Magna pure compact kit

1 mL of each extract was centrifuged at 5000× g for 10 min at 4 °C, and the pellet was subjected to DNA extraction using five commercial methods. Each condition was performed in triplicate. The methods used were: (1) QIAmp BiOstic Bacteremia kit (BI, Qiagen, Hilden, Germany); (2) QIAmp DNA Microbiome kit (MI, Qiagen); (3) AMPure XP (MB, Beckman-Coulter, Brea, CA, USA); (4) DNeasy Blood and Tissue kit (BT, Qiagen); (5) MagNA Pure Compact Kit (MA, Roche, Meylan, France) (Table 1). Manufacturers’ protocols were performed with the following details: for the BT kit, the Gram-positive optional steps were performed; for the MA kit, all the optional lysis steps were performed, including the addition of Bacterial Lysis Buffer (Roche), enzymatic cocktail, and proteinase K, and the boiling step; for the MB kit, the same optional lysis steps as for the MA kit were performed. For all kits, DNA was eluted in 50 µL of elution buffer or water, as per the manufacturers’ recommendations. For each condition, extracted DNA was quantified on a Qubit 2.0 fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Genetic analyses were performed for three patients with DPAGT1-CDG. RNA and/or genomic DNA was extracted from patient-derived fibroblasts or whole blood using the MagNA Pure Compact Kit (Roche Applied Sciences) according to the manufacturer’s instructions. Genetic analysis was performed by conventional Sanger sequencing, or by DNA massive parallel sequencing of either the whole exome or of a customised panel of 43 CDG-associated genes (SureSelect, Agilent, Santa Clara, California). All massive parallel sequencing was performed in a Hiseq2000 sequencer (Illumina, San Diego, California). After alignment of the reads and annotation of single nucleotide variants (SNVs), the latter were filtered by segregation analysis, population frequency and bioinformatic analysis to select pathogenic candidates.
DPAGT1 transcriptional profile analysis was performed using specific primers for amplifying the entire coding DPAGT1 cDNA and employing the SuperScript^®Vilo^™ cDNA Synthesis Kit (Life Technologies, Carlsbad, California). The primers used for RT-PCR amplification or Sanger sequencing can be provided upon request.
The Ethics Committee of the Universidad Autónoma de Madrid approved the present study. Written informed consent from the parents or their guardians was obtained prior to analysis.