Big dye terminator cycle v1.1 sequencing kit

The poorly covered regions from NGS were amplified and verified by Sanger sequencing. The variants identified by multiplex genetic sequencing were also validated with Sanger sequencing. In brief, primers were designed using Primer3 software [16 (link)]. Primer sequences for validation are listed in S2 Table. Identified variants, including WRN (c.4108DelA), DICER1 (c.A3334G), and ELAC2 (c.A248G), were amplified in duplicate from genomic DNA of the six family members by using Hot FirePol DNA polymerase (Solis BioDyne, Tartu, Estonia). Sanger sequencing was performed using the Big Dye Terminator Cycle v1.1 Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) and ABI Prism 3130 Genetic Analyzer (Applied Biosystems).

Most likely disease-causing variants were confirmed by Sanger sequencing in the patient and the available family members. Primers were designed using Primer3 algorithm[14 (link)] and purchased at Microsynth AG (Balgach, Switzerland). All target regions were amplified in duplicate from genomic DNA of the patients and available family members using Hot FirePol^® DNA Polymerase (Solis BioDyne, Tartu, Estonia). PCR products were purified by treating them with ExoSAP reagent (Affymetrix, Santa Clara, CA) and sequenced using the Big Dye Terminator Cycle v1.1 Sequencing Kit (Applied Biosystems, Carlsbad, California, USA) and ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Carlsbad, California, USA). Sanger sequencing data analysis was performed using the Sequencing Analysis Software v5.4, SeqScape v2.6 (Applied Biosystems, Carlsbad, California, USA), MutationSurveyorV5.0.0 (Soft Genetics, Pennsylvania, USA) and Chromas (Technelysium, South Brisbane, Australia) to identify the likely disease causing mutations. Mutation is defined as previously described[15 (link)].

Primers were designed using Primer3 software [25] and purchased at Microsynth AG (Balgach, Switzerland). Exon 42 of COL18A1 was amplified in duplicate from genomic DNA of the patient and available family members using Hot FirePol DNA Polymerase (Solis BioDyne, Tartu, Estonia) and the following primers: forward 5′-GTGTCTGGCAGAAGCAGCAT-3′ and reverse 5′-TCACAGGTCAGGGGAGAGTT-3′. Sanger sequencing was performed using the Big Dye Terminator Cycle v1.1 Sequencing Kit (Applied Biosystems, Carlsbad, California, USA) and ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Carlsbad, California, USA). 192 randomly collected DNAs from the general population were used to assess the frequency of the mutation. Sanger sequencing data was analyzed using SeqScape v2.6 (Applied Biosystems, Carlsbad, California, USA).