Big dye terminator cycle sequencing ready reaction kit 3

All available family members were tested for co-segregation of the candidate CCA-causing variant with the disease phenotype by Sanger sequencing. PCR primers were designed to contained the mutation sites and their flanking regions (PCR primers, PCR reaction conditions are available upon request) using Primer3 [23 (link)]. An ABI Prism 3730xl DNA Sequencer and the Big Dye Terminator Cycle Sequencing Ready Reaction Kit 3.1 (Applied Biosystems, Foster City, CA, USA) were used for sequencing. Sequences were assembled and analyzed with Mutation Surveyor software (SoftGenetics, State College, PA, USA). We also tested the DNA of 200 healthy controls.

We performed direct Sanger sequencing to screen the coding sequence of the CD36 gene in 184 French individuals of European ancestry. The protocol was carried out using the automated ABI Prism 3730xl DNA sequencer in combination with the Big Dye Terminator Cycle Sequencing Ready Reaction kit 3.1 (Applied Biosystems). PCR conditions and primers sequences are available on request. Samples from the FREX project have been sequenced using the Agilent V5 + UTR exome capture kit and genotyped on Illumina Core Exome SNP-chip. Full details of data processing, variant calling, filtering process and variant annotation in ExAC have been previously described^{34 (link)}. GnomAD was quality controlled and analyzed using the Hail open source framework (https://github.com/hail-is/hail). This data set can be accessed via the gnomAD Browser (http://gnomad.broadinstitute.org/).