Mutation surveyor software

The peripheral venous blood samples from 113 patients were prepared for DNA extraction using the Blood Extraction Kit (QIAGEN, Germany). The cardiac tissue samples from 95 patients were prepared for cDNA extraction. All of the regions covered by TES and all primers for smyd4 exon sequencing in cDNA are listed in S5 Table. Variant analysis was performed using the Mutation Surveyor software (Softgenetics, USA). All variants were screened in public databases, including the 1000 Genome database, the dbSNP database, and the ExAC database, and an internal database in the molecular diagnosis laboratory at the Children’s Hospital of Fudan University. A risk analysis of SNVs was performed using SIFT, Polyphen2, and Mutation Taster to predict the possible effects on protein function. A 3D structure analysis of the wild-type and mutant proteins was performed on the SWISS MODEL website (https://www.swissmodel.expasy.org/).

To determine length polymorphism of dinucleotide GT_n of intron 1 COL1A2 gene, the raw sequencing data were submitted to Mutation Surveyor software version 5.0.1 (www.softgenetics.com). This program can directly compare chromatograms with genomic DNA of reference sequence of COL1A2 (NT_007933_94023373). Alignment of the sequences was carried out using BioEdit software version 8.0.5 and ClustalW algorithm (Thompson et al., 1994 (link)). Sequences obtained (Hall, 1999 ) were thoroughly checked, cleaned, and aligned to identify homologies among sites, and also to perform other phylogenetic analysis including the determination of variability index and genetic diversity as well as the parameters of genetic differentiation. Genetic variability parameters (number of polymorphic sites, total number of haplotype, average number of nucleotide difference K) were obtained through DnaSP 5.10 software (Librado and Rozas, 2009 (link)) and MEGA 7.0.26 (Kumar et al., 2016 (link)). To estimate genetic variation according to epidemiological parameters, the factor of genetic differentiation (Fst) and the analysis of molecular variance (AMOVA) were determined with Arlequin software version 3.5.1.3 (Excoffier and Lischer, 2010 (link)). Values of P less than 0.05 are considered significant at a 5% confidence interval.

The basic structure of OPN1LW and OPN1MW gene cluster was captured and amplified by PCR, then screened with Sanger sequencing as previously described^{17 (link),18 (link)}. Briefly, OPN1LW and OPN1MW ’s exons were captured using specifically designed primers as first step (Tables S2 and S3), then amplified for Sanger sequencing using Step 2 primers shown in Table S3. The data were analyzed by using Mutation Surveyor software (SoftGenetics, USA).

Genomic DNA from all the available members in the family and 150 unrelated female sporadic Chinese cases with high myopia were isolated from peripheral blood using RelaxGene Blood Kit (TIANGEN, Beijing, China), following the manufacturer’s instructions. The exons and exon–intron boundaries sequence of ARR3 gene were downloaded from Ensembl Genome Browser. The primers were designed using the Primer-BLAST (Table S1). All the coding and splice site regions of ARR3 gene were sequenced for all individuals. The data were analyzed by using Mutation Surveyor software (SoftGenetics, USA).

For the discovery phase, genomic DNA was extracted from peripheral blood of the NL family using a modified salting out protocol (Miller et al. 1988 (link)) and screening was performed by bi-directional Sanger sequencing and analyzed using Mutation Surveyor Software (v5.0, SoftGenetics LLC State College, PA, USA). The NL proband was Sanger sequenced for all pathogenic variants causing hearing loss in this genetically isolated population, followed by select autosomal dominant genes matching the proband’s (PID IV-7) audiometric data with that of reference audioprofiles of 34 autosomal dominant deafness loci (Audiogene v4.0) (Hildebrand et al. 2009 (link)).
We next performed genome-wide single nucleotide polymorphism (SNP) genotyping using the Illumina 610Quad genotyping chip (Illumina Inc., San Diego, CA, USA) on multiple family members. Starting with a set of > 500,000 high quality SNP markers, informative SNPs (n = 17,407) were imported into Superlink (v1.7) (Fishelson and Geiger 2004 (link)) and linkage analysis performed under an autosomal dominant model with 99% penetrance and a disease allele frequency of 0.0025.

DNA was extracted from snap-frozen human PDO cell pellets by the Sample Processing Lab using an AllPrep kit (Qiagen). Primers sequences for amplification and sequencing of exons of the KRAS gene that contain the G12/13 codons are listed below:
KRAS G12/13 forward: 5′-CTGGTGGAGTATTTGATAGTG-3′
KRAS G12/13 reverse: 5′-CTGTATCAAAGAATGGTCCTG-3′.
The following PCR conditions were used as previously described^{54 (link)} and specifically noted in that paper: ‘94 °C for 2 min; three cycles of 94 °C for 30 s, 64 °C for 30 s and 72 °C for 30 s; three cycles of 94 °C for 30 s, 61 °C for 30 s and 72 °C for 30 s; three cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s and three cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, followed by 72 °C for 5 min and a hold at 4 °C’. PCR products were purified using a QIAquick PCR purification kit and sent to and sequenced by Eurofins. The resulting sequences were analyzed using Mutation Surveyor software (SoftGenetics).