Figure 1. Example coverage of the  IDT xGen Exome Hyb Panel v2 vs. competitor. Two different users performed hyb captures on different days in different locations using different aliquots (lots) of the IDT xGen Exome Hyb Panel v2. Captures were performed using 100 ng Coriell DNA NA12878 in a singleplex reaction (n=3). Bioinformatics analysis was done for 3-way coverage correlation comparison, a representative pair from each vendor is demonstrated here. The IDT xGen Exome Hyb Panel v2 shows a linear regression line that mimics the predicted one-to-one correlation line with an r2 value of 0.76.   In this example, IDT outperformed the competitor.

Figure 2. The IDT whole exome sequencing workflow has higher on-target percentage in comparison to alternate suppliers. Sequencing libraries were prepared using enzymatic shearing and ligation-based library preparation using the library prep protocols from each vendor. Libraries were captured and multiplexed according to the vendor’s exome panel capture protocol (X-8, R-8, and A-8, where “8” signifies 8-plex captures, n=2). For IDT, we show both 1-plex (n=3) and 12-plex (n=2) captures (IDT-1 and IDT-12, respectively). Enriched libraries were sequenced on a NextSeq® instrument (Illumina) in high output mode using 2 x 100 bp paired-end reads for analysis. On-target bases were determined with Picard (percent selected bases) using 5 Gb per library. 

Figure 3. Consistent uniform sequence coverage with the xGen Exome Hyb Panel v2. DNA libraries were created from 100 ng of human genomic DNA (Coriell) and enriched either as 1-plex captures (n=3) or as a single 12-plex (n=2) capture using the xGen Exome Hyb Panel v2. The enriched libraries were sequenced (2 x 100 bp reads) on a NextSeq® instrument (Illumina) and subsampled to 5 Gb. The data shows uniform coverage with a flanked on-target rate of 94.7%, mean target coverage of 64.5X, and a duplication rate of 3.3% (calculated with Picard).

Figure 4. Coverage profile of the xGen Exome Hyb Panel v2 closely resembles whole genome data. (A) Comparing the coverage depths of different exome panels (8-plex, n=1) to coverage depth from whole genome sequencing (WGS) of a matched library shows that the xGen Exome Hyb Panel v2 closely matches WGS. Analysis of guanine-cytosine content (GC) shows that the xGen Exome Hyb Panel has a more effective capture of GC content (compare dark red and red dots in the right and left panels). (B) RB1 exons 1 and 2 show extremes of GC content with ~76% in exon 1 and ~38% in exon 2. A comparison of capture between IDT and supplier A shows a higher read depth across exon 1 for the xGen Exome Hyb Panel v2 on the Integrative Genomics Viewer (Broad Institute), whereas supplier A coverage has a lower number of reads of this exon with high GC content.

Figure 5. Exome coverage of the xGen Exome Hyb Panel v2. (A) 100 ng Coriell gDNA hybridization capture libraries (n=2) were sequenced with 5 Gb per sample while the percent of exons covered end-to-end at each read depth were calculated. The xGen Exome Hyb Panel v2 (IDT) shows the highest percentage of exons covered at each indicated depth (compared to suppliers X, R, and A). (B) The xGen Exome Hyb Panel v2 provides end-to-end RefSeq gene coverage at ≥20X. Individual samples were subsampled at different read depths (2 x 100 bp read length). The percentage of genes that were covered for every base of every exon at ≥20X was calculated at each read depth and plotted.

Figure 6. xGen Exome Hyb Panel v2 can increase the number of samples loaded into a flowcell. DNA libraries were created from 100 ng of human genomic DNA (Coriell) and enriched either as 8-plex, n=1 (supplier R), 8-plex, n=2 (suppliers X and A) or 12-plex, n=2 (IDT) captures. The enriched libraries were sequenced (2 x 100 bp) on a NextSeq® instrument (Illumina) and the number of reads required to achieve 75X mean target coverage (Picard) per sample was estimated along with the number of samples that would fit on a NovaSeq™ S2 flowcell (Illumina). 

Figure 1. Coverage uniformity obtained with xGen  AML Cancer Panel. Greater than 0.2X mean coverage is observed for >98% of targets. Illumina TruSeq® HT libraries (n=8) were enriched using the xGen AML Cancer Panel and sequenced on a NextSeq 550 system using 2 x 150 paired-end reads. Each sample was sub-sampled to 4M total reads. 

Figure 2. Deep coverage of targeted regions using xGen AML Cancer Panel. Eight genomic DNA libraries were enriched using the xGen Acute Myeloid Leukemia Cancer Panel and sequenced on a NextSeq 550 system using 2 x 150 paired-end reads. Total reads for each sample were sub-sampled to 4M total reads. In all samples, there was >1X coverage for 99.3% of targets and >30X coverage for 98.7% of targets.

Figure 3. Consistent results obtained with xGen AML Cancer Panel.  Genomic DNA libraries were enriched with two lots of the xGen AML Cancer Panel (n=8 for each lot). The enriched samples were sequenced on the NextSeq 550 sequencing platform using 2 x150 paired-end reads. All samples were sub-sampled to a level of 4M total reads. The probe-by-probe target coverage was averaged for eight replicate samples for each lot of the xGen AML Cancer panel. A comparison of probe-by-probe target coverage between two lots show consistent results between lots, with an R2 value of 0.9887.

Figure 1. An example of, deep coverage of targeted regions using xGen Pan-Cancer Hyb Panel. Genomic DNA libraries (n=8) were enriched using the xGen Pan-Cancer Hyb Panel and sequenced on a NextSeq® 550 system using 2 x 150 paired-end reads. Total reads for each sample were sub-sampled to 4M total reads. In all samples, there was >1X coverage for 99.9% of targets and >30X coverage for 99.7% of targets.

Figure 2. An example of high coverage uniformity obtained with xGen Pan-Cancer Hyb Panel. Greater than 0.2X mean coverage is observed for >99.3% of targets. Genomic DNA Illumina TruSeq® HT libraries (n=8) were enriched using the xGen Pan-Cancer Hyb Panel and sequenced on a NextSeq® 550 system using 2 x 150 paired-end reads. Each sample was sub-sampled to 4M total reads. 

Figure 3. An example of lot to lot consistency obtained with xGen Pan-Cancer Hyb Panel. Genomic DNA libraries were enriched with two lots of the xGen Pan-Cancer Hyb Panel (n=8 for each lot). The enriched samples were sequenced on the NextSeq 550 sequencing platform using 2 x150 paired-end reads. All samples were sub-sampled to a level of 4M total reads. The probe-by-probe target coverage was averaged for eight replicate samples for each lot of the xGen Pan-Cancer Hyb Panel. A comparison of probe-by-probe target coverage between two lots showed excellent consistency, with an R2value of 0.8378.

Figure 1. Example showing the spike in of the xGen Human ID Hyb Panel does not impact on-target performance of recipient panel. DNA libraries were prepared from NA12878 genomic DNA. 500 ng of the libraries were enriched with a custom panel comprised of 509 xGen Hyb Probes, with (n=8) and without spike in (n=3) of the xGen Human ID Research Panel. The enriched libraries were sequenced on a MiSeq® System (Illumina) using 2 X 150 paired-end reads. Each sample was sub-sampled to 0.5M total reads. The average of on-target rate (with 150 bp flank) across experiments are shown.

Figure 2. Example of uniform coverage of targeted regions using xGen Human ID Hyb Panel. Genomic DNA libraries were enriched using the xGen Human ID Hyb Panel spiked into a custom panel containing 509 probes (n=8). Enriched samples were sequenced on a MiSeq system using 2 x 150 paired-end reads. Total reads for each sample were sub-sampled to 0.5M total reads. The average normalized target coverage ranged between 0.60X and 1.51X for the regions targeted by the human ID Panel. Normalized target coverage bounds at 0.5X and 1.5X are denoted by the doted lines.