High contiguity de novo genome assembly and DNA modification analyses for the fungus fly, Sciara coprophila, using single-molecule sequencing

Abstract

Background The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. Results We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. Conclusions We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.

Gene-rich germline-restricted chromosomes in black-winged fungus gnats evolved through hybridization

“…We annotated 41,418 genes in our B. coprophila genome assembly: 17,802 on the autosomes, 4,277 on the X chromosome, and 15,812 on the GRCs (Table 1). The number of genes that we annotated on the autosomes and X chromosome are comparable to the recently published reference genome for B. coprophila [43] (Table 1), which did not contain GRC sequence.…”

“…To disentangle the origins of GRCs in Sciaridae, we sequenced germline and somatic tissue from B. coprophila and were able to unambiguously identify GRC scaffolds in a single genome assembly generated from both tissue types by comparing scaffold coverage and k-mer distributions between the 2 sequence types (with the idea that GRC sequences will be present in the germline but not in the soma). We also use the recently published reference genome for B. coprophila [43], which was produced from male early embryos after GRC elimination (i.e., is not expected to contain GRC sequence) to perform downstream analyses to compare the gene-content between GRCs, autosomes and the X chromosome in B. coprophila. We find that the 2 GRCs are gene rich and carry many homologs to the core genome.…”

“…To better understand the origins of the GRCs in Sciaridae, we conducted a reciprocal blast search with the amino acid sequence of all annotated genes with the goal of understanding whether the GRCs have homologs primarily on one chromosome in the genome (indicating their likely origin) and whether the divergence level of homologs can give us an idea of whether gene traffic has occurred from the core chromosomes to the GRCs after their formation. We also conducted a collinearity analysis to identify larger homologous blocks in the PLOS BIOLOGY genome, in which we identified collinear blocks of 5 or more genes anchored to the reference assembly (for autosomal and X-linked genes) [43] or an assembly we generated with long-read data from male germline tissue (for GRC genes, see S2 Text for methods). This allowed us to increase the continuity of our assembly and to anchor genes within our assembly to known chromosomes (autosomes A-II, A-III, A-IV, and the X chromosome) in the reference genome.…”

“…Expected [46] This study Urban and colleagues [43] This study Chromosome sizes are compared to microspectrophotometry estimates for B. coprophila [46], and gene number is compared to the reference genome assembly [43], which is not expected to contain GRC genes. See S2…”

DNA Damage Responses during the Cell Cycle: Insights from Model Organisms and Beyond

“…For example, since the 1930s it was noticed that it was difficult to recover X-radiation-induced mutants in the fungus gnat Sciara coprophila, due to an increased radiation tolerance compared to Drosophila [ 167 , 168 , 169 , 170 ]. The mechanism behind such radiation resistance remains unknown, yet the Sciara genome was recently fully sequenced [ 171 ]. Future efforts should endeavor to incorporate a more diverse array of model systems to fill in our knowledge of DDR mechanisms.…”