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Background: Immortalized cell lines are widely used model systems whose genomes are often highly rearranged and polyploid. However, their genome structure is seldom deciphered and is thus not accounted for during analyses. We therefore used linked short- and long-read sequencing to perform haplotype-level reconstruction of the genome of a Drosophila melanogaster cell line (S2-DRSC) with a complex genome structure.

Results: Using a custom implementation (that is designed to use ultra-long reads in complex genomes with nested rearrangements) to call structural variants (SVs), we found that the most common SV was repetitive sequence insertion or deletion (> 80% of SVs), with Gypsy retrotransposon insertions dominating. The second most common SV was local sequence duplication. SNPs and other SVs were rarer, but several large chromosomal translocations and mitochondrial genome insertions were observed. Haplotypes were highly similar at the nucleotide level but structurally very different. Insertion SVs existed at various haplotype frequencies and were unlinked on chromosomes, demonstrating that haplotypes have different structures and suggesting the existence of a mechanism that allows SVs to propagate across haplotypes. Finally, using public short-read data, we found that transposable element insertions and local duplications are common in other D. melanogaster cell lines.

Conclusions: The S2-DRSC cell line evolved through retrotransposon activity and vast local sequence duplications, that we hypothesize were the products of DNA re-replication events. Additionally, mutations can propagate across haplotypes (possibly explained by mitotic recombination), which enables fine-tuning of mutational impact and prevents accumulation of deleterious events, an inherent problem of clonal reproduction. We conclude that traditional linear homozygous genome representation conceals the complexity when dealing with rearranged and heterozygous clonal cells.

Repetitive DNA, represented by transposons and satellite DNA, constitutes a large portion of eukaryotic genomes, being the major component of constitutive heterochromatin. There is a growing body of evidence that it regulates several nuclear functions including chromatin state and the proper functioning of centromeres and telomeres. The 1.688 satellite is one of the most abundant repetitive sequences in Drosophila melanogaster, with the longest array being located in the pericentromeric region of the X-chromosome. Short arrays of 1.688 repeats are widespread within the euchromatic part of the X-chromosome, and these arrays were recently suggested to assist in recognition of the X-chromosome by the dosage compensation male-specific lethal complex. We discovered that a short array of 1.688 satellite repeats is essential for recruitment of the protein POF to a previously described site on the X-chromosome (PoX2) and to various transgenic constructs. On an isolated target, i.e., an autosomic transgene consisting of a gene upstream of 1.688 satellite repeats, POF is recruited to the transgene in both males and females. The sequence of the satellite, as well as its length and position within the recruitment element, are the major determinants of targeting. Moreover, the 1.688 array promotes POF targeting to the roX1-proximal PoX1 site in trans Finally, binding of POF to the 1.688-related satellite-enriched sequences is conserved in evolution. We hypothesize that the 1.688 satellite functioned in an ancient dosage compensation system involving POF targeting to the X-chromosome.