3730xl genetic analyzer

The amplified genes from six varieties were subjected to bidirectional Sanger sequencing with gene-specific primers on an ABI 3730xl Genetic Analyzer using a BDT v3.1 Cycle sequencing kit. The reverse primer nucleotide sequence was converted into a reverse complementary sequence. A consensus sequence was created by identifying an overlapping sequence between forward and reverse complementary sequences. The GT 104 consensus sequence was used as a query sequence in NCBI-BLASTn analysis [34 (link)]. Furthermore, to determine sequence polymorphism, all sequences were subjected to CLUSTALW multiple sequence alignment [35 ]. For gene prediction, Eukaryotic GeneMark.hmm version 3.54 [36 (link)] was used to locate exons and introns in sequences of reasonable length. Open reading frames (ORFs) of all six varieties were created by joining all exons and then translated to predict protein sequences with BioEdit [35 ]. These sequences have been submitted to the NCBI under the accession numbers ON711024 to ON711029 (Supplementary Material).

Trimmomatic was used to remove adapter contamination and trim low-quality reads to obtain clean reads (15 (link)). Then, the cleaned reads were aligned to the human reference genome (hg19) using the Burrows–Wheeler Alignment tool (16 (link)). DNA variants were called following the Genome Analysis Toolkit software best practices (17 (link)). Then, variants were annotated using Variant Effect Predictor (VEP) (18 (link)–22 (link)). Multiple computational predictive tools were applied to predict the pathogenicity of the detected variants (23 (link)–29 (link)). Further, variants with an allele frequency (AF) of <0.1% were retained for downstream analysis. According to the ACMG/AMP guidelines, all retained variants were classified into pathogenic (P), likely pathogenic (LP), variants with unknown clinical significance (VUS), likely benign (LB), or benign (B) (30 (link)). The putative diagnostic variants were experimentally validated by Sanger sequencing (ABI 3730xl Genetic Analyzer) and real-time PCR.

Total genomic DNA was extracted from fecal samples using QIAamp DNA stool mini kits (Qiagen, Germany), according to the manufacturer's instructions. We used seven tetra‐microsatellite loci to distinguish among individuals. These were as follows: GPL‐60, gpz‐20, GPL‐29, gpz‐6, GPL‐53, GPL‐44, and gpz‐47 (Huang et al., 2015). The probability of identity across these loci in the target population was estimated using GIMLET 1.3.3 (Valière, 2002). PCR amplifications were carried out in 25 μl reaction mixtures comprising approximately 50 ng of template DNA, 2 mm MgCl₂, 200 μmol of dNTP each, 15 pmol of each primer, 1.0 μg of bovine serum albumin (BSA), and 0.3 units of Hotstart DNA polymerase (Takara). Amplifications were performed using the following PCR procedure: an initial denaturation step for 5 min at 95°C, followed by 35 cycles of 95°C for 45 s, 30 s at locus‐specific annealing temperature and 50 s at 72°C，and a final elongation for 10 min at 72°C. For genotyping, the PCR amplification products were separated by capillary electrophoresis using a denaturing acrylamide gel matrix on an ABI 3730xl Genetic Analyzer. Alleles were detected using Genemapper 3.2 software.

Genomic DNA of Alport syndrome fibroblasts and iPSCs were extracted using the Blood and Tissue Kit (Qiagen, Cat NO. A1120). Exons 47, 26, and 10 of the COL4A5 gene were amplified by PCR. Three pairs of primers were used. COL4A5 Exon 47 forward primer: 5'-TGTTTTGTCAATATCCATAAGAGTGG-3'; COL4A5 Exon 47 reverse primer: 5'-GGCCAAGGCTACTCTAGAACC-3'; COL4A5 Exon 26 forward primer: 5'-GGGTGGATCATCCTTATTCG-3'; COL4A5 Exon 26 reverse primer: 5'-CAGCAAGCCAACATCACG-3'; COL4A5 Exon 10 forward primer: 5'-AGAGCAGAATTCCAATGACG-3'; and COL4A5 Exon 10 reverse primer: 5'-TTATGAAGCCCTGCTTTTGC-3'. The PCR products were subsequently sequenced with an ABI 3730XL Genetic Analyzer.

A selection of environmental and clinical strains were analyzed by microsatellite polymorphism genotyping using three multiplex PCRs. A total of nine microsatellite markers consisting of di-, tri-, or tetranucleotide short tandem repeats (STR) were used [56 (link)]. Fungal DNA extraction was performed by freeze-drying the cultures and mechanically breaking the cells by bead-beating. DNA was then extracted using the ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research) following the manufacturer’s instructions. Genotyping was performed by Genoscreen (Lille, France) using the PCR conditions described by De Valk et al. [56 (link)] with the fluorophores 6FAM/HEX/NED. The size of the amplicons was determined with a ABI 3730XL genetic analyzer using the GeneScan 500 ROX size standard (ABI) and the GeneMapper v5.0 software. The size of each microsatellite fragment was measured to determine the number of repetitions for each marker according to de Valk et al. [56 (link)]. All results are reported as repeat numbers. The relatedness of the strains was estimated by a minimum spanning tree analysis in Bionumerics 8.0 (Applied Maths, St-Martens-Latem, Belgium). The discriminatory power of the microsatellite markers was calculated using the Simpson index of diversity (Hunter 1990).