Schematic view of EXaCT-1 assay workflow. (a) (1) Slides are assessed by pathologist for neoplastic content, and tumour tissue marked and macrodissected. (2) DNA from fresh or FFPE tumour tissue and matched normal control specimen is extracted. (3) DNA is then enriched for exome sequences (357,999 exons corresponding to 21,522 genes) with HaloPlex technology described in this protocol in four major steps: fragmentation by restriction enzyme digestion, hybridisation to HaloPlex probes and introduction of Illumina sequence motifs, solid-phase capture and DNA ligation, and amplification of targeted fragments by PCR, followed by (4) sequencing on an Illumina HiSeq 2500 rapid mode, four samples per lane. (5) Paired-end reads are aligned to the human genome and (6) variant calls are made. (7) Variants are annotated and classified by our internally developed informatics pipeline, using publicly available and our own developed knowledge base. (8) The case is reviewed and results interpreted by a molecular pathologist who also signs it out in the LIS. (9) A report is then automatically generated and (10) dispensed to medical records. EXaCT-1 data analysis pipeline. (b) Schematic view of EXaCT-1 assay validation. (c) DNA derived from matched normal/tumour pairs from either fresh or FFPE specimens as well as standardised commercial DNA material were used to demonstrate the accuracy, sensitivity, specificity, reproducibility and precision of the assay using bioinformatics process for the detection of numerous types of variants (SNV, indel and CNV gain), according to NYS-DOH NGS guidelines for somatic genetic variant detection. IPM, Institute for Precision Medicine.