The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4,400 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the genera, subgenera and species groups in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus dataset of 17 genes from 704 species of Drosophilidae. We then inferred a species tree with highly supported groups for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

Y chromosomes are widely believed to evolve from a normal autosome through a process of massive gene loss (with preservation of some male genes), shaped by sex-antagonistic selection and complemented by occasional gains of male-related genes. The net result of these processes is a male-specialized chromosome. This might be expected to be an irreversible process, but it was found in 2005 that the Drosophila pseudoobscura Y chromosome was incorporated into an autosome. Y chromosome incorporations have important consequences: a formerly male-restricted chromosome reverts to autosomal inheritance, and the species may shift from an XY/XX to X0/XX sex-chromosome system. In order to assess the frequency and causes of this phenomenon we searched for Y chromosome incorporations in 400 species from Drosophila and related genera. We found one additional large scale event of Y chromosome incorporation, affecting the whole montium subgroup (40 species in our sample); overall 13% of the sampled species (52/400) have Y incorporations. While previous data indicated that after the Y incorporation the ancestral Y disappeared as a free chromosome, the much larger data set analyzed here indicates that a copy of the Y survived as a free chromosome both in montium and pseudoobscura species, and that the current Y of the pseudoobscura lineage results from a fusion between this free Y and the neoY. The 400 species sample also showed that the previously suggested causal connection between X-autosome fusions and Y incorporations is, at best, weak: the new case of Y incorporation (montium) does not have X-autosome fusion, whereas nine independent cases of X-autosome fusions were not followed by Y incorporations. Y incorporation is an underappreciated mechanism affecting Y chromosome evolution; our results show that at least in Drosophila it plays a relevant role and highlight the need of similar studies in other groups.

Genome assembly depends critically on read length. Two recent technologies, from Pacific Biosciences (PacBio) and Oxford Nanopore, produce read lengths >20 kb, which yield de novo genome assemblies with vastly greater contiguity than those based on Sanger, Illumina, or other technologies. However, the very high error rates of these two new technologies (∼15% per base) makes assembly imprecise at repeats longer than the read length and computationally expensive. Here we show that the contiguity and quality of the assembly of these noisy long reads can be significantly improved at a minimal cost, by leveraging on the low error rate and low cost of Illumina short reads. Namely, k-mers from the PacBio raw reads that are not present in Illumina reads (which account for ∼95% of the distinct k-mers) are deemed sequencing errors and ignored at the seed alignment step. By focusing on the ∼5% of k-mers that are error free, read overlap sensitivity is dramatically increased. Of equal importance, the validation procedure can be extended to exclude repetitive k-mers, which prevents read miscorrection at repeats and further improves the resulting assemblies. We tested the k-mer validation procedure using one long-read technology (PacBio) and one assembler (MHAP/Celera Assembler), but it is very likely to yield analogous improvements with alternative long-read technologies and assemblers, such as Oxford Nanopore and BLASR/DALIGNER/ Falcon, respectively.

Genome assembly depends critically on read length. Two recent technologies, PacBio and Oxford Nanopore, produce read lengths above 20 kb, which yield genome assemblies that are vastly superior to those based on Sanger or short-reads. However, the very high error rates of both technologies (around 15%-20%) makes assembly computationally expensive and imprecise at repeats longer than the read length. Here we show that the efficiency and quality of the assembly of these noisy reads can be significantly improved at a minimal cost, by leveraging on the low error rate and low cost of Illumina short reads. Namely, k-mers from the PacBio raw reads that are not present in the Illumina reads (which account for ~95% of the distinct k-mers) are deemed as sequencing errors and ignored at the seed alignment step. By focusing on ~5% of the k-mers which are error-free, read overlap sensitivity is dramatically increased. Equally important, the validation procedure can be extended to exclude repetitive k-mers, which avoids read miscorrection at repeats and further improve the resulting assemblies. We tested the k-mer validation procedure in one long-read technology (PacBio) and one assembler (MHAP/ Celera Assembler), but is likely to yield analogous improvements with alternative long-read technologies and overlappers, such as Oxford Nanopore and BLASR/DAligner. "Thm: Perfect assembly possible iff a) errors random b) sampling is Poisson c) reads long enough 2 solve repeats." Myers, 2014 "One chromosome, one contig." Koren et al., 2012 the following real example (throughout this manuscript we set k=16, which is a typical value). The genome of the bacterium E. coli strain K-12 MG1655 has been fully sequenced and finished to high quality years ago, using Sanger reads (Blattner et al. 1997). More recently, it has been sequenced using Illumina and PacBio technologies at high coverage (77x and 94x respectively; (Kim et al. 2014); https://basespace.illumina.com). The genome itself has 4.64 Mbp, and hence contains approximately 4.64million distinct k-mers, the vast majority of them occurring only once (bacterial genomes have few repetitive regions). The PacBio reads contain a total of 436 million k-mers (4.64 million k-mers times 94fold coverage); if there were no sequencing errors, these k-mers would correspond to 4.64 million distinct k-mers, each one occurring on average 94 times. However, these reads actually contain 292,687,635 distinct k-mers (~293 millions); among these, 4,513,248 (1.5%) are correct (i.e., present in the finished E. coli genome), and the remaining 288,174,387 are sequencing errors ("error k-mers"; see Methods). As expected, the correct k-mers show up repeatedly, and their proportion among the total k-mers is 16.6%.On the other hand, most error k-mers are unique, because the chance that random errors create twice the same 16-mer sequence (or a pre-existing 16-mer) is small. Fig. 1 shows a graph of the k-mer frequency spectrum of the PacBio reads and also, for comparison, of Illumina reads. It is easier to consider fi...

Three North American cactophilic Drosophila species, D. mojavensis, D. arizonae, and D. navojoa, are of considerable evolutionary interest owing to the shift from breeding in Opuntia cacti to columnar species. The 3 species form the “mojavensis cluster” of Drosophila. The genome of D. mojavensis was sequenced in 2007 and the genomes of D. navojoa and D. arizonae were sequenced together in 2016 using the same technology (Illumina) and assembly software (AllPaths-LG). Yet, unfortunately, the D. navojoa genome was considerably more fragmented and incomplete than its sister species, rendering it less useful for evolutionary genetic studies. The D. navojoa read dataset does not fully meet the strict insert size required by the assembler used (AllPaths-LG) and this incompatibility might explain its assembly problems. Accordingly, when we re-assembled the genome of D. navojoa with the SPAdes assembler, which does not have the strict AllPaths-LG requirements, we obtained a substantial improvement in all quality indicators such as N50 (from 84 kb to 389 kb) and BUSCO coverage (from 77% to 97%). Here we share a new, improved reference assembly for D. navojoa genome, along with a RNAseq transcriptome. Given the basal relationship of the Opuntia breeding D. navojoa to the columnar breeding D. arizonae and D. mojavensis, the improved assembly and annotation will allow researchers to address a range of questions associated with the genomics of host shifts, chromosomal rearrangements and speciation in this group.