Phage Immuno-Precipitation Sequencing (PhIP-Seq) is an emerging technique that enables the medium-to-high throughput characterization of the antibody repertoire of an individual against a library of epitopes. PhIP-Seq is based on a library of bacteriophages displaying antigens of choice, which are then bound by antigen-specific immunoglobulins from the individual’s samples. Antibody-bound viruses are immune-precipitated, and the corresponding epitopes are identified through sequencing. Here, we describe the design and synthesis of BoviScan, a T7 bacteriophage library that encodes the pan-proteome of 295 M. bovis strains. A set of 557 genome assemblies was used to extract 421,793 predicted ORFs. The amino-acid sequence encoded by each ORF was then split into 56 amino-acids segments (or “tiles”), with a 26 amino-acids overlap between each tile. To reduce redundancy, the 5,140,114 tiles generated were then clustered at 98% sequence identity, forming a set of 37,027 unique tiles. A set of 258 control tiles (antigens for which specific antibodies are available) were added, yielding the final set of 37,285 tiles. A pool of synthetic DNA oligonucleotide encoding each tile was ordered from Twist, and subsequently converted into double-stranded DNA. After sequencing of the DNA pool for quality control, it was cloned by restriction-ligation into the left and right arms of the T7 genome. The resulting assembled viral genomes were rescued by in vitro packaging, and after expansion the final BoviScan library was recovered. Sequencing of the library showed that more than 99.85% of the expected tiles are present, with a uniform abundance (95% of tiles within 1 log). We are currently adapting the immune-precipitation protocol to bovine immunoglobulins, and will use BoviScan to study a broad range of samples (serum, milk, BAL fluid, etc…) from a broad range of animals (infected naturally, infected in laboratories, non-infected, vaccinated, etc…).< Réduire