Transcript Isoform Diversity of Y Chromosome Ampliconic Genes of Great Apes Uncovered Using Long Reads and Telomere-to-Telomere Reference Genome Assemblies

Abstract

Y chromosomes of great apes harbor Ampliconic Genes (YAGs) - multi-copy gene families (BPY2, CDY, DAZ, HSFY, PRY, RBMY, TSPY, VCY, and XKRY) that encode proteins important for spermatogenesis. Previous work assembled YAG transcripts based on their targeted sequencing but not using reference genome assemblies, potentially resulting in an incomplete transcript repertoire. Here we used the recently produced gapless telomere-to-telomere (T2T) Y chromosome assemblies of great ape species (bonobo, chimpanzee, human, gorilla, Bornean orangutan, and Sumatran orangutan) and analyzed RNA data from whole-testis samples for the same species. We generated hybrid transcriptome assemblies by combining targeted long reads (Pacific Biosciences), untargeted long reads (Pacific Biosciences) and untargeted short reads (Illumina)and mapping them to the T2T reference genomes. Compared to the results from the reference-free approach, average transcript length was more than two times higher, and the total number of transcripts decreased three times, improving the quality of the assembled transcriptome. The reference-based transcriptome assemblies allowed us to differentiate transcripts originating from different Y chromosome gene copies and from their non-Y chromosome homologs. We identified two sources of transcriptome diversity - alternative splicing and gene duplication with subsequent diversification of gene copies. For each gene family, we detected transcribed pseudogenes along with protein-coding gene copies. We revealed previously unannotated gene copies of YAGs as compared to currently available NCBI annotations, as well as novel isoforms for annotated gene copies. This analysis paves the way for better understanding Y chromosome gene functions, which is important given their role in spermatogenesis.

The complete sequence and comparative analysis of ape sex chromosomes

“…We first collected annotations from the NCBI annotation pipeline, CAT, and Liftoff. To these annotations, we added mappings from human and species-specific gene sequences onto the latest assemblies and included Iso-seq reads 109 and Iso-seq transcripts 110 . To combine these annotations, we first performed an interval analysis to find all annotated, mapped, or predicted copies, with one or more sources of evidence and then manually curated the final set of protein-coding and pseudogene copies for each of these genes (Supplementary Table 45).…”