Hipure tissue blood dna kit

Genomic DNA of the gonads from male, female, and intersex eels were extracted using the HiPure Tissue & Blood DNA Kit (Magen). The concentration and quality of DNA were detected using an Agilent 2100 Bioanalyzer (Agilent Technologies) and the integrity was detected using 1% agarose gel electrophoresis. Two microgrammes of gDNA was repaired using a NEB Next FFPE DNA Repair Mix kit (New England Biolabs). The ONT template prep kit (Oxford Nanopore Technologies) was used to prepare the template according the manufacturer’s instructions. The library was sequenced using the ONT sequencing reagent kit (Oxford Nanopore Technologies) according to the manufacturer’s instructions on the ONT PromethION48 platform with PromethION Flow Cells [20 (link)]. The fast5 format data were converted to fastq format for QC analysis using Basecall with ONT’s Guppy software [21 ]. The fastq data were further filtered to remove the adapters, short reads (length < 500 bp), and low-quality reads (MeanQual < 6).

Genomic DNA was extracted from peripheral blood samples of the three patients and family members of Patient 1 using a HiPure Tissue & Blood DNA Kit (Magen, Guangzhou, China) according to the manufacturer’s instructions. All three FGF23 exons, 10 GALNT3 coding exons, and five KL exons including intron-exon boundary regions were PCR-amplified with specific sets of upstream and downstream primers (12 (link), 13 (link)). Primer sequences are shown in Table 1. PCR amplification products were purified and sequenced using an automated sequencer (ABI 3730XL; Applied Biosystems, Foster City, CA, USA).
The obtained sequences were aligned against the human genome reference sequence hg38 (GRCh38; https://blast.ncbi.nlm.nih.gov/Blast.cgi). The possible functional significance of the potential candidate variant was assessed using Mutation Taster (http://www.mutationtaster.org/) and Polymorphism Phenotyping (PolyPhen-2; http://genetics.bwh.harvard.edu/pph2/). The impact of the variant on protein structure was further predicted by SWISS-MODEL (https://www.swissmodel.expasy.org). The variant was compared with the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar) to exclude previous reports.