Comparison of single-cell sequencing technologies for allele-specific expression analysis in rabbit spermatids
Gene drives are meiotic transmission distorters that can transmit specific genes to more than 50% of the progeny. The t-haplotype in mice is a classic example for a naturally occurring gene drive. Its mechanism of action involves biased allele expression of t-haplotype-associated genes with impaired transcript sharing between developing sperm cells. For invasive pest species, there is interest in co-opting naturally occurring meiotic drives to spread transgenes that impact fertility or sex ratios as a means for population suppression or eradication. It is unknown whether similar natural gene drives exist in the European rabbit, one of the most detrimental pest species in Australia. In this work, we analysed the allele-specific expression (ASE) in rabbit spermatids at the single cell level to identify genes that could potentially be further investigated for genetic biocontrol purposes. We utilised both short-read (Illumina) and long-read (Pacific Biosciences and Oxford Nanopore) technologies and performed a comparative analysis to identify the most suitable workflow. While Illumina sequencing was unsuitable for the ASE analysis due to short read length and uneven transcript coverage, both long-read sequencing platforms demonstrated a similar performance with a slightly higher number of genes identified with Pacific Biosciences sequencing technology. Several genes with ASE were identified for further assessment as targets for genetic biocontrol. For example, the SPINK2 gene that plays an important role in sperm development and fertility, consistently showed biased allele expression towards one of the alleles in all samples tested. Furthermore, two serine/threonine-protein kinases were found to display a bimodal allele expression that could be indicative of impaired transcript sharing between the spermatids. Future work is warranted to assess suitability of the identified genes for genetic biocontrol.