Ez1 advanced xl instrument

Clinical samples were obtained from patients with symptoms related to SARS-CoV-2 infection, admitted to the Emergency Department (ED) of University Hospital “Città della Salute e della Scienza di Torino” (Torino, Italy). A nasopharyngeal swab was collected from each patient and analyzed by the Microbiology and Virology Unit of the aforementioned hospital. Samples were collected in 3 mL UTM^® swabs (Copan, Brescia, Italy). Viral RNA extraction was performed using an EZ1^® Advanced XL instrument (Qiagen, Germantown, MD, USA) with EZ1^® DSP Virus Kit (Qiagen, Germantown, MD, USA); starting volume was 200 µL, while elution volume was 90 µL. For the direct RT-PCR, 100 µL of UTM diluents swab from primary samples were heated at 95 °C for 10 min and then cooled at 4 °C. Subsequently, samples were immediately amplified.

In this retrospective study, genetic results were compiled from all 861 referrals for clinical genome sequencing at our center due to suspected neuromuscular disorders including patients with ataxia and/or spastic paraparesis from January 2016 to October 2022. The cohort included patients of all ages from prenatal cases to elderly patients. Genomic DNA was isolated from whole blood using QIAsymphony (QIAGEN, Hilden, Germany) and the QIAsymphony DSP DNA Midi Kit (cat. no. 937255, QIAGEN, Hilden, Germany) according to manufacturer's protocol. For the prenatal cases, DNA was extracted from a chorion villi biopsy or fetal tissue using the EZ1 Advanced XL instrument (QIAGEN, Hilden, Germany) and the EZ1 DNA Tissue Kit (cat. no. 953034, QIAGEN, Hilden, Germany) following manufacturer's protocol. Individuals that were analyzed clinically before December 2019 (n = 455) were previously presented by Stranneheim et al. (13 (link)). Over the years, the number of genes in the NMD panel was updated regularly. Furthermore, bioinformatic pipelines were continuously improved (13 (link)). Therefore, prior to compiling the results, all negative cases were reanalyzed with the latest NMD gene panel and bioinformatic pipeline as outlined in Figure 1 and described below.

Material from buccal swabs was collected on FTA cards from 12 Danes and 1 Argentinian individual. The DNA was extracted from two 3 mm punches using the Trace Tip Dance (TD) procedure of the EZ1 DNA Investigator Kit (Qiagen, Hilden, Germany) and the EZ1 Advanced XL Instrument (Qiagen). The extracted DNA was eluted in 50 µL water.
Blood samples from 10 Italians, 2 Ethiopians and 7 individuals of Eastern European, Moroccan, Iraqi, Indian, Korean, Chinese and Brazilian origin, respectively, were used in this study. The DNA was extracted from 200 µL blood using the spin protocol of the QIAamp® DNA Mini Kit (Qiagen). The DNA extractions were quantified using the Qubit 3.0 instrument (Thermo Fisher Scientific, Waltham, MA, USA).

DNA extraction was performed with the EZ1 Advanced XL instrument, Qiagen® from colonies isolated in 4 to 10 days culture at 25°C. A portion of colonies on culture medium was introduced into a bead tube containing 700 μl of lysis buffer G2 (supplied with the EZ1 DNA Investigator kit, Qiagen®). This was followed by mechanical lysis with FastPrep®-24: 2 runs and by centrifugation at 10,000 rpm for 1 minute. Finally, 200 μl of the supernatant was removed and placed in an EZ1 flat tube before launching the EZ1 Advanced XL instrument according to the instructions of the manufacturer. Genomic DNA was extracted, eluted in 200 μl elution buffer, and stored at −20°C for downstream analyses.

Total nucleic acid (TNA) was extracted from 400 µL of Universal Transport Media containing the NP and OP specimens and eluted into 110 µL using the EZ1 Advanced XL Instrument with the EZ1 Mini Viral 2.0 Kit (Qiagen) following the manufacturer's instructions. Seven specimens were extracted at a time with an extraction control consisting of nuclease‐free water.

Tape strips were transferred into Lysing Matrix E tubes (MP Biomedicals), and 500 μL of ATL Buffer (Qiagen) was added. Samples were subjected to bead-beating with FastPrep-24 Instrument (MP Biomedicals) at a speed of 6.0 m/s for 40 s. Subsequently, samples were centrifuged at 16,000 g for 5 min and 200 μl of supernatant was treated with 10 μl of Proteinase K (Qiagen) and incubated at 56 °C for 15 min. DNA was extracted using EZ1 Advanced XL Instrument (Qiagen) with the EZ1 DNA Tissue Kit (Qiagen) with an elution volume of 100 μl and was quantified using Qubit dsDNA HS Assay Kit (Life Technologies) and later stored in a − 20 °C freezer.

RNA was isolated from blood or serum samples using the EZ1 Virus Mini Kit v2.0 on an EZ1 Advanced XL instrument (Qiagen, Hilden, Germany). DNA was removed using the Turbo DNA-free Kit (Thermo Scientific Inc.) according to the manufacturer’s instructions. cDNA was synthesized with the Maxima H minus double-stranded cDNA synthesis kit (Thermo Fisher Scientific Inc.) using random hexamers. In the second step of cDNA synthesis, we extended the incubation at 16 °C to 2 h. NGS libraries were prepared and sequenced in the same manner as for amplicon sequencing.