Haloplex kit

Sequencing data from 95 CRC patients fulfilling the Amsterdam criteria but without identified germline mutation (previously tested for MLH1, PMS2, MSH6, MSH2, APC and MUTYH) was investigated for the POLE mutation. The library was prepared according to the manufacturer’s instructions using a custom Haloplex kit (Agilent Technologies) and was subsequently sequenced on a HiSeq 2500 (Illumina) with 2 × 100 bp paired end sequencing.

Briefly, genotypes of donors and recipients were assayed by exome sequencing (Illumina TruSeq enrichment kit for the Discovery Cohort and Agilent Haloplex kit for the Cornell Validation Cohort and the French Validation Cohort). Reads were aligned to the human genome with the Last [9 (link)] aligner integrated as a plugin in GobyWeb [8 (link)]. Genotype calls were made with Goby [10 (link)] and GobyWeb [8 (link)]. Prediction of polymorphism impact on the protein sequence were performed with the Variant Effect Predictor [27 (link)]. Genes that contain at least one transmembrane segment were identified using Ensembl Biomart [28 ].

Targeted enrichment and sequencing were performed on 225 ng of DNA extracted from the cell lines. Enrichment was performed using a custom HaloPlex Kit (Agilent, Santa Clara, CA, USA) targeting 41 genes. Sequencing was undertaken on a MiSeq sequencer (Illumina Inc, San Diego, CA, USA), following the manufacturer’s protocols.

DNA enrichment was performed using HaloPlex kit (Agilent Technologies, Santa Clara, CA, USA) and sequencing was conducted on a MiSeq system sequencer (Illumina, San Diego, CA, USA) with 150 bp paired-end reads. A total of 463407 bp were included in the capture design, covering the entire ST18 gene (chr8: 53,023,399–53,373,519, GRCh37/hg19 assembly) as well as 10 kb downstream and 50 kb upstream to the gene and an additional 2 Mb located upstream and downstream to the gene and predicted to harbor putative regulatory regions (https://www.encodeproject.org).
The sequencing data were processed using MiSeq Reporter 2.0.26 and Casava softwares (Illumina, San Diego, CA, USA) and analyzed for quality control using FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were aligned to the Genome Reference Consortium Human Build 37 (GRCh37/hg19) using Burrows-Wheeler Aligner [40 (link)] and variant detection was achieved using The Genome Analysis Toolkit [41 (link)]. Variants were annotated by ANNOVAR [42 (link)] and the frequency of each variant was determined using data from dbSNP138, the 1000 Genome Project and an in-house database. Case-control association test for variants was performed with chi-square, and permutation test, using the Caucasian population from the 1000 Genome Project data (http://www.1000genomes.org) as a control.

Mutation screening was performed for 110 patients clinically diagnosed with 46,XY DSD. We analyzed 11 major causative genes for 46,XY DSD (AR, CBX2, DHH, GATA4, MAP3K1, NR5A1, SOX9, SRY, SRD5A2, WT1, and ZFPM2) using a custom made NGS gene panel. The library was constructed using a HaloPlex kit (Agilent Technologies, Santa Clara, CA, USA) and sequence data were obtained using MiSeq or HiSeq 2000 (Illumina, San Diego, CA, USA). The sequencing reads were mapped by BWA (version 0.7.12) using Human GRCh37/hg19 (UCSC Genome Browser) as the reference. Base quality calibration was carried out by GATK (version 3.5). Functional consequences of missense variants were assessed using four in silico programs, i.e., Combined Annotation Dependent Depletion (CADD, https://cadd.gs.washington.edu/), PolyPhen-2 (https://genetics.bwh.harvard.edu/pph2/), Sorting Intolerant From Tolerant (SIFT, https://sift.jcvi.org/), and MutationTaster (https://www.mutationtaster.org/). Variants of interest were subjected to Sanger sequencing. The frequency of variants in the general population was examined by the Genome Aggregation Database (genomAD, https://gnomad.broadinstitute.org/), the Human Genetic Variation Database (https://www.hgvd.genome.med.kyoto-u.ac.jp/) and the 2,049 Japanese genome reference panel (2KJPN)^{30 (link)}.