Strong bias in long-read sequencing prevents assembly of Drosophila melanogaster Y-linked genes.

Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) are generally considered free from sequence composition bias, a key factor, alongside read length, that explains their success in producing high-quality genome assemblies. Indeed, there had been very few reports of bias, the clearest one against GA-rich repeats in the human genome. However, our study reveals a systematic failure of both technologies to sequence and assemble specific exons of Drosophila melanogaster genes, indicating an overlooked limitation. Namely, multiple Y-linked exons are nearly or completely absent from raw reads produced by deep sequencing with state-of-the-art Nanopore (10.4 flow cells, 200× coverage) and PacBio (HiFi 50×). The same exons are accurately assembled using Illumina 67× coverage. We find that these missing exons are consistently located near simple satellite sequences, in which sequencing fails at multiple levels: read initiation (very few reads start within satellite regions), read elongation (satellite-containing reads are shorter on average), and basecalling (quality scores drop as sequencing enters a satellite sequence). These findings challenge the assumption that long-read technologies are unbiased and reveal a critical barrier to assembling sequences near repetitive regions. As large-scale sequencing projects move toward telomere-to-telomere assemblies in a wide range of organisms, recognizing and addressing these biases will be important to achieving truly complete and accurate genomes. Additionally, the underrepresented Y-linked exons provide a valuable benchmark for refining those sequencing technologies while improving the assembly of the highly heterochromatic and often neglected Drosophila Y Chromosome.