W29: Demultiplexing QC/Good Practices — Hidden Surprises and Illumina Sequencing

Almost all Illumina high-throughput sequencing today involves multiplexing of samples within lanes. High selectivity and sensitivity of the necessitated post-sequencing demultiplexing is important for experimental success. However, there are several under-appreciated/surprising aspects of multiplexing on the Illumina platform, and common analysis workflows do not provide strong signals of all problems at this level, leading to a widespread impression that demultiplexing is a trivial topic, when it is not. This workshop will discuss multiplexing on the Illumina platform (including single and dual indices, unique duals, and UMIs), use of the Illumina BCL2FASTQ2 tool (on the campus Hoffman2 cluster), and QC steps (via command line tools) that should be considered for all experiments (even if you typically rely on a Core’s nominal demultiplexing). A focus will be on specific classes of problems actually observed by the Instructor’s demultiplexing without typical assumptions of more than 600 diverse NovaSeq lanes from the full spectrum of BSCRC Sequencing Core users.