xGen® Lockdown® panels are designed to integrate into different workflows in order to accommodate the various preferences and goals of researchers. IDT strives to ensure that these panels are compatible with not only different next generation sequencing (NGS) platforms, but also different library prep methods. IDT researchers and bioinformaticians evaluate the panels based on the metrics described in our earlier blog post, How Important are Those NGS Metrics?, and set new benchmarks as we improve the protocols and panel designs.
At IDT, we use Lockdown Panels to enrich libraries for regions of interest before sequencing them on the Ion Torrent Personal Genome Machine® (PGM®) and Illumina MiSeq® platforms. We prepare the libraries using human genomic DNA and library preparation kits that are compatible with the sequencing platform. Our scientists also frequently collaborate with organizations such as Washington University and the Emory Genetics Laboratory to assess the efficacy of these panels in other researchers’ hands. Our collaborators will generally use their own sequencing platforms. such as the HiSeq® 2500, to assess the panels.
Library Preparation Kits
The number of kits and methods available for preparing sequencing libraries is growing steadily with the increasing popularity of NGS. IDT scientists evaluate different library prep kits to determine their efficacy at producing satisfactory input material for Lockdown Panels. We prepare sequencing libraries from human genomic DNA using the various kits, and then enrich the libraries using IDT Lockdown Panels. The enriched library is sequenced and assessed using established metrics to ensure that the capture is successful.
Metrics to Determine Panel Success
To evaluate panel performance, IDT bioinformaticians rely on the metrics described in our previous post. Generally, we look for high on-target percentages of the reads, which are an indication of a panel’s specificity. On-target percentage is dependent on the panel size, but IDT scientists have achieved nearly 70% on target (and over 80% with flanking regions) using the xGen Acute Myeloid Leukemia Panel. This performance sets a high standard for every other panel we offer.
The xGen® Acute Myeloid Leukemia (AML) Panel achieves ~70% on-target reads, and >80% when flanking regions are included. The data show percent on target for 4 replicate capture reactions using the AML panel. The target sequence included 150 bp flanking regions.
Our bioinformaticians calculate the on-target percentage and determine uniformity of coverage of the panel. We calculate the proportions of regions that have depth of coverage within 25, 50, and 100% of the mean. A high proportion within 25% of the mean indicates high uniformity and we typically find that >98% of regions have depth of coverage greater than 25% of the mean.
As demonstrated in the figure, we typically find that >98% of regions captured have coverage depth >25% of the mean. This confirms the high uniformity that is achieved with our xGen® target capture panels. The data show target region coverage for 4 replicate capture reactions using the AML panel.
We also evaluate the reproducibility of the panels by preparing multiplexed libraries for enrichment and looking for differences between the multiplexed libraries, and then repeating the experiment to ensure that results of each multiplexed experiment can be reproduced. Further results of panel evaluations described here are available on the relevant panel page, which can be accessed through www.idtdna.com/xgen.
Our bioinformaticians are currently exploring other metrics of the panel, such as percentage of repeats captured and bias due to GC content. This research is important for continual improvement of xGen Lockdown Panels and raises the standard for evaluation, leading to better NGS products for our customers.
Rami Zahr is NGS Product Manager at IDT.