Searching for somatic immune gene diversification regulatory sequences in individual sea urchin cells using MinION nanopore long reads

Vertebrates adaptive immunity relies on a somatic diversification mechanism, which recombines V, D, and J gene segments to generate a broad array of antibodies. A similar somatic immune gene diversification mechanism has not been found so far in invertebrates. Recently, we showed that a sea urchin SpTrfimmune gene family is subjected to somatic gene deletions, duplication, and single point mutations in individual cells. In spite of the frequent sequence alternations observed in the SpTrf family, all sequenced gene members had complete open reading frames. We, therefore, hypothesize that the SpTrfdiversification mechanisms are highly regulated. In our research, we aim to uncover and characterize these diversification mechanisms. To begin to address this goal, we looked for diversification regulatory sequences around and within the alternated regions in the SpTrfgenes. This will be achieved by the comparison of genomes of individual somatic cells and their germline counterparts. The methodology included single-cell isolation with FACS, whole-genome amplification (WGA) followed by MinION nanopore sequencing and a dedicated bioinformatics pipeline for the filtering and assembly of Trf sequence-containing reads. Our results confirm that single coelomocytes contain modified SpTrfgene repertoires in single coelomocytes compared with germline. Candidate diversification regulatory sequences, including two types of short tandem repeats, have been identified successfully using MinION nanopore long-read sequencing. These results contribute to our understanding of the ways invertebrates diversify their immune gene repertoires. The mechanisms underlying the diversification processes are yet to be uncovered.