Qiasymphony dna mini kit

This study was performed using samples collected for the REDS-III Brazil SCD cohort study (9) eligible participants were randomly selected that included institutions in four Brazilian states: São Paulo (Hospital das Clínicas), Minas Gerais (Hemominas), Rio de Janeiro (Hemorio) and Pernambuco (Hemope).
The study was approved by the local ethical review committee of participating institutions,namely, the Pro-Sangue foundation, Hemominas foundation, Hemope foundation and Hemorio blood bank. Also, the study was approved by the REDS-II collaborating centers (Blood Systems Research Institute/University of California at San Francisco, San Francisco, CA) and data-coordinating center (Westat, Inc.) in the United States.
The samples were collected in an EDTA tube, centrifuged at 3500rpm, and plasma was separated from cells. Both components were frozen and shipped to the central laboratory at the University of Sao Paulo for further testing.
DNA extraction was performed using the QIAsymphony apparatus (Qiagen, Germany) and the QIAsymphony DNA Mini Kit (Qiagen, Germany), following the manufacturer's instructions and protocol.

DNA extraction from all sample types was performed using either the Qiagen DNeasy Kit (for tissue DNA extraction) or QIAsymphony^® SP robot (QIAGEN, Valencia, CA, USA) and QIAsymphony^® DNA Mini Kit (for blood and blood culture DNA extractions), per the manufacturer’s recommended protocols. DNA quality and concentration for blood, tissue, and spiked blood samples was assessed by 260/280 nm OD measurement, as described previously [18 (link)].

DNA was extracted from whole blood samples using QIAsymphony DNA Mini Kit at QIAsymphony automated system (both Qiagen, Valencia, CA, United States). The extracted DNA was eluted into a final volume of 90 µl with an elution buffer. Genomic DNA purity was evaluated using NanoDrop 2000 (thresholds: 260/280 ratio ≈1.8 and 260/230 ratio between 1.8 and 2.2). DNA quantification was performed with Qubit^® 4 fluorometer using the Qubit^® dsDNA HS assay (both Life Technologies, Carlsbad, CA, United States). If sample input does not reach the desired quality, we reject it and request sample recollection.
Genomic DNA was fragmented into 350 bp inserts by Covaris ME220 ultrasonicator (Covaris, Woburn, MA, United States), with the following treatment settings–DNA input: 1 µg (final volume of 55 μl, completed with resuspension buffer); peak incident power: 50 W, duty factor: 20%, cycles per burst: 200, duration: 65 s, and temperature set point: 20 C. The identity of all specimens remained unknown to wet lab staff throughout the workflow.

GMO quantifications were performed by qPCR according to norms ISO 21569, 21570, 21571 and 24276. 100g of samples were grinded and homogenized in a GM 200 grinder (Retsch, Germany). 200 mg were used to extract DNA with QIAsymphony DNA mini kit (Qiagen, Germany) and each subsample was amplified twice by real-time PCR Rotor Gene (Qiagen, Germany). LOD and LOQ were respectively 0.01 and 0.1% (determined on IRMM or AOCS standard material). The global uncertainty of measurements was calculated by adding uncertainty of measurements of the methods (calculated with the standard deviation obtained on certified materials) and standard deviation of the 4 repeats per sample. These global uncertainties ranged from 20 to 33%. Plant contents were first determined by a screening PCR for taxon-specific markers (HMG for maize, lectin for soy, acc for oilseed rape, gluA3 for sugar beet, Qgene for wheat, acp1 for cotton, UGPase for potato). Then the presence of GM material was assessed by a screening PCR for generic recombinant markers (CAMVp35S, Tnos, FMVp35S) and 22 GM-specific events (GMOs authorized in diets imported in European Union) were searched in maize (12) and soy (2) for all the diets, and in addition oilseed rape (3), potato (1), sugar beet (1) and non-commercialized GMOs like rice and wheat, in the 3 diets containing the highest proportion of GMOs.

Genomic DNA was extracted from peripheral blood using a QIAsymphony DNA Mini Kit (Qiagen, Hilden, Germany). A custom capture panel (Dxome, Seoul, Republic of Korea) targeting coding exons and intron-exon boundaries of 497 genes related to hematologic disorders (Supplementary Table S1) was used. Prepared libraries were hybridized with capture probes and sequenced as paired-end reads (2 × 150 bp) using NextSeq 550Dx (Illumina, San Diego, CA, USA). NGS data analysis was performed with DxSeq Analyzer (Dxome). Single-nucleotide variants, small insertion and deletions, and copy number variants were identified [13 (link), 14 (link)]. All variants were classified and reported as a 5-tier system according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines [15 (link)]. Genes included in our panel that are associated with HHAs are classified according to their relevant phenotypes in Table 1.
Table 1

Genes included in our panel classified according to phenotype

Phenotype	Genes included in the panel
Membranopathy	ANK1, SPTB, SPTA1, SLC4A1, EPB42, EPB41, PIEZO1, RHAG
Enzymopathy	G6PD, PKLR, AK1, AK2, ALAS2, GPI, NT5C3A, GCLC, GPX1, GSR, GSS, HK1, BPGM, TPI1, PFKL, PFKM, PGK1, ALDOA
Hemoglobinopathy	HBB, HBA1, HBA2, HBD