Repurposing a HiSeq 2500 for Immunofluorescence Methods

The HiSeq 2500 system has been obsolesced by new sequencers and Illumina has announced
they will stop supporting the instrument and supplying the reagents in early 2023. The HiSeq was a
legacy workhorse sequencing system, that had a large installation base around the world.
The cost of acquiring this system is falling drastically because sequencing centers are decommissioning
their HiSeqs and some are even available to purchase on eBay. The instrument itself is a powerful
system of scientific equipment that includes precise XYZ stages, fast imaging, and integrated fluid
handling. Several methods to spatially map multiplexed transcripts and/or proteins at subcellular
resolution across tissue sections such as merFISH, STARmap, CODEX, 4i, and immuno-SABER have been
published using automated confocal microscopes with integrated fluidics that usually take days to run.
We assessed the feasibility of repurposing a HiSeq 2500 instead of a dedicated confocal microscope
system with integrated fluidics to perform spatial transcriptomics/immunofluorescence methods on
tissue. We wrote software in Python, called PySeq2500, to control the stages, pumps, valves, optics, and
cameras of the HiSeq. Custom flow cells were developed from off the shelf components to mount tissue
sections and to interface with the HiSeq fluidics. Only minor modifications to the HiSeq stage were made
to accommodate the custom flow cell and to the fluidic plumbing to minimize dead volume. PySeq2500
protocols were developed to automate various spatial transcriptomic/immunofluorescence methods on
the HiSeq 2500 and map targeted biomolecules across tissue sections. Adjacent tissue sections were
also mapped on a confocal microscope with integrated fluidics. The HiSeq and confocal systems were
compared in terms of data quality, performance, and ease-of-use. We show that HiSeqs that have been
opened up after being decommissioned for sequencing are still valuable systems that can perform
advanced imaging based spatial mapping of biomolecules in tissue.