(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life [1]

['Bernard Y. Kim', 'Department Of Biology', 'Stanford University', 'Stanford', 'California', 'United States Of America', 'Hannah R. Gellert', 'Samuel H. Church', 'Department Of Ecology', 'Evolutionary Biology']

Date: 2024-07

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Funding: SGB was funded by the National Science Foundation (NSF) GRFP. SHC was funded by NSF PRFB DBI 2109502. KNC was funded by the National Institutes of Health (NIH) R35 GM137834. SED and RAM were funded by The Expanding Horizons Initiative, Case Western Reserve University. SD, SD, and ZS were funded by the NSF DEB-1811805. MBE was funded by the Howard Hughes Medical Institute. TF was funded by the Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Research Fellow (22J11897). JAH was funded by the NIH NHGRI 5T32HG000044-27. JH was funded by the Czech Ministry of Education, Youth and Sports grant ERC CZ LL2001. MK was funded by the Academy of Finland 322980. BYK was funded by the NIH NIGMS F32 GM135998. AK was funded by the NIH NIGMS R35 GM122592. TM was funded by the Grant-in-Aid for Research Activity Start-up 22K20565. DM was funded by the NIH NIGMS R35GM148244. PMO was funded by NSF DEB2030129, DEB1839598, and DEB1241253. DJO was funded by the UK Biotechnology and Biological Sciences Research Council BB/T007516/1. DAP was funded by the NIH NIGMS R35GM118165. AT was funded by the JSPS KAKENHI JP23H02530. KHW was funded by the NIH NIGMS K99GM137041. TW was funded by the NSF DOB/DEB-1737877 and the Huron Mountain Wildlife Foundation (Michigan Tech Agreement #1802025). National Science Foundation: https://www.nsf.gov/ ; National Institutes of Health: https://www.nih.gov/ ; The Expanding Horizons Initiative, Case Western Reserve University: https://artsci.case.edu/expanding-horizons-initiative/ ; Howard Hughes Medical Institute: https://www.hhmi.org/ ; Japan Society for the Promotion of Science: https://www.jsps.go.jp/ ; Czech Ministry of Education, Youth and Sports: https://www.msmt.cz/ ; Academy of Finland: https://www.aka.fi/ ; UK Biotechnology and Biological Sciences Research Council: https://www.ukri.org/councils/bbsrc/ ; Huron Mountain Wildlife Foundation: https://www.hmwf.org/ The funders did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All new data generated for this work are deposited at NCBI SRA and GenBank under BioProject PRJNA1020440. The whole-genome alignment is archived at Dryad (DOI: dx.doi.org/10.5061/dryad.x0k6djhrd ). NCBI accessions and citations for public data used for this study are provided in S1 Table . Illumina-only assemblies generated from publicly available datasets (i.e., not generated by this work, S1 Table ), RepeatModeler2 libraries, variant calls, diploid assemblies, genomes, and phylogenetic trees are archived at Zenodo (DOI: dx.doi.org/10.5281/zenodo.11200891 ). Raw Nanopore signal data (fast5, pod5) will be provided upon email request due to their large file sizes. Online locations of data are summarized in S8 Table .

Copyright: © 2024 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we present the outcome of that work and another major step towards unbiased comprehensive genomic study of an entire insect family: 183 new genome assemblies of 179 drosophilid species representing many of the various genera, species groups, and subgroups across Drosophilidae. With this study, genome sequences of 360 drosophilid species (plus 1 new outgroup genome from Family Diastatidae) are available for public download. Of just the new genomes, 121 are from single adult flies and 62 are from laboratory strains. Genomes were predominantly sequenced with a hybrid ONT R9.4.1 (87 genomes) or R10.4.1 (80 genomes) and Illumina approach, except for a small fraction of samples where sample quality issues or low gDNA yield limited our sequencing. The new data include several R10.4.1 runs for community testing and benchmarking, including 378× depth of coverage of the D. melanogaster reference [ 1 , 20 ] strain. We present an updated wet lab protocol and genome assembly pipeline that work for sequencing even the most miniscule of the drosophilids (e.g., some Scaptomyza spp. individuals that we estimate to be less than 1/2 the length of adult D. melanogaster and that yield 35 ng gDNA from 1 adult) at a per-sample cost of US $150 including short reads. Even at this lower limit, our single-fly sequencing approach regularly produces contiguous (>1 Mb contig N50), complete (>98% BUSCO), and accurate (>QV40 genome-wide with ONT R10.4.1) genomes. Finally, we present a reference-free, whole-genome alignment of 298 drosophilid species to facilitate comparative genomic studies of this model system.

While single-specimen insect genome assembly methods [ 17 , 18 ] circumvent the laboratory culture step and can address the taxonomic sequencing bias, the utility of Nanopore sequencing approaches for this purpose has not been thoroughly explored. It is indeed possible to obtain high-quality genome assemblies from single small organisms like drosophilids [ 17 , 19 ]; however, the aforementioned low-input assembly protocols typically increase the material for library construction by employing some form of amplification, and offset contiguity decreases from shorter reads by scaffolding contigs with Hi-C. These methods, while effective on a genome-by-genome basis, can introduce amplification bias but more importantly increase costs and add complexity to the process. Applying these methods at the scale of hundreds or thousands of genomes is not currently feasible without the resources of a large consortium. Furthermore, these methods still require fairly fresh (ideally, flash-frozen) samples. Collections held by field biologists, systematists, and museums are a largely untapped resource for addressing taxonomic bias in available genomes, but the efficacy of long-read sequencing for these types of specimens is similarly untested. We therefore sought out both freshly collected and ethanol-preserved (up to 2 decades old) specimens to test the limits of amplification-free Nanopore sequencing approaches for single flies.

Correcting the taxon sampling bias in genome assemblies is an important step on the path towards a comprehensive genomic study of family Drosophilidae [ 7 , 11 ], but there are technical challenges to address. Establishment of a laboratory culture is a typical first step of generating a drosophilid genome due to the comparatively demanding sample input requirements for long-read sequencing. Recommended genomic DNA (gDNA) inputs range from a few hundred nanograms to several micrograms of high-quality, high molecular weight (HMW) gDNA, often exceeding the amount that can be extracted from an average-sized or smaller drosophilid. Pooling multiple wild individuals increases total gDNA yield but increases the number of haplotypes in the data, leading to inflated consensus error rates and lower assembly contiguities [ 8 , 15 , 16 ]. This is further exacerbated by the high genetic diversity of many insect populations, as well as issues arising from pools containing mis-identified individuals of similar species.

We previously assembled 101 genomes of 93 different drosophilid species in a major step towards the comprehensive genomic study of Drosophilidae [ 8 ]. A hybrid Oxford Nanopore (ONT) long-read and Illumina short-read assembly proved to be an inexpensive and efficient approach to this task, at the time enabling us to build genomes in under a week at the low cost of US $350 per genome. Since then, the number of drosophilid genomes has increased significantly: as of writing (August 2023), 468 genome assemblies for 167 drosophilid species are available to the public through NCBI (representative genomes are listed in S1 Table ). While impressive, the genomic resources for the Drosophila system have major gaps: one of the most obvious is the sampling bias towards species predisposed to culture in a laboratory environment [ 7 , 11 ]. As a result, large sections of interesting drosophilid biodiversity are almost entirely unstudied with modern genomic tools. This includes the Scaptomyza-Hawaiian Drosophila clade, which may be one of the best examples of an adaptive radiation in nature and contains about a fifth of the species in the family [ 12 – 14 ], as well as many lesser-studied species or groups that may provide important context to the currently known evolutionary history of drosophilids.

Species in the model system Drosophilidae (vinegar or fruit flies) have long served to showcase the power of genomics as a tool for understanding evolutionary pattern and process. Drosophilid species were represented among the very first metazoan genomes [ 1 , 2 ], comparative genomic datasets [ 3 , 4 ]; population genomic datasets [ 5 ]; and more recently, single-cell atlases [ 6 ]. These community-built resources have served as a foundation for subsequent discoveries across many fields of scientific inquiry. A logical next step would be the exhaustive sequencing of the >4,400 species [ 7 ] in this biologically diverse family, with the ultimate aim of creating a framework for connecting micro- to macro-evolutionary processes through the powerful lens of the Drosophila system. This seemingly daunting goal is made entirely feasible by cost effective long-read sequencing approaches [ 8 – 10 ], which greatly simplify the process of genome assembly. Further, the genomic tractability of drosophilids (genome sizes approximately 140 to 500 Mbp), their worldwide abundance, and scientific importance of this model system, allows us to rapidly sequence new species while building upon the extensive scaffold of existing scientific resources and knowledge [ 11 ].

Results and discussion

Taxon sampling We selected additional species for sequencing with the primary objective of improving the taxonomic diversity of genomes of species across the family Drosophilidae (Fig 1). Robust inference of historical evolutionary relationships among species and higher taxonomic groups is a key first step that lays the foundation for future study into drosophilid evolution. To date, the largest molecular phylogeny of the group is based on 17 genes from 704 species [7]. While these data are by far the most comprehensive in the number of species surveyed, many deep branches in the phylogeny and many of the exact relationships of species within species groups and subgroups are not confidently resolved. More loci are needed to resolve the species tree, particularly in the presence of extensive introgression and incomplete lineage sorting [21]. Whole-genome sequencing, particularly long-read sequencing, makes thousands of orthologous loci immediately accessible and addresses these issues. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Cladogram of drosophilid species with whole-genome data, with some groups collapsed (gray triangles). Species relationships were inferred from 1,000 orthologs (see Methods). Node values are the local posterior probabilities reported by ASTRAL-MP [26]. Counts of described species for each group were obtained from the TaxoDros database [22]. Values in the colored boxes indicate, as of August 2023, the number of species with whole-genome sequences for each taxon. The count of short-read and long-read datasets, and data available before this study (including [8]) and new genomes presented here are shown separately. *Note that Scaptodrosophila, Hirtodrosophila, Zaprionus, immigrans, and histrio groups are potentially rendered polyphyletic by these samples. The positions of nigrosparsa and pinicola groups are currently considered to be uncertain. The data underlying this figure can be found in https://doi.org/10.5281/zenodo.11200891. https://doi.org/10.1371/journal.pbio.3002697.g001 Sampling was conducted across the family as follows. From the TaxoDros database [22], family Drosophilidae is split into the lesser studied subfamily Steganinae, for which we sequenced 9 species from 5 genera (Stegana, Leucophenga, Phortica, Cacoxenus, and Amiota) and the better known subfamily Drosophilinae. Within the subfamily Drosophilinae, we sequenced 8 species from 4 genera (Colocasiomyia, Chymomyza, Scaptodrosophila, and Lissocephala) that are outgroups to the large, well-studied, and paraphyletic genus Drosophila. Previous studies (e.g., [7,14,23,24]) have long noted this paraphyly, but a taxonomic revision has not occurred in part due to potential effects on the nomenclature of model organisms and due to uncertainty about the placement of many taxa. We therefore sampled 22 species from 14 genera that render the genus Drosophila paraphyletic (Collessia, Dettopsomyia, Dichaetophora, Hirtodrosophila, Hypselothyrea, Liodrosophila, Lordiphosa, Microdrosophila, Mulgravea, Mycodrosophila, Phorticella, Sphaerogastrella, Zygothrica, and Zaprionus). Finally, we sampled new species from the testacea, quinaria, robusta, melanica, repleta, and Hawaiian Drosophila species groups. For the Scaptomyza-Hawaiian Drosophila radiation specifically, we present 63 new genomes from most major groups, including multiple subgenera of the genus Scaptomyza (Exalloscaptomyza, Engiscaptomyza, Elmomyza) and representatives of the picture-wing, haleakale, antopocerus, modified tarsus, ciliated tarsus, and modified mouthpart species groups and their subgroups. We also included 2 species, D. maculinotata and D. flavopinicola, considered to be close relatives of the Scaptomyza+Hawaiian Drosophila lineage [7,25].

Highly accurate genomes with Nanopore R10.4.1 sequencing Oxford Nanopore sequencing hardware and chemistry have seen major upgrades in the shift to R10.4.1 and are now able to read DNA fragments at >99% single-read accuracy (the “Q20 chemistry”). To assess consensus sequence accuracy of drosophilid assemblies using these reads, we extracted HMW gDNA from male adult flies of the D. melanogaster Berkeley Drosophila Genome Project iso-1 strain and generated 54.4 Gbp (378× depth) of ONT R10.4.1 data, with a read N50 of 28,261 bp and containing 11.2 Gbp (79× depth) of reads 50 kb in length or longer. About 40× depth of publicly available short-read data [10] were downloaded from NCBI. Using the full dataset, we assembled (Methods) 142.1 Mbp of genomic sequence in 93 contigs, with a contig N50 of 21.7 Mbp and a genome-wide consensus accuracy (QV46.0) comparable to the dm6 [27] reference genome (QV43.4). Of all the 17,867 features annotated in the dm6 reference genome, we were able to transfer with LiftOff [28] 17,627 (98.7%) to the ONT assembly, and of the subset of 13,967 protein-coding genes, 13,870 (99.3%) were successfully lifted over to the ONT assembly. Next, we assessed the performance of R10.4.1 for more practical levels of sequencing coverage. We downsampled the original reads to 10 replicate datasets for each of 20×, 25×, 30×, 40×, 50×, and 60× depths of coverage and ran our genome assembly pipeline both with and without additional Illumina polishing (using all 40× of the short-read data each time). Gene annotations were again lifted from the dm6 reference genome to each new assembly, and consensus quality scores were evaluated for the entire genome, then separately for each of the major chromosome scaffolds, coding sequences, introns, and intergenic regions (Fig 2 and S2 and S3 Tables). To assess which genomic regions were best and most consistently assembled, we mapped each assembly to a reference genome and computed alignment coverage over each major chromosome (S2 Fig). The D. melanogaster Y chromosome (estimated to be approximately 40 Mbp) is composed of repeat-rich heterochromatin and is poorly assembled, even in the current dm6 reference assembly (approximately 4 Mbp). We used an alternative, heterochromatin-enriched assembly with an additional 10.6 Mbp of Y-linked sequences [29] for these reference alignment-based assessments, reasoning that it would provide a better, albeit still limited, evaluation of the completeness of the Y chromosome. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. High genome-wide consensus accuracy with Nanopore R10.4.1 sequencing. The Phred-scaled consensus accuracy (left axis) and per-base consensus error rate (rate) are shown for genomes built with 20× to 60× coverage of ONT reads. Dashed gray lines show consensus accuracy estimates for R9.4.1 + Illumina [8] and the dm6 reference genome [20]. The data underlying this figure can be found in https://doi.org/10.5281/zenodo.11200891. https://doi.org/10.1371/journal.pbio.3002697.g002 Even for lower (<30×) coverage ONT-only datasets, genome-wide consensus accuracy (Fig 2) exceeds both the standard recommended by the Vertebrate Genomes Project (QV40) [30] and the genome-wide consensus accuracy we previously reported (QV37.15) for a D. melanogaster R9.4.1 and Illumina hybrid assembly [8]. Consensus accuracy is fairly stable past 30× depth but increases in total by about QV1 from 30× to 60× depth of coverage (Fig 2). As we previously observed, consensus accuracy varies across both chromosomes and genomic elements (S2 and S3 Tables), where autosomes are more accurate than sex chromosomes and coding sequences (>QV55 at ≥30× depth) are significantly more accurate than the rest of the genome. Of the ~23 Mbp of coding regions in the D. melanogaster genome we assembled with 60× ONT data, we detect an average of 47 errors (or QV56.9) for R10.4.1-only assemblies and 32 errors (or QV58.6) for Illumina-polished assemblies. For most genomic elements, additional polishing with Illumina data slightly improved consensus accuracy by ~QV1-2 except for contigs mapping to the dm6 Y chromosome, which were not improved by short-read polishing. The depth of ONT sequencing coverage had little impact on assembly completeness for the major D. melanogaster chromosomes (S2 Fig), roughly following the patterns exhibited by the consensus accuracy estimates. The major euchromatic chromosome arms (2L, 2R, 3L, 3R, 4, and X) were well assembled and exhibited similar high degrees of completeness (all approximately 90% or above) across the entire range of downsampled coverages, even though we expected about half coverage on the sex chromosomes relative to the autosomes (e.g., 10× X/Y versus 20× autosome) from male flies. Similarly, the Y chromosome was always poorly assembled (about 10% complete) irrespective of coverage. While this result is expected given previous efforts to assemble the Y chromosome [27,29], our results further indicate that a modest increase in read lengths (approximately 28 kb read N50 in this study versus approximately 14 kb read N50 in [29]) and increasing sequencing coverage have little effect on improving assembly quality, particularly for repeat-rich heterochromatic sequences. More optimistically, these results demonstrate the effectiveness of even modest long-read datasets for assembling the majority of the genome. While we will not delve further into assembly of the D. melanogaster reference genome here, our benchmarking clearly shows the R10.4.1 ONT and Illumina hybrid approach is a cost-effective way to generate high-quality assemblies that meets current standards for reference genomes. We also note a significant increase in R10.4.1 chemistry sensitivity over R9.4.1, in other words, that about 1/5 to 1/10 (by mass) of loaded library is needed to achieve similar pore occupancy to R9.4.1 for libraries of the same fragment size distribution. This indicates great potential for low input ONT library preps and is the critical factor that makes it possible to sequence the more challenging samples presented shortly. Despite the relatively small improvements to sequence accuracy provided by short-read polishing, we still recommend Illumina sequencing for Nanopore-based genome assembly projects of drosophilid species. The input requirements (1 to 10 ng gDNA) and the per-genome costs of Illumina sequencing (US $35 for 30× coverage of a 200 Mbp fly genome) are minimal. Short-read data provide an avenue for assessing assembly accuracy in a less biased reference-free manner [31,32] and are easier to integrate into downstream population genomic analyses within a single variant calling pipeline for genomes from wild-collected individuals.

Haplotype phasing improves the accuracy of single-fly assemblies One of the recent major developments in genome assembly is the class of methods that consider linkage information contained in long reads from a diploid sample to construct a phased diploid assembly, rather than a single haploid assembly. Diploid assembly methods can incorporate phasing during assembly [31], phase while polishing a haploid draft genome [15,33], or correct long reads prior to genome assembly in a haplotype-aware manner [34]. These methods all aim to minimize issues arising from random haplotype switches in a haploid consensus of a diploid organism, which may have downstream effects on read mapping, variant calling, and out-of-frame errors in coding regions [15]. Further, switch errors between variants in close proximity may introduce novel k-mers into the reference genome that will be counted as errors. While these genomes are not completely phased with respect to the parents and may still randomly switch parental phase within contigs, we find that the diploid assembly approach (Methods) improves assembly consensus accuracy by up to an order of magnitude (from QV29.7-QV37.3 to QV40.3-QV58.2) in a head-to-head comparison with a subset of our single-fly genome samples (Fig 3). Variants called separately with Illumina and ONT reads are in general highly (>90%) concordant for 90 out of 101 tested samples (S4 Table), further indicating the effectiveness of even modestly long reads for variant calling and phasing here. These results also imply that diploid assembly of single flies is a superior strategy to assembly from pools of wild individuals. Phased diploid assembly of single flies will therefore be the primary strategy for genome assembly with wild-collected flies in this manuscript and in future work. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Consensus accuracy for single-fly genomes is greatly improved by diploid assembly. Phred-scaled consensus quality (QV) is shown for a subset of 25 R10.4.1 and Illumina hybrid single-fly genomes assembled with haploid (left) and diploid (right) pipelines. The data underlying this figure can be found in https://doi.org/10.5281/zenodo.11200891. https://doi.org/10.1371/journal.pbio.3002697.g003

183 New drosophilid whole-genome sequences Here, we present 183 new genomes for 179 drosophilid species. Of this total, 163 genomes were assembled with a hybrid ONT and Illumina approach, 4 were assembled with only ONT R10.4.1, and 16 were assembled only from Illumina paired-end reads. Sixty-two genomes were assembled with material from a laboratory stock, and the other 121 genomes were assembled from material extracted from a single adult fly. As described, these data improve the depth of sampling for key taxa, such as the Hawaiian Drosophila, multiple mycophagous taxa, and the repleta group, but also capture at least 1 species from many, but not all, drosophilid clades without a sequenced representative (Fig 1). High-quality genome assemblies were generated from these data following our pipeline for haploid assembly from laboratory lines [8], or a diploid assembly workflow for single, wild-caught flies (see Methods for details). Most of the long-read assemblies are highly contiguous, complete, and accurate (Fig 4), in most cases exceeding the QV40 and 1 Mb contig N50 minimum standards proposed by the Vertebrate Genomes Project [30]. We note that genome-wide sequence quality for R9.4.1 drosophilid hybrid assemblies usually does not exceed QV40 even though we have previously demonstrated coding sequences in R9.4.1 hybrid assemblies to be highly accurate [8]. The full details on the samples underlying these assemblies and the corresponding genome QC metrics are provided in S4 Table. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. The distribution of genome quality metrics for 168 new long-read assemblies. Distributions of genome N50, the percentage of complete dipteran BUSCOs [35], and Phred-scaled QV are plotted separately for R10.4.1 and R9.4.1 assemblies from lab strains and from single flies. The black dashed line is the value computed for the D. melanogaster dm6 reference genome. The 16 samples that were only sequenced with Illumina are omitted from these plots. Data underlying this figure and additional sample information is provided in S4 Table. https://doi.org/10.1371/journal.pbio.3002697.g004 The major factors limiting assembly quality were contamination and sample quality. Freshly collected samples had minimal issues during the library prep steps, but microbial (e.g., bacterial, nematode) sequences were abundant in wild-collected specimens (particularly in quinaria group), reducing on-target read coverage of both Illumina and Nanopore reads and thus genome contiguity, accuracy, and concordance of SNPs called separately with both types of reads. For some of these samples (specifically, D. subquinaria, D. suboccidentalis, D. recens, and D. rellima), we made multiple attempts at a single-fly genome assembly and present the best one here. Older ethanol-preserved specimens were also challenging to prepare and assemble. Heavily fragmented and degraded gDNA (e.g., Zaprionus obscuricornis and Phortica magna) limited our ability to remove shorter gDNA fragments with size selection buffers during the ONT library prep. The co-purification of unknown contaminants (frequently present in some of the older Hawaiian picture-wing flies like D. quasianomalipes) required us to perform additional sample purification steps that led to significant sample loss. All these factors negatively impacted read throughput and consensus quality. Genome quality metrics reported in S4 Table are generally lower for these samples. Limited yield and/or highly fragmented gDNA limited us to Illumina sequencing for 16 specimens. The lack of Nanopore data for these gDNA-limited specimens is noted in S4 Table. They tended to be older (collected between 2008 and 2012, with the exception of P. flavipennis in 2017) and smaller flies and were all stored long term in absolute ethanol. For these datasets, a simple draft assembly was generated from paired-end reads with only a contaminant removal post-processing step. While the utility of the latter type of assembly for population or comparative genomics is limited, the sequences are useful for phylogenetic inference [36,37].

Comparative resources based on whole-genome data We present a number of additional resources to assist users of these data with downstream comparative genomic analyses. First, we provide a curated (current as of August 2023) list of representative whole-genome datasets of 360 drosophilid species and 4 outgroups (1 agromyzid, 2 ephydrids, and 1 diastatid) in S1 Table. This table provides a convenient reference for readers to keep track of the genomes incorporated into the resources we present. This can be especially challenging in such a rapidly changing field. Updated versions of this list will be presented in future work. We inferred species relationships of these 364 genomes using 1,000 dipteran BUSCO genes [35,38] identified as complete and single copy across the most genomes (S1 Fig). As expected, the relationships of the major species groups in our phylogeny remains mostly consistent with previous work [7,24]. The differences also reflect our much larger set of orthologs: deep-branching relationships between the clade containing Mulgravea, Hirtodrosophila, Zygothrica, and Mycodrosophila; Dichaetophora; Dettopsomyia; and the Drosophila and Siphlodora subgenera are confidently resolved, as well as the more recent evolutionary relationships between nearly all individual species. Interestingly, we still observe some uncertainty (local posterior probability <0.9) for a few branches in the phylogeny. The cause of increased gene tree-species tree discordance at these branches is currently unknown but will be investigated using a complete set of orthologous markers in future work. A more detailed discussion of the taxonomic implications of whole-genome sequencing will be reserved for this forthcoming study. Lastly, the phylogeny was scaled by the substitution rate at 4-fold degenerate sites in the BUSCO genes to provide a guide tree for whole-genome alignment. A Progressive Cactus [39] reference-free whole-genome alignment was computed for a subset of 298 genomes (S5 Table) out of the 364 species listed in S1 Table. The Cactus alignments and HAL file format utilities [40] are part of a large suite of comparative genomics tools that allow users to quickly perform liftover of genomic features, comparative annotation [41], compute evolutionary rate scores [42], and more. To minimize issues from low-quality assemblies, genomes for the alignment were selected based on minimum contiguity (N50 > 20 kb) and minimum completeness (BUSCO >95%) filters. For species with multiple assemblies present, we deferred to the NCBI representative genome unless our new genome was a major improvement. Alignment coverage of D. melanogaster genomic elements in other species is depicted in Fig 5, showing that most of the protein-coding genome aligns across Drosophilidae. The inferred ancestral drosophilid genome is 33.5 Mbp in size, about 10 Mbp larger than the sum of D. melanogaster coding sequences and contains 97.3% of the dipteran BUSCO genes as complete and single-copy. This suggests the ancestral assembly is enriched for functional sequences and provides an upper bound for the total amount of functional sequence conserved across drosophilid genomes. Moreover, the estimated average substitution rate at 4-fold degenerate sites is 44.8 substitutions/site (S1 Fig), suggesting a complete saturation of substitutions at every neutrally evolving site in the alignable genome and base-level resolution of comparative genomics approaches for evolutionary rate estimation [43]. Together, these results demonstrate the immense potential for comparative genomics provided by this dataset even across deep evolutionary timescales. We note that fitting branch lengths with 4-fold sites alone may mis-estimate deeper branch lengths due to substitution saturation and will reexamine this problem with more complex codon substitution models in forthcoming work on the Drosophilidae Tree of Life. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. Proportion of D. melanogaster genomic elements aligning to other species as a function of 4-fold divergence from D. melanogaster. Each dot represents 1 species. Alignment coverage is defined as the proportion of genomic elements in D. melanogaster that uniquely map to another species. The data underlying this figure can be found in https://doi.org/10.5281/zenodo.11200891. https://doi.org/10.1371/journal.pbio.3002697.g005

Remaining challenges As another major step towards clade-scale genomics of the family Drosophilidae, we have demonstrated that assembling genomes from individual flies, even those preserved in ethanol for up to 2 decades, is now feasible. We are still far from sequencing every, or even most, species in the family. We are faced with several immediate challenges as this resource grows, and it is important for users to be aware of them. There is significant variation in genome quality and completeness despite the generally optimistic genome quality metrics we have reported. Read lengths for single-fly library preps are usually around an order of magnitude shorter than those from inbred lines due to sample material limitations. This difference is particularly egregious for the most fragmented gDNA extractions from the oldest ethanol samples, which are close to the lower limits of what is acceptable for ONT sequencing and assembly (1,000 bp read N50). The reconstruction of repetitive sequences and other complex genomic regions will be severely limited from such short sequences, although these data are still superior to paired-end short reads for genome assembly. We urge that comparative genomic analyses, particularly those of structural variation, carefully consider these possibilities and do not readily consider absence of sequence as evidence of absence. As this resource grows beyond the most commonly studied drosophilids, ambiguity in species identification and taxonomy may become significant issues. All wild-collected samples are, to the best of our ability, identified through key morphological characters prior to sequencing [44,45]. It is nevertheless inevitable that we will misidentify or sequence ambiguous specimens, especially ones that belong to rare and/or cryptic species (e.g., D. colorata) or those with historically inconsistent taxonomic placement (e.g., D. flavopinicola). In a few cases, we have discovered through sequencing that even laboratory and stock center lines were misidentified or contaminated (usually by D. melanogaster, see Materials and methods). We try to minimize these issues by checking genome assemblies against known marker sequences on the Barcode of Life [46] and NCBI Nucleotide databases (Methods), but even then there are many species, some not formally described, for which basic genetic markers are not readily available. It is possible for these markers to come from incorrectly identified samples too. Whole-genome sequencing approaches applied alongside comprehensive field collections will help address the majority of these taxonomic and data quality problems. We will continue to update and correct records as new datasets are generated or if errors in identification are detected.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002697

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.