Abstract Introduction: Genomic profiling of tumors is an important clinical tool for biomarker discovery and therapy selection. Tumor profiling often relies on formalin-fixed, paraffin-embedded (FFPE) tissue which is challenging because of fragmentation, cross-linking, and nucleotide modifications that arise from the fixation process. AVENIO Tumor Tissue Analysis Kits (For Research Use Only. Not for use in diagnostic procedures) provide an end-to-end solution for FFPE tumor tissue genomic profiling using hybrid-capture next-generation sequencing (NGS). Three panel options (Targeted, Expanded, and Surveillance Kits) provide flexibility in gene coverage with accurate detection of four major types of cancer mutations: single nucleotide variants (SNVs), insertions/deletions (indels), fusions and copy number variations (CNVs). AVENIO Tumor Tissue Analysis Kits were verified and validated on the Illumina NextSeq 500/550 sequencers but there is a need to show comparable performance on alternative NGS platforms. Confirmation of equivalent performance on the HiSeq 4000 allows for sequencing of up to sixteen multiplexed library pools, and simultaneous sequencing of samples analyzed using the AVENIO Tumor Tissue Analysis Kits and the AVENIO ctDNA Analysis Kits. Methods: In this study, we compared the performance of the three panel options for the AVENIO Tumor Tissue Analysis Kits (For Research Use Only. Not for use in diagnostic procedures) across three Illumina NGS platforms: Illumina NextSeq 500, Illumina HiSeq 4000, and Illumina HiSeq 2500. Ninety libraries containing variants at targeted limit of detection (5% allele frequency for SNV, Fusions, and Indels, and 4-5 copies for CNV) were prepared, sequenced, and analyzed using an internal analysis pipeline equivalent to the commercially available AVENIO Oncology Analysis Software. The total number of reads per sample were normalized across sequencers, and sequencing metrics and variant calls were compared. Results: The libraries that were prepared using the AVENIO Tumor Tissue Analysis Kits (For Research Use Only. Not for use in diagnostic procedures) and sequenced on the Nextseq 500 demonstrate sensitivities of >99% for SNVs, >99% for Indels, >99% for CNVs, and >99% for Fusions and >99% specificity for all variant types. We found that overall sensitivity and specificity performance were comparable when the samples were subsampled at the same level across the three Illumina platforms. Key sequencing metrics such as sequencing depth, on-target rate, error rate, and uniformity were also similar across the platforms. This study demonstrates that the high assay performance of the AVENIO Tumor Tissue Analysis Kits is consistent across multiple Illumina sequencing platforms. Citation Format: Isabel Soto, Lewyn Li, Cindy Choi, Garima Kushwaha, Jingchuan Li, Bosun Min, Laura Qin, Seng Salee, Liang Feng, Gregory Turenchalk, Jing Li. AVENIO Tumor Tissue Analysis Kits performance across Illumina sequencing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 755.