Exome sequencing targets only the protein-coding regions of the genome. Since exons represent only 1% of the human genome, a method of separating these regions from noncoding DNA is important to study mutations that could be important for disease research. The captured genomic material must also be suitable for sequencing to a satisfactory depth of coverage for research on variant alleles and sensitive applications like copy number variation (CNV) detection. To provide this increased depth of coverage and enable high multiplexing of samples, the xGen Exome Hyb Panel v2 targets only the coding sequences (CDS) of human coding genes in the RefSeq 109 database.
Our xGen Exome Hyb Panel v2 consists of 5′ biotin–modified oligonucleotide probes that are individually synthesized and analyzed by electrospray ionization-mass spectrometry (ESI-MS) and optical density (OD) measurement. The probes are normalized before pooling to ensure that each probe is represented in the panel at the correct concentration. Probes that fail quality control are resynthesized. This manufacturing process gives the xGen Exome Hyb Panel v2 a unique advantage over array-derived pools in which missing or truncated probes cannot be identified before sequencing. IDT proprietary synthesis methods enable challenging probes—such as those with high GC and AT content—to be appropriately represented in the panel. The individual probes are synthesized on a larger scale than is used for array manufacturing, allowing IDT to produce a single, large lot of material that is aliquoted over time, providing consistent content from one aliquot to the next.
The xGen Exome Hyb Panel v2 consists of 415,115 probes that spans a 34 Mb target region (19,433 genes) of the human genome and 39 Mb of probe space—the genomic regions covered by probes. Our probes are designed using a new “capture-aware” algorithm and assessed with proprietary off-target analysis. All probes in the panel are manufactured under ISO 13485 standards, and then, mass spectrometry and dual quantification measurements of each probe are performed before they are pooled into the xGen Exome Hyb Panel v2. These measures ensure the quality of the probe and its appropriate representation in the final panel.