Data S3: Whole Exome Sequencing and Touchdown Polymerase Chain Reaction (PCR) Conditions

Data S3: Whole exome sequencing and touchdown polymerase chain reaction (PCR) conditions

Whole exome sequencing and data analysis

DNA was captured using the Agilent SureSelect v4 (51Mb) kit (Santa Clara, CA) and sequenced on the Illumina HiSeq 2000 platform (San Diego, CA). Three samples were multiplexed per flowcell lane using 2 x 150bp paired-end chemistry to give an overall coverage of 100 times. Sequencing reads were processed using the GATK (McKenna et al 2010) recommended best practice of alignment to the reference human genome (human_g1k_v37) using the Burrows-Wheeler Aligner (Li and Durbin 2009) with default settings, followed by sorting and duplicate marking using Picard tools, GATK data processing tools, base quality score recalibration and indel realignment to refine the alignment. SNP and indel discovery and genotyping was performed using the GATK variant discovery pipeline including variant calling using Unified Genotyper followed by variant quality score recalibration (DePristo et al 2011). The resultant VCF files were filtered using Ingenuity Variant Analysis software (Redwood City, CA). Variants were assessed to ensure they were not located within a segmental duplication, and BAM files were inspected using Integrative Genomics Viewer (Broad Institute) (Robinson et al 2011) software to confirm call quality. Mutation(s) were confirmed by Sanger sequencing of genomic DNA.

PCR reaction mix (2mM MgCl2):

Component Volume (µL) dH2O 16.35 Buffer 6

MgCl2 (25 mM) 2.4 dNTPs 0.6 Forward Primer (5 µM) 1 Reverse Primer (5 µM) 1 5% DMSO 1.5 Taq (Go Taq DNA polymerase [5 U/µl] 0.15 DNA [75 ng/µl] 1 Total 30 Primer sequences available on request.

Touchdown PCR conditions:

Step Conditions 1 95°C for 3 minutes 2 95°C for 30 seconds 3 64°C for 30 seconds (-1°C per cycle) 4 72°C for 1 minute 5 Repeat steps 2-4 9 times for a total of 10 cycles 6 95°C for 30 seconds 7 54°C for 30 seconds 8 72°C for 1 minute 9 Repeat steps 6-8 24 times for a total of 25 cycles 10 71°C for 7 minutes

References Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25(14):1754- 60.

McKenna A, Hanna M, Banks E, et al (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next- generation DNA sequencing data. Genome Res 20(9):1297-303.

DePristo MA, Banks E, Poplin R, et al (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43(5):491-8.

Robinson JT, Thorvaldsdóttir H, Winckler W, et al (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24-6.