Maxwell rsc whole blood dna kit

Genomic DNA was isolated from EDTA blood samples with the Maxwell RSC Whole Blood DNA Kit using a Maxwell RSC instrument (Promega, Dübendorf, Switzerland). DNA from 12 affected and 8 unaffected animals was genotyped on illumina_HD canine BeadChips containing 220,853 markers (Neogen, Lincoln, NE, USA). The raw SNV genotypes are available in File S1. We did not have complete pedigree information on all 20 dogs that were genotyped on the SNV arrays. Some of the dogs were closely related, including, for example, one complete family with two affected full siblings and one healthy puppy, which was used for parametric linkage analysis.

Genomic DNA was isolated from blood using either the Gentra PureGene blood kit (Qiagen) or the Maxwell RSC whole blood DNA kit (Promega). All dogs were genotyped for an ARHGEF10 deletion (i.e., the LPN1 allele) as previously described [11 (link)], and only dogs that were homozygous wild type or heterozygous for the ARHGEF10 allele were selected for genotyping on the SNP arrays. The phenotypic characterization of PN in Leonberger dogs has been described elsewhere [5 (link)] and the previously established criteria to select cases and controls were applied [11 (link)]. Samples from a total of 7455 Leonbergers, including 922 dogs with detailed phenotype records (Additional file 1) were used during this study. Initially, a cohort of 314 dogs was used for the GWAS; later a follow-up cohort of 608 dogs with detailed phenotype information was collected, in addition to 6533 dogs that were submitted without known phenotype status. A total of 56 PN-affected Leonberger dogs homozygous for the deletion in ARHGEF10 were excluded from the mapping studies.

DNA was extracted from peripheral blood samples using an automated extraction method (Maxwell RSC Whole Blood DNA Kit, Promega, Madison, WI, USA) and following the manufacturer’s instructions.
A targeted gene sequencing panel was designed to evaluate the reported genes associated with HHT and other arteriovenous malformation related syndromes (ACVRL1, BMPR2, BMP10, BMPR1A, ELMO2, ENG, EPHB4, GDF2, PIK3CA, RASA1, SMAD1, SMAD4, SMAD6, TEK). Nonacus Cell3™ Target Enrichment System (protocol v1.2.2, Nonacus, Birmingham, UK) was used. This method is based on the enzymatic fragmentation of 100 ng of genomic DNA followed by end-repair and A-tailing. Adapter sequences for massive parallel sequencing are then joined to the DNA fragments. In addition, unique molecular indexes (UMIs) are added to identify individual samples and allow sample multiplexing. All the DNA fragments from selected individuals are then mixed to create the sequencing library. The fragmented genomic DNA library is hybridized to the biotinylated capture customized probes. Subsequently, streptavidin beads are used to recover those DNA fragments that are attached to the biotin probes. The library generated was sequenced with a MiSeq sequencer (Illumina, San Diego, CA, USA), following the protocol developed by the company, and generating 2 × 150 pair-end reads.

DNA was extracted in the Precision Genomics CAP-certified clinical laboratory from DNA Genotek ORAcollect buccal swab samples using the Promega Maxwell RSC Whole Blood DNA kit on the Promega Maxwell RSC system. Libraries were prepared using Illumina TruSight and/or Nextera Flex reagents and sequenced using the Illumina Miseq and Nextseq platforms to at least 100× of 2 × 150 bp.
Resultant sequence data were demultiplexed using bcl2fastq [28 ] and aligned to the reference human genome sequence GrCh37 using BWA-MEM v0.7.17 [29 (link)]. Sequencing quality was assessed using Picard v2.18.21+ [30 ] and FastP v0.20.0 [31 (link)]. Indel variant and single-nucleotide variants (SNVs) were called using the Genome Analysis Tool Kit (GATK v4.0.12+) [32 (link)] and copy number variants (CNVs) were called using DECoN v1.0.2 [33 (link)]. The genetic test was validated to detect single nucleotide polymorphisms (SNPs), and insertions and deletions (Indels) up to 20 bp in length and copy number variants (CNVs) of at least one exon in length. Variants >20 bp and less than one exon in length may have been detected with reduced sensitivity. CNVs were confirmed by MLPA and SNVs and indels with coverage <30× or <30% minor allele frequency were confirmed by Sanger sequencing.

For the determination of polymorphisms, blood was collected in a Vacutainer containing EDTA any time during the participant’s first two treatment cycles and stored at -20° Celsius until further processing. Genomic DNA was purified from whole blood samples using the Maxwell® RSC Whole Blood DNA kit (Promega, Italy). DNA was amplified using the TaqMan® Genotyping Master Mix (Thermo Fisher Scientific, USA) and analyzed according to manufacturer’s instructions for the presence of selected SNP allele variants by real-time PCR technique (ABI-7900; Applied Biosystems, Italy) using TaqMan SNP Genotyping assays (Thermo Fisher Scientific, USA) specific for each gene of interest. Additional file 1: Table S1 shows the SNPs analyzed, selected based on their reported association with neuropathy induced by anticancer agents. Real-time PCR was carried out in 384-wells plates prepared with automatic liquid handling (epMotion 5075; Eppendorf, Italy). Completed PCR plates were analyzed using the TaqMan® Genotyper Software (Thermo Fisher Scientific, USA).

From each person, 2 mL of peripheral venous blood was collected, and DNA was extracted using the Maxwell RSC Whole Blood DNA Kit (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. A Pearl Nanophotometer (Implen GmbH, Munich, Germany) was used in determining the DNA concentration and purity.