Blood dna kit

Genomic DNA (gDNA) was extracted from the whole blood samples using the Blood DNA kit (TIANGEN BIOTECH, Beijing, China), and 1 ug of purified gDNA fragmented to 200–300 base pairs using an ultrasonoscope (Covaris S2, Massachusetts, USA). End-repair, adenylation and adapter ligation were performed for library preparation following the Illumina's protocol. The same amount of library were pooled then hybridized to the customized capture array (NimbleGen, Roche) including exons, splicing sites and immediate flanking intron sequences of 29 genes for non-syndromic autosomal dominant hearing loss and TNC, a novel causative gene for ADNSHL identified in our previous research (Table S1). Sequencing was carried out on Illumina HiSeq2000 to generate paired end reads (90 bps at each end) [7] (link).
Raw image files were processed by Illumina Pipeline (version 1.3.4) for base-calling with default parameters. Reads were aligned to NCBI37/hg19 assembly using the BWA (Burrows Wheeler Aligner). SNPs and indels (inserts and deletions) were detected using the GATK software [8] (link).

Genomic DNAs of the probands, their parents were extracted from peripheral blood samples using the Blood DNA kit (TIANGEN BIOTECH). We used a targeted genomic enrichment platform to simultaneously capture exons, splicing sites, and immediate flanking intron sequences of 127 known deafness genes point mutations, micro‐indels and duplications (<20 bp) could be detected simultaneously. Targeted gene capture and high throughput sequencing have been described in detail previously. Sanger sequencing was used to identify probands and their two parents segregating causative variants in the candidate gene (Guan et al., 2018). The data were not analyzed for copy number variants (CNVs).

Genomic DNA was extracted from whole blood samples using a Blood DNA kit according to the standard protocol (TIANGEN BIOTECH, Beijing, China). PCR and Sanger sequencing were performed on the propositus and then on all available members from Family 304 to determine whether the potential mutation the in causative gene co-segregated with the disease phenotype in the family. The direct PCR products were sequenced using BigDye terminator v3.1 cycle sequencing kits (Applied Biosystems. Foster City, CA, USA) and analyzed using an ABI 3700XL Genetic Analyzer. The primer sequences and PCR cycles used are provided in Supplementary Fig. S5.

The genomic DNA (gDNA) of peripheral blood was extracted from all participants in the four families using a Blood DNA kit according to the standard protocol (TIANGEN BIOTECH, Beijing, China). Polymerase chain reaction (PCR) and Sanger sequencing were performed on the propositus and available members to determine whether the potential variant cosegregated with the disease phenotype in the family. Direct PCR products were sequenced using BigDye terminator v3.1 cycle sequencing kits (Applied Biosystems. Foster City, CA) and analyzed using an ABI 3700XL Genetic Analyzer. The primer sequences and PCR cycle used are provided in Appendix S2. We also selected 1,700 blood samples from healthy Chinese individuals with normal hearing as controls.

The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shandong Eye Institute (Qingdao, China). Written informed consent was obtained from all participants (or guardians). Patients diagnosed with KC were recruited from Qingdao Eye Hospital, Shandong Eye Institute (Qingdao, China). The diagnosis of KC was based on clinical examination (corneal stromal thinning, Vogt’s striae, Fleischer’s ring, Munson’s sign, signs of videokeratography, and refractive errors). In total, the family of one pair of monozygotic twins and 200 sporadic KC patients were collected. The pedigree (Fig. 1A) suggested that the inheritance pattern of these twins’ family was either autosomal recessive or de novo mutation. Two hundred unrelated healthy individuals of Chinese origin were used as control. Peripheral blood samples from all participating individuals were collected in EDTA tubes. Genomic DNA was extracted with Blood DNA Kit (Tiangen Biotech Co., Beijing, China).

Genomic DNA was extracted from the whole blood samples using the Blood DNA kit (TIANGEN BIOTECH, Beijing, China). Mutation screening of all exons and flanking splicing sites of GJB2 was performed by PCR amplification and bidirectional sequencing as previously described [11] (link). The dominant mutations identified in this study were also screened in 100 ethnically-matched normal hearing controls.