Eurostars project, MTP in situ sequencer, 9877, Stockholm University

Purpose and goal

SU engages in development, testing - and if applicable adaptation of in situ sequencing assays to microfluidic tissue processor prototypes that will enable automated in situ sequencing. Performance comparisons and protocol adaptations have been performed for sequencing by ligation steps and rolling circle amplification. Respective comparisons and adaptations are currently ongoing for remaining protocol steps to generate in situ sequencing substrates, i.e reverse transcription, cDNA anchoring, RNA removal, padlock probe hybridisation & ligation.

Expected results and effects

Spatially resolved highly-multiplexed profiling of biomarkers is expected to improve our understanding of highly heterogenous tissues such as brain as well as shedding light on the biology of complex diseases including cancer. Full automation of in situ sequencing is expected to make this spatial profiling technology more available to researchers and clinicians who might use in situ sequencing as a diagnostic tool in the future, similar to how simple histological and immunochemical stains are used today.

Planned approach and implementation

We have prioritised the adaptation of those steps, which are most relevant to be automated, i.e. the steps that benefit from automation the most. Performing the sequencing chemistry of in situ substrates is the most time consuming part of the current implementation of this technology since it involves sequential partially manual staining, imaging and stain-removal cycles, which are performed in different instruments. Our second priority is to automate library preparation, which we are currently focusing on. A research assistant has been exclusively assigned to the project.