Single-molecule long-read sequencing reveals a conserved intact long RNA profile in sperm

Sperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to impact offspring phenotypes, our knowledge of the sperm transcriptome, especially the composition of long RNAs has been limited by the lack of sensitive, high-throughput experimental techniques that can distinguish intact RNAs from fragmented RNAs, known to abound in sperm. Here, we integrate single-molecule long-read sequencing with short-read sequencing to detect sperm intact RNAs (spiRNAs). We identify 3,440 spiRNA species in mice and 4,100 in humans. The spiRNA profile consists of both mRNAs and long non-coding RNAs, is evolutionarily conserved between mice and humans, and displays an enrichment in mRNAs encoding for ribosome. In sum, we characterize the landscape of intact long RNAs in sperm, paving the way for future studies on their biogenesis and functions. Our experimental and bioinformatics approaches can be applied to other tissues and organisms to detect intact transcripts. We used PacBio IsoSeq and Oxford Nanopore Technologies to sequence mouse sperm and testis RNAs, as well as human sperm RNAs. We also used Illumina short reads to sequence human sperm RNAs and mouse sperm RNAs. The Summary.MousePacBio.ONT.xlsx table contains both PacBio and ONT data for mouse: Mouse Sperm and Testis PacBio: Summary.MousePacBio.ONT.xlsx Mouse Sperm ONT: Summary.MousePacBio.ONT.xlsx The following three data files are linked to the corresponding sample records: HumanSpermPacBio: Summary.HumanPacBio.xlsx RNAseq.HumanSperm.cauda: RNAseq.HumanSperm.cauda.salmon.sf RNAseq.MouseSperm.cauda.adult: RNAseq.MouseSperm.cauda.adult.salmon.sf The following three GTF files are results of our PacBio assembly. They were based on both PacBio and Illumina sequencing data: TranscriptomeAssembly.MouseTestis.mm10.gtf TranscriptomeAssembly.spiRNA.HumanSperm.hg38.gtf TranscriptomeAssembly.spiRNA.MouseSperm.mm10.gtf These three fasta files are the full-length non-chimeric (FLNC) CCS reads for downstream analysis: SpermPacBio_FLNC.fasta TestisPacBio_FLNC.fasta HumanSpermPacBio_FLNC.fasta