Tianamp blood genomic dna purification kit

Genomic DNA was extracted and purified from peripheral blood of the two patients and their parents using TIANamp Blood Genomic DNA Purification Kit (Tiangen Biotech, Beijing, China). Whole-exome sequencing was applied to test mutation of genes in both patients. Approximately 3 µg of genomic DNA was randomly fragmented. An exome enriched kit (Agilent, Santa Clara, CA) was used to obtain the coding exons and flanking intronic regions. The sequencing was performed using HiSeq2000 sequencer (Illumina, San Diego, CA). The obtained mean exome coverage was over 99.2%, and average sequencing depth of each sample was 100%. Raw data obtained from the sequencer were further analyzed including read alignment, variant calling, and annotation by SinoPath Enterprise Ltd (Beijing, China). Low-quality reads (quality score ≤ 20 and sequencing depth ≤ 5) in the raw data were removed. Filtered reads were aligned to the human reference genome (UCSC hg19 Feb.2009) using the Burrows-Wheeler Aligner (Raney et al., 2014 (link)). Single-nucleotide variants (SNVs) and small insertions/deletions (indels) can be detected. Annotation was carried out by ANNOVAR for gene information, protein functional predictions, and population allele frequencies (Wang et al., 2010 (link)). Variants outside of coding regions and greater than 1% MAF (minor allele frequency) in the population were excluded.

After obtaining written informed consent from all participating individuals, peripheral blood samples and an amniotic fluid sample were collected from the two families. Genomic DNA was extracted using a DNA extraction kit (TIANamp Blood Genomic DNA Purification Kit; Tiangen Biotech, Beijing, China). The coding region and the exon–intron boundaries of TYR, p, TYRP1, and SLC45A2 were amplified by PCR. Mutation screening was performed in the two families using direct DNA sequencing.

Genomic DNA was extracted from peripheral blood samples of the probands and their parents using TIANamp Blood Genomic DNA Purification Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. Potential RNA contamination was removed by RNaseA (TIANGEN, Beijing, China). The DNA was quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). The genomic DNA was genotyped using the Affymetrix Human Genome-Wide SNP Array 6.0 or Affymetrix CytoScan HD Arrays (Affymetrix, Santa Clara, Calif., USA). DNA digestion, ligation, fragmentation, labeling, hybridization, staining and scanning were performed following the manufacturer’s protocols (Affymetrix, Santa Clara, CA).