Accel-Amplicon™ 56G Oncology Panel v2 for Illumina

Targeted Sequencing Panel for Illumina® Platforms

All-in-One Oncology Panel with Sample Identification:

The Accel-Amplicon 56G Oncology Panel v2 offers comprehensive and hotspot coverage of 56 clinically-relevant oncology-related genes. This panel utilizes a 263-amplicon design, covering over 16,000 COSMIC mutations (Forbes et al. Oxford Journals. 2014), to generate targeted libraries compatible with Illumina sequencing platforms and now includes 104 exonic and gender Sample_ID amplicons spiked in at a low percentage (2-4% of reads) for tracking tumor-normal pairs and samples in longitudinal studies.

The Accel-Amplicon 56G Oncology Panel v2 is compatible with short DNA fragments from both FFPE and cfDNA samples, and is well-suited for detection of clinically-relevant allele variants in DNA from circulating tumor cells (CTCs). This product is a complete kit that includes all components necessary for generating ready-to-sequence libraries, including primer pairs and indexed sequencing adapters.

Features:

Single-tube assay to interrogate 56G and Sample_ID targets
Ready-to-sequence libraries in 2 hours
Inputs as low as 10 ng
Limit of detection as low as 1%
Complete library generation in a single kit

Benefits:

On-target specificity and coverage uniformity > 95%
Average amplicon size of 138 bp for compatibility with cfDNA and FFPE
Leverage a power of discrimination over 1 in 100,000
Easily track samples within and between studies
Validate tumor/normal pairs and track samples throughout longitudinal studies
Compliment WGS or exome sequencing for sample tracking

Workflow:

The Accel-Amplicon workflow uses a fast, single-tube approach consisting of a 90-minute target enrichment amplification step and a 10-minute adapter ligation step, yielding a 2-hour start-to-finish procedure.
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The single-tube workflow includes two brief incubations to generate the multiplex amplicon targets and add a unique combination of Illumina-compatible indexed adapters, creating up to 96 uniquely-indexed libraries for multiplexing on a single sequencing run.

Product Specifications:

Decrease Input, Not Sensitivity

Like the Accel-Amplicon Comprehensive TP53 Panel, the Accel-Amplicon 56G Oncology Panel v2 offers high sensitivity variant detection from input amounts from 10-25 ng. The kit utilizes Illumina-compatible dual-indexed adapter sequences and has been validated on the MiSeq® and HiSeq® platforms.

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*As quantified by qPCR. Qubit® represents amplifiable DNA content more accurately than NanoDrop®, however is still not as accurate as the qPCR assay. For sample types with more consistent high quality DNA including whole blood, fresh frozen samples, and cultured cells, quantification by Qubit is a reliable indicator of amplifiable content.

Genes Represented in the 56G Oncology Panel v2 and Number of Amplicons

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The Accel-Amplicon 56G Oncology Panel v2 includes both clinically relevant hotspot loci and regions of contiguous coverage, depending on the allele distribution across each target gene. The table depicts the genes represented, followed by the number of amplicons for each gene. Contiguous, overlapping coverage is included for APC, ATM, EGFR, FBXW7, FGFR3, HNF1A, KIT, MSH6, PIK3CA, PTEN, SMAD4, and TP53. Comprehensive coding exon coverage is included for TP53.

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The Accel-Amplicon 56G Oncology Panel v2 includes coverage of both clinically relevant hotspot loci and regions of contiguous coverage, as well as Sample_ID targets spiked-in at low percentage (2-4% of reads). This allows for somatic mutation detection using high depth of coverage and sample identification using low coverage depth of germline targets. Analysis of the Sample_ID targets included with the new version of the panel are optional and will have a minimal impact on your sequencing results if they are left out of the analysis pipeline.

Performance Data:

Performance on the Illumina Platform

The data below demonstrate the performance of the Accel-Amplicon 56G Oncology Panel on a variety of sample types.

High Coverage Uniformity Across Sample Types

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10 ng of input DNA from a variety of sample types was used to generate libraries with the Accel-Amplicon 56G Oncology Panel. The coverage uniformity, as the percentage of the bases covered at least 20%, 30%, 40%, or 50% of the average depth, was determined across four sample types. The percentage of reads on target was > 95% for all sample types.

Reproducible Variant Calling from Q-Seq HDx™ Quantitative Standards

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The Accel-Amplicon 56G Oncology Panel consistently detected validated variants at the expected frequency in replicates from 10 ng of the Horizon Diagnostics Quantitative Multiplex DNA Reference Standards HD701. The variants were called by LoFreq 2.1.1 (Genome Institute of Singapore) and GATK HaplotypeCaller (Broad Institute). When examining uncommon variants between the 10 replicates, the majority of background variants were present at less than 0.6%. No sporadic variants greater than 0.6% were detected.