Abstract 319: Demonstrating the use of Archer VARIANTPlex
TMand FUSIONPlex
TMassays in observing variants at sub-0.1% allele frequencies and low transcript number fusions in acute myeloid leukemia relevant genes
Authors: Johnson Laura A. , Washburn Michael , Kalavakur Paula , Legare Dhruti 
Published: March 2024

The presence of acute myeloid leukemia (AML) residual disease is valuable for understanding cancer progression. Studies have shown that variants detected at allele frequencies as low as 0.01% are useful for stratifying acute myeloid leukemias [1]. Both low limits of detection and high specificity are required for an assay to be useful in this context. Therefore, labs must choose an assay that has inherently low levels of background noise and that employs robust error suppression techniques.

Here we demonstrate the use of IDT’s Archer Next Generation Sequencing-based VARIANTPlex and FUSIONPlex assays (RUO) and accompanying software, Archer Analysis, for the application of detecting low allele frequency and low transcript number fusions with high levels of sensitivity and specificity. Currently available Archer assays and software solutions target AML-relevant variants and are compatible with multiple sequencing platforms. Using Archer technology, key AML-related mutations were detected at allele frequencies less than 0.1%, and low transcript numbers of myeloid relevant fusions were identified. In addition, greater than 95% of the bases in the targeted region of interest were powered to detect variants at allele frequencies of less than 0.1% in the VARIANTPlex libraries. Finally, error correction and noise filtering techniques remove many false positive variants in order to reduce the false positive variant calling rate.

To generate the input material, 1,000 ng total gDNA or 200 ng total RNA from commercially available cell lines containing myeloid relevant variants was diluted into a background of wild type gDNA at a mass ratio of 1:100 or RNA at a mass ratio of 1:20 to assess analytical sensitivity. Libraries were prepared using the catalog panels VARIANTPlex AML Focus or the FUSIONPlex Pan Heme assay. Libraries were sequenced on Illumina and Element Biosciences sequencing platforms. Data were analyzed with Archer Analysis using a pipeline which includes read cleaning, deduplication, error correction, variant calling, and reporting steps.

In conclusion, the VARIANTPlex AML Focus assay used with Illumina or Element sequencers demonstrated the ability to detect variants down to allele frequencies of 0.05%, and the FUSIONPlex Pan Heme assay was used to detect low transcript number fusions which may be useful for AML minimal residual disease research.

1. Dillon et. al. JAMA. 2023;329(9):745-755.

Citation Format: Laura A. Johnson, Michael Washburn, Paula Kalavakur, Dhruti Legare. Demonstrating the use of Archer VARIANTPlex TMand FUSIONPlex TMassays in observing variants at sub-0.1% allele frequencies and low transcript number fusions in acute myeloid leukemia relevant genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 319.