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Accel-Amplicon™ 56G Oncology Panel v2

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

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.

amplicon-workflow-100416

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.

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.
Product Specifications

Amplicon_56G_Table_Specs

*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

Amplicon_Genes Rep Table

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.

custom-panel-with-spike-in

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 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

Amplicon_56G Coverage Uniformity

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

amplicon_table_genes

 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.

The level of multiplexing is directly correlated to the size of the specific Accel-Amplicon Panel and the flow cell chemistry utilized for sequencing of the libraries. The table below provides an example of the number of libraries from the Accel-Amplicon Comprehensive TP53 and 56G Oncology Panels which can be multiplexed on a single MiSeq® flow cell. Accel-Amplicon panels currently include up to 96 indexing combinations and are consistently increasing.

*Please inquire by email to Tech Support if you require more than 96 unique indices.

Amplicon_Table_Level of Multiplexing

Lower than expected yields most likely relate to the quality and quantity of the sample, as well as how it was quantified. If possible, for severely compromised samples including FFPE, use 25 ng of qPCR-quantified input and extend the incubation time for the Indexing Step from 20 minutes to 60 minutes to improve yields. It is very important to quantify cfDNA or FFPE with qPCR as opposed to Qubit or NanoDrop to ensure there is a minimum of 10 ng of amplifiable content in the sample.

It is not recommended to use a Bioanalyzer or Qubit for quantifying libraries because there is no PCR enrichment of the library following the Indexing Step, so the Bioanalyzer or Qubit will not accurately quantify fully adapted library vs. other DNA. In addition, the Accel-Amplicon library adapters have secondary structure which exhibit migration artifacts on the Bioanalyzer.

Low cluster density is typically related to an error in library quantification. If the final library is quantified by methods other than qPCR, this will lead to determining an inaccurate value for library concentration. It is not recommended to use a Bioanalyzer or Qubit for quantifying libraries because there is no PCR enrichment of the library following the Indexing Step, so the Bioanalyzer or Qubit will not accurately quantify fully adapted library vs. other DNA.

In addition, the Accel-Amplicon library adapters have secondary structure which exhibit migration artifacts on the Bioanalyzer. When diluting the library for loading the flow cell and sequencing, if the dilution is based off an erroneous quantification the cluster density will not be optimal.

Swift is pleased to accept custom Accel-Amplicon Panel requests. We will use a custom design pipeline to generate the primer pools and provide a functionally-tested pilot kit, with pricing decided on a case-by-case basis dependent on reaction volumes and assay complexity. Please contact us by email at Tech Support and specify:

  • Number of genes to cover, with gene symbols
  • Hotspot (i.e., SNP) or whole-gene coverage requirements
  • Sample type(s) to be used with the custom panel such as FFPE, cfDNA, or high quality gDNA
  • Expected number of reactions required for the custom panel

There are multiple factors which determine the LOD, the most important being the number of copies of the variant-containing DNA actually present in the sample and the depth of sequencing performed.

There is also some variation in the reasonable LOD depending on the variant of interest. In general, the Accel-Amplicon technology is capable of a LOD down to 1% for most base substitution variants when working with 10-25 ng input DNA as quantified by a qPCR input assay.

It is very important to quantify cfDNA or FFPE with qPCR as opposed to Qubit or Nanodrop to ensure there is a minimum of 10 ng of amplifiable content in the sample for this LOD to be achieved, since for 10 ng this will be represented by ~30 mutant copies detected in ~3000 total copies.

It is also critical to achieve adequate sequencing depth to obtain sufficient mutant copy number detection. The table below illustrates observable allele frequencies for Accel-Amplicon libraries at 10 ng and 1 ng input amounts.

Please note that Swift Biosciences currently supports inputs down to 10 ng.

Amplicon_Table_Allele Frequencies

For maximal (lowest) LOD, increasing the input amount to 25 ng, if possible, will provide more confidence due to increased copy number ( > 6000 total copies and therefore > 60 copies of a 1% variant).

It is also important to note that the LOD inherent in Illumina technology can be discussed in terms of bases rated at Q30. Most bases are read with a rate of 1 error in 1000, or 0.1%. Some bases have a lower quality score and therefore the background false positive noise level is between 0.1-0.6%.

Using a base quality filter during the variant calling steps is one way to address this issue.

Swift does not currently offer a proprietary data analysis software package. Please view our technical note “Accel-Amplicon Panels: Bioinformatics Guidelines“, which includes a tutorial for using open-source Linux-based tools to perform the required primer trimming step, as well as general recommendations for the alignment and variant calling analysis.

While this can be caused by multiple factors, one of the most common explanations is that the Sample Sheet was not set to automatically trim the Illumina adapter sequences when generating the FASTQs prior to the primer trimming requirement of Accel-Amplicon Panels.

Make sure that the “Use adapter trimming” and “Use adapter trimming Read 2” are selected during the sample sheet setup. It is possible to re-run this analysis even after the sequencing run has been performed if these selections were not made during the initial sample sheet setup.

Place Order

First, select a product and quantity:

Catalog No. Description Price QTY
AL-56248 Accel-Amplicon 56G Oncology Panel v2 (48 rxns) $3600.00

$3,600.00 Add to cart

The below solution is included with your product. If you require extra, please add now:

Catalog No. Description Price QTY
90196  PEG NaCl Solution (96 rxns) $20.00

$20.00 Add to cart