Abstract Massively parallel DNA sequencing is a critical tool for genomics research and clinical diagnostics. Here, we describe the Association of Biomolecular Resource Facilities (ABRF) Next-Generation Sequencing Phase II Study to measure quality and reproducibility of DNA sequencing. Replicates of human and bacterial reference DNA samples were generated across multiple sequencing platforms, including well-established technologies such as Illumina, ThermoFisher Ion Torrent, and Pacific Biosciences, as well as emerging technologies such as BGI, Genapsys, and Oxford Nanopore. A total of 202 datasets were generated to investigate the performance of a total of 16 sequencing platforms, including mappability of reads, coverage and error rates in difficult genomic regions, and detection of small-scale polymorphisms and large-scale structural variants. This study provides a comprehensive baseline resource for continual benchmarking as chemistries, methods, and platforms evolve for DNA sequencing.

Multi-Platform Assessment of DNA Sequencing Performance using Human and Bacterial Reference Genomes in the ABRF Next-Generation Sequencing Study