A Customizable Suite of Methods to Sequence and Annotate Cattle Antibodies

Studying the antibody response to infection or vaccination is essential for developing more ef-fective vaccines and therapeutics. Advances in high throughput antibody sequencing technolo-gies and immunoinformatic tools now allow fast and comprehensive analysis of antibody rep-ertoires at high resolution in any species. Here we detail a flexible and customisable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to anti-body sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy-light chain pairs. When combined with the Ig-sequence Multi-species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96 and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy-light chain pairs, respectively. Each method has strengths and limitations in terms of throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the prin-ciples outlined here can be applied to study antibody responses in other mammalian species