon must be > 150 bp. The standard clean up protocol depletes libraries < 500 bp. Therefore, Illumina recommends that amplicons < 500 bp undergo a 1.8 x sample purification bead volume ratio to supernatant during Clean Up Libraries on page 11. Shorter amplicons can otherwise be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end. More information can be found here: https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf

The XBI BioLab for life science experiments at the European XFEL

The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.

Electrochemiluminescent Screening for Methamphetamine Metabolites

The abuse of methamphetamine (MA) is to date detected and subsequently verified through the monitoring of MA and its metabolites within biological specimens. Current approaches require complex sample purification strategies...

A Novel Method for Determination of the Natural Toxin Ptaquiloside in Ground and Drinking Water

Ptaquiloside (PTA) is a carcinogenic compound naturally occurring in bracken ferns (Pteridium aquilinum). It is highly water soluble and prone to leaching from topsoil to surface and groundwaters. Due to possible human exposure via drinking water, PTA is considered as an emerging contaminant. We present a sensitive and robust method for analysis of PTA and its degradation product pterosin B (PtB) in groundwater. The method comprises two steps: sample preservation at the field site followed by sample pre-concentration in the laboratory. The preservation step was developed by applying a Plackett–Burman experimental design testing the following variables: water type, pH, filtering, bottle type, storage temperature, transportation conditions and test time. The best sample preservation was obtained by using amber glass bottles, unfiltered solutions buffered at pH 6, transported without ice, stored at 4 °C and analysed within 48 h. The recovery was 94% to 100%. The sample purification step had a pre-concentration factor of 250, and the recovery percentages of the entire method were 85 ± 2 (PTA) and 91 ± 3 (PtB). The limits of detection (LOD) of the full method were 0.001 µg L−1 and 0.0001 µg L−1 for PTA and PtB, respectively. The method enables sensitive monitoring of PTA and PtB in groundwater. Carcinogenic PTA was detected in one groundwater well (0.35 µg L−1).

Solvent front position extraction with semi-automatic device as a powerful sample preparation procedure to quantitatitation of tryptophan in human plasma

In the paper the results of the tryptophan determination in human plasma samples prepared with the novel Solvent Front Position Extraction (SFPE) technique are presented. The SFPE procedure is used for preparation of real biological sample for the first time. The results obtained using SFPE are compared with those using the classical sample preparation procedure. Under the optimal conditions, tryptophan and its internal standard were separated from other plasma compounds (matrix) as a small common zone/spot on a chromatographic plate using semiautomatic device equipped with moving pipet, which distributed developing solvent on the adsorbent layer. Tryptophan and the internal standard were evenly distributed within the small common zone from that the both substances were extracted and the solution obtained was transferred to quantitation with LC–MS and MS techniques. The determination results are satisfactory, the percentage values of relative error and RSD relative standard deviation do not exceed 5%. The procedure is characterized by simplicity, high analysis throughput, very good sample purification and seems to be easy applicable to other biological samples with these advantages mentioned.

A Simple GC-MS/MS Method for Determination of Smoke Taint-Related Volatile Phenols in Grapes

Volatile phenols (VPs) derived from smoke-exposed grapes are known to confer a smoky flavor to wine. Current methods for determination of VPs in grape berries either involve complex sample purification/derivatization steps or employ two analytical platforms for free and bound VP fractions. We report here a simple gas chromatography-tandem mass spectrometry (GC-MS/MS) method for quantification of both free and bound VPs in grapes, based on optimized (1) GC-MS/MS parameters, (2) an analyte extraction procedure, and (3) phenol glycoside hydrolysis conditions. Requiring neither sample cleanup nor a derivatization step, this method is sensitive (LOD ≤ 1 ng/g berries) and reproducible (RSD < 12% for repeated analyses) and is expected to significantly reduce the sample turnover time for smoke taint detection in vineyards.

Sequential In-Situ Carbonation Process for the Preparation of Hand Sheets with Waste Lime Mud

In the pulp and paper industry, the white liquor obtained from the causticizing green liquor in the smelting process mostly contains NaOH and Na2S. These chemicals are returned to the digester for reuse in the pulping process. The lime mud (LM) material is obtained from the reaction of the causticization process in paper industries. It is mainly composed of CaCO3; it appears with a green color with a high moisture content; and it has a small proportion of impurities such as non-process elements, for example Fe, Na, Mg, Al, Si, P, and S oxides and other toxic metals. This lime mud has poor whiteness with less efficiency due to its contaminated with impurities. The recycling or reutilizing process for lime mud and solid wastes are minimizing its toxic effect on the environment. The present study proposed to improve the whiteness of the waste lime mud by the calcination and hydration process at high temperatures and reutilizing it for hand sheets, making the process improve the paper brightness. In this study, we used a lime mud sample for calcination at 1000 °C and 1200 °C for 2 h and hydration at different times (3–24 h) with different temperatures (30–80 °C) and measured the powder whiteness and hand sheet brightness. The results indicated that after the calcination and hydration process, the lime mud sample whiteness was improved and that re-utilization with pulp for making hand sheets also can improve the paper brightness. It can be concluded that waste lime mud sample purification and the re-utilization process are more advantageous in paper industries.

Chemical Bleaching to Minimize Fluorescence Interference in Raman Spectroscopic Measurements for Sulfonated Polystyrene Solutions

Auto-fluorescence is a significant challenge for Raman spectroscopic analyses. Since fluorescence is a much stronger phenomenon than Raman scattering, even trace fluorescent impurities can overwhelm the Raman signal. Strategies to minimize fluorescence interference in Raman measurements include either an instrumental-based approach or treatment of the sample itself to minimize fluorescence. Efforts focused on sample-based treatments to reduce fluorescence interferences have generally focused on sample purification and photobleaching methodologies. In this work, we present a sample treatment approach based upon chemical bleaching to remove fluorescence from Raman measurements of aqueous solutions of sulfonated polystyrene (SPS). Synthetic batches of SPS are characterized by a wide variation in fluorescence from minimum to a catastrophic level, which greatly limits the use of Raman spectroscopy. We systematically investigate the efficacy of various sample-based treatments of the SPS samples. An important acceptance criterion is that the procedure effectively and reliably removes fluorescence without damaging the SPS component. The chemical bleaching, which involves the addition of hydrogen peroxide and incubation at 60 ℃, is found to be highly effective. The parameters affecting the bleaching efficacy are studied, including temperature, hydrogen peroxide dosage, and bleaching time. Classification models are then developed based on the drastically diverse fluorescence background levels in Raman spectra of SPS to help optimize bleaching time for each specific sample. This work serves as an example of using chemical bleaching to remove fluorescence, which is inexpensive and readily available. It can facilitate a broader use of Raman spectroscopy as a quantitative qualitative control method in industrial settings.

Deproteinization as a Rapid Method of Saliva Purification for the Determination of Carbamazepine and Carbamazepine-10,11 Epoxide

Saliva is a valuable diagnostic material that, in some cases, may replace blood. However, because of its different composition, its use requires the development of new, or the modification of existing, extraction procedures. Therefore, the aim of the study was to develop a method of saliva purification that would enable the determination of carbamazepine and its metabolite, carbamazepine-10,11 epoxide. When comparing two methods of sample purification (Solid Phase Extration (SPE) and deproteinization), it was found that the second method yielded more favorable results. A 1% formic acid solution in acetonitrile was used for extraction. The samples were shaken and centrifuged, and the supernatant obtained was evaporated and dissolved in a mobile phase, then chromatographically analyzed. The developed method was validated by determining its linearity in the range of 10–5000 ng/mL for both analytes. Intra- and inter-day precision did not exceed 14%. In order to check the usefulness of the method, both analytes were determined in the saliva samples from 20 patients treated with carbamazepine. The content of both analytes was detected and determined in all of the tested samples of saliva. It was found that the method developed is rapid, sensitive, reliable, and can be used to monitor the concentration of carbamazepine and metabolite in patients’ saliva.