xGen™ Hybridization Capture Core Reagents

Figure 1. Reduce hybridization capture time and maintain data quality. To evaluate the performance of the new buffer system in xGen Hybridization and Wash Buffer v3 we set up a series of experiments to assess hybridization time with two different panels. The DNA libraries were prepared using xGen DNA EZ Library Prep kit with 25 ng Coriell gDNA NA12878. The DNA libraries were subsequently enriched using 400 ng of input with either the (A) xGen AML Cancer Hyb Panel or (B) xGen Exome Hyb Panel v2 for either overnight (ON), 2 hours, or 1 hour.

Captured libraries were sequenced on a NextSeq 2000 system (Illumina®) and subsampled to 5 M total reads per sample. A standard analysis pipeline was run to produce Picard metrics. The flanked-on target percentage and fold 80 base penalty was consistent for both panels between hybridization incubation times. (A) The mean target coverage and the GCskew were consistent for the xGen AML Cancer Panel. (B) A variation in the GC skew metric was observed between the different hybrization incubation times using the xGen Exome Hyb Panel v2.

Figure 2. Consistent capture between xGen Hybridization and Wash Buffer v2 and v3 across a range of library inputs. To evaluate the performance of the v3 buffer system in xGen Hybridization and Wash Buffer v3 was compared to the xGen Hybridization and Wash Buffer v2 with different library inputs. The DNA libraries were prepared using the xGen cfDNA & FFPE DNA Library Prep v2 MC kit with 25 ng Coriell gDNA NA12878. The DNA libraries were subsequently enriched using 100 ng, 500 ng, 2500 ng, or 6000 ng of library input with the xGen Exome Hyb Panel v2 for overnight hybridization. Captured libraries were sequenced on a NextSeq 2000 system (Illumina®) and subsampled to 30 M total reads per sample. Standard analysis pipeline was run to produce Picard metrics. Despite the low sequencing data (30 M reads), the Mean Target Coverage was consistent and robust across conditions.

Figure 3. Impact of hybridization time on SNV detection in degraded samples. Three commercially available samples with known SNVs at measured frequencies, and with differing degrees of DNA damage (as measured by DIN score), were used as test library prep input. One hundred nanograms of DNA from each sample was used with the xGen cfDNA & FFPE DNA Library Prep Kit following IDT’s published protocol. Each sample was prepared in quadruplicate. Five hundred nanograms of library was used in a singleplex capture for various times of hybridization (overnight, 2 hours, and 1 hour). Samples were sequenced to approximately 100 M total reads and analyzed, to create ssConsensus data that was interpreted by VARDICT. The measured allele frequency for each of the known SNVs is indicated for different hybridization times. The following samples were used: Horizon discovery standards - HD803 (fcDNA severe), HD799 (fcDNA moderate), and HD798 (fcDNA mild).

Figure 4. An example of improved on-target rate delivered by xGen Universal Blockers across Illumina TruSeq® (A) and Illumina Nextera® (B) adapter styles. (A) 1 µg input DNA libraries were prepared from human genomic cell line NA12878 (Coriell) using either 8 nt or 10 nt adapters and enriched using the xGen AML Cancer Hyb Panel (single-plex, n = 3 for each condition) with xGen™ Universal Blockers TS, xGen™ Universal Blockers 10bp TS or without blockers. Sequencing was performed on a NextSeq 500 System (Illumina) to generate 2x150 bp, paired-end reads. (B) 50 ng cell line NA12878 (Coriell) gDNA was used for Nextera library preparation. Amplified libraries were enriched in 4-plex reactions (n = 3) using the xGen AML Cancer Hyb Panel, or in 12-plex (n = 1) using the xGen Human ID Hyb Panel, with xGen Universal Blockers NXT or without blockers (n = 1). Sequencing was performed on a NextSeq System (Illumina) to generate 2x75 bp paired-end reads. Libraries and captures from both figures were amplified using KAPA 2x HiFi PCR Mix, as this data was generated prior to release of xGen 2x HiFi PCR Mix.

Figure 5. Consistent coverage in multiplexed captures and improved on-target rates with the addition of xGen Universal Blockers. (A) Example of consistent results with multiplexed samples using the xGen Universal Blockers 10bp TS. DNA libraries (500 ng input per sample) from cell line NA12878 (Coriell) were enriched in single (n = 8) or multiplex reactions (n = 3) using the xGen AML Cancer Hyb Panel. The libraries were created using 10 bp TruSeq adapters with unique dual indexes. Sequencing was performed on a NextSeq 500 System (Illumina) with 2x150 bp paired-end reads. Coverage was consistent across all multiplexing levels tested. (B) Example of improved on-target rates delivered by xGen Universal Blockers 10bp TS. DNA libraries (500 ng input per sample) from cell line NA12878 were enriched in single-plex reactions (n = 3) using the xGen AML Cancer Hyb Panel with and without xGen Universal Blockers 10bp TS. Sequencing was performed on a NextSeq 500 System to generate 2x150 bp, paired-end reads. On-target values (with 150 bp flank) were averaged across experiments. Libraries and captures from both figures were amplified using KAPA 2x HiFi PCR Mix, as this data was generated prior to release of xGen 2x HiFi PCR Mix.