Comparison of Mendeliome exome capture kits for use in clinical diagnostics

Abstract

Next generation sequencing has disrupted genetic testing, allowing far more scope in the tests applied. The appropriate sections of the genome to be tested can now be readily selected, from single mutations to whole-genome sequencing. One product offering within this spectrum are focused exomes, targeting ~5,000 genes know to be implicated in human disease. These are designed to offer a flexible platform offering high diagnostic yield with a reduction in sequencing requirement compared to whole exome sequencing. Here, we have undertaken sequencing of control DNA samples and compare two kits, the Illumina TruSight One and the Agilent SureSelect Focused Exome. Characteristics of the kits are comprehensively evaluated. Despite the larger design region of the Agilent kit, we find that the Illumina kit performs better in terms of gene coverage, as well as coverage of clinically relevant loci. We provide exhaustive coverage statistics for each kit to aid the assessment of their suitability and provide read data for control DNA samples to allow for bioinformatic benchmarking by users developing pipelines for these data.

Figure 1

Euler diagram showing size (Mb) of regions as defined by the vendor provided target locations (A) and RefSeq transcripts covered to ≥ 20 X in our data without regard for vendor defined targets (B).

Figure 2

Capture biases for the two kits. The degree of GC bias seen is greater for the SSFE data (r² = 0.56 vs. r² = 0.09 for SSFE and TSO respectively, p < 2.2×10⁻¹⁶ for both, Pearson’s correlation).

Figure 3

Proportion of genes covered to varying levels in downsampled datasets representing 12–48 samples being included in a single sequencing run. A reduction in the number of genes covered to a high level can be seen with the data down-sampling.

Figure 4

Overview of laboratory processes for the two capture kits. For SSFE physical fragmentation is followed by DNA fragment repair and ligation of adapter sequences; hybridisation of patient DNA with the baits and pulldown is then performed, followed by library indexing and pooling. For TSO, combined fragmentation and adapter ligation is performed enzymatically, followed by sample amplification, indexing and pooling. Two iterations of bait hybridisation and pulldown are then performed, with a final pooled library amplification.