Library preparation and hybridization capture
To assess the xGen Exome Hyb Panel v2, twelve libraries were prepared using 100 ng of human genomic DNA (Coriell Institute). Then, xGen Stubby Adapters and Unique Dual Indexing primers (UDIs) were added with 6 cycles of PCR. The libraries were quantified
using Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific). Indexed DNA libraries were captured and multiplexed according to the xGen Hybridization Capture of DNA Libraries Protocol. Then, to test the flexibility of the xGen Exome Hyb
Panel v2 in library capture strategies, 500 ng of a DNA library was used to generate four single-plex captures and one 8-plex capture (5 total captures). Each pool underwent a 4 h hybridization reaction. PCR was performed on each capture using settings
selected for the panel size and the number of pooled libraries in each capture (10 PCR cycles for single-plex captures; 7 PCR cycles for 8-plex captures).
Whole Exome Sequencing
The final enriched library was normalized to 4 nM and sequenced on a NextSeq® 550 instrument (Illumina®) using 2 x 150 paired-end (PE) reads. The library was subsampled to 50 M total reads before analysis against the product’s
target space in the human reference genome, hg38. Sequencing metrics were calculated using Picard analysis tools .
xGen DNA Library Prep EZ libraries in combination with the xGen Exome Hyb Panel v2 results in comprehensive and quality coverage
On-target rates give information regarding precision of bases or reads aligning with a targeted region, so a higher percentage of on-target reads indicates better capture precision. Using the xGen DNA Library Prep EZ kit and the xGen Exome Hyb Panel v2
an on-target rate of >90% was obtained for both single-plex and 8-plex captures (Figure 2A). In addition, with this WES workflow, 97% of the target bases reached >20X coverage depth with no noticeable drop-off in resolution with 8-plex captures
Uniform coverage of both high and low GC regions indicates more comprehensive coverage of targeted regions and fewer dropouts. To evaluate the xGen whole exome sequencing solution against this metric, Exon 1 (GC-rich) and Exon 2 (AT-rich) of the RB1 gene target space were analyzed. The sequences generated using the xGen Exome Hyb v2 panel uniformly covered both regions (Figure 3). Meaning that with the xGen whole exome sequencing solution, researchers get the uniform, comprehensive breadth of
coverage as well as sufficient coverage depth that is necessary for reliable variant calling.