P073 Multiplexing a variety of clinicaaximize capacity of ion torrent next generation sequencing

Abstract

Aim We have established the Ion-torrent based Next Generation Sequencing (NGS) system in our clinical laboratory. The chief use of the combination Ion-ChefTM and Ion-S5 is for high resolution HLA typing. However, the full sequencing capacity of the system is not always reached with the HLA typing workflow. In this study, we validated the practicality of combining library pools of different clinical tests into a single NGS run. Methods HLA library pools of 24–32 patient samples were generated with NXType™ Ion-Torrent NGS for Class I (HLA-A, -B, and -C) and Class II (HLA-DR, HLA-DQ, and HLA-DP). These libraries were run alone and then separately combined with library pools of other clinical NGS-based assays. These included: (1) our lab-generated custom tests (APOL1 genotyping (single gene, three SNPs) and Cholestasis genotyping (15 genes, multiple SNPs and INDELS), and (2) commercial assays (16S-Metagenomics, Thermo Fisher Scientific, Inc.) and Lymphotrack, T and B cell clonality (Invivoscribe). All library pools were run on Ion-Chef™ and Ion-S5. HLA data was analyzed with TypeStream™ software v1.1.0.11. The concentration of HLA library pools was maintained at 100 pM. Importantly, the concentration of library pools of other tests were adjusted between 5 and 20 pM, depending on the test (number of genes and size of amplicons) and the number of samples in the pool. Exported data of APOL1, Cholestasis and 16S-Metagenomics were analyzed with Ion Reporter™ software. Lymphotrack data was exported and analyzed with Lymphotrack data analysis software. Results The number of reads covering HLA genes were slightly reduced when runs were combined with libraries of other tests, however, the results of HLA library pools run alone and combined were 100% concordant. The coverage and read numbers obtained for the other tests were sufficient or exceeded the minimal required. Conclusions To our knowledge, this is the first report showing that multiplexing different assays in a single run of Ion-S5 was achievable. We demonstrated that mixing sequencing samples of different genotyping tests in a single NGS run generated accurate results of each individual test. The findings of this works have a positive impact reducing turnaround times and sequencing costs.