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Hybridization Capture Sequencing

Targeted sequencing is a cost-effective alternative to whole genome sequencing since higher depth of coverage for specific regions can be obtained from less sequencing. Whole exome sequencing utilizing hybridization capture-based methods remains a predominant process for discovery because entire protein coding regions of genes constitute the majority of known disease causing variants. Alternatively, smaller disease specific or custom-based panels provide increased sensitivity and more efficient data analysis by focusing on targeted regions most likely to contain variants of interest. In either case, an unbiased NGS library must be generated in order to maximize the potential sensitivity in the selected hybridization capture panel.

Swift Biosciences offers a solution to construct bias-free libraries for target enrichment utilizing hybridization-based capture technologies. The Accel-NGS® 2S Hyb DNA Library Kit produces libraries with exceptional complexity and uniform coverage from high quality gDNA, FFPE, and cfDNA samples, regardless of input, to improve confidence in data analysis throughout the target region. Libraries generated with the kit are compatible with a variety of commercially available hybridization capture technologies, including Agilent SureSelectXT and SureSelectXT2, NimbleGen™ SeqCap™ EZ, and IDT® xGen® Lockdown® Probes for whole exome sequencing, disease specific or custom-generated panels.

SWIFT PRODUCT LINES COMPATIBLE WITH HYBRIDIZATION CAPTURE SEQUENCING:

Accel-NGS 2S Hyb DNA Library Kit

The Swift advantage:
  • Compatible with multiple capture technology suitable for a diverse set of panels.
  • Highly efficient adapter ligation provides meaningful data from low input samples.
  • Exceptional library complexity results in more unique molecules available for capture.
  • No adapter titration or heat steps make the kit readily automatable.

Performance with Limiting DNA on Illumina® Platforms

The data below demonstrate the performance of the Accel-NGS 2S Hyb DNA Library Kit with various hybridization capture panels, including Agilent SureSelect, IDT xGen Lockdown Probes and NimbleGen SeqCap EZ using a variety of sample types and input quantities.

Sequencing Low Input DNA from FFPE Tumor and Matching Circulating cfDNA

Detection and monitoring of cancer status via liquid biopsy is an attractive and feasible alternative to tumor biopsy, and enables detection of mutant alleles that may be missed in a section biopsy of heterogeneous tumor or those occurring in metastatic disease. In collaboration with Q2 Solutions, we evaluated the performance of our Accel-NGS 2S Hyb DNA Library Kit with the Agilent SureSelectXT Comprehensive Cancer Panel. The metrics below represent % duplication and mean bait coverage of 10 ng and 20 ng FFPE samples and their corresponding cfDNA.

AP_Hyb Cap_Table_cfDNA and FFPE

The Accel-NGS 2S Hyb DNA Library Kit performance with the Agilent SureSelectXT Comprehensive Cancer Panel was evaluated with 10 and 20 ng DNA from FFPE tumor sample and corresponding cfDNA. FFPE libraries were normalized to 3M reads, and cfDNA libraries were normalized to 4M reads.

The Integrative Genomic Viewer (IGV) figure below depict comparisons of the allele frequencies between a 20 ng FFPE tumor sample and its corresponding cfDNA for a COSMIC variant ALK.

AP_Hyb Cap_Image_IGV

The Accel-NGS 2S Hyb DNA Library Kit performance with the Agilent SureSelectXT V5 Comprehensive Cancer Panel was evaluated with 20 ng DNA from FFPE tumor samples and corresponding cfDNA. IGV figure demonstrates detection of chr2:294,164,81 (COSMIC1130802; p.K1491R) in ALK in FFPE and cfDNA from the same tumor at varying allele frequencies at 38% and 56%, respectively.
Exceptional Coverage Uniformity Achieved for Low Input Coriell Samples

The Accel-NGS 2S Hyb DNA Library Kit performance with the Agilent SureSelectXT V5 Exome Panel was evaluated with 60 ng DNA from two distinct high quality Coriell DNA samples. The metrics below represent outstanding evenness of coverage which is critical for a robust data analysis.

AP_Hyb Cap_Table_Cov Uniformity Coriell

The Accel-NGS 2S Hyb DNA Library Kit performance with the Agilent SureSelect V5 Exome Panel was evaluated with 60 ng DNA from two distinct high quality Coriell DNA samples. Libraries were sequenced on a HiSeq® 2500 Rapid Run with 100 bp Paired End reads.
Formalin-Compromised DNA Samples with the IDT Pan-Cancer Panel

AP_Hyb Cap_Table_Formalin DNA Pan-Cancer

Accel-NGS 2S Hyb libraries were constructed from 5 and 1 ng of Horizon Discovery reference standards. HD701 is genomic DNA, HD-C749 is formalin-compromised I, and HD-C751 formalin-compromised II. Libraries were enriched with the IDT xGen Pan-Cancer Panel.
Superior Performance with the NimbleGen MedExome Panel

The Accel-NGS 2S Hyb DNA Library Kit and Kapa Library Preparation Kit performance was compared with the NimbleGen SeqCap EZ MedExome Panel. High quality Coriell NA12878 gDNA at 100, 10, 1 ng inputs were evaluated. Reads were normalized to 39M for comparison of coverage metrics. SNP concordance with the NIST GIAB truth list in high-confidence regions was 99% for all inputs and library preparation methods, with the exception of Kapa 1 ng, which was 98% (Zook et al Nature Biotechnology 2014). Kapa Library Preparation performance drops significantly at 1 ng, illustrated by duplication rates, mean coverage depth, and percentage of the target area covered at least 20X as shown below.

AP_Hyb Cap_Table_MedExome Panel

Libraries were made from 100, 10, and 1 ng of HapMap DNA NA12878 (Coriell) with both the Swift Accel-NGS 2S Hyb Kit and the Kapa Library Preparation kit, followed by the NimbleGen SeqCap EZ MedExome Panel. All libraries were normalized to 39M reads.