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Genome assembly naming

For each completed assembly of an individual, we gave that assembly an abbreviated name with the following rules: Lineage/GenusSpecies/Individual#.Assembly#. The first letter, in lowercase, identifies the particular lineage: m, mammals; b, birds; r, reptiles; a, amphibians; f, teleost fish; and s, sharks and other cartilaginous fishes. The next three letters (first in caps) identify the species scientific genus name; the next three letters (first in caps) identifies the specific species name. In the last position is the genome identifier, where integers (1, 2, 3, …) represent different individuals of the same species, and decimals (1.1, 1.2, 1.3, …) represent different assemblies of the same individual. For example, the first submission of the curated Anna’s hummingbird (Calypte anna) assembly is bCalAnn1.1, and an updated assembly for the same individual is bCalAnn1.2. When the abbreviated lineage or genus and species names for two or more species were identical, we replaced the subsequent letters (fourth, fifth and so on) of the genus or species name until they could be differentiated. We have created abbreviated names for all 71,657 vertebrate species (http://vgpdb.snu.ac.kr/splist/; https://id.tol.sanger.ac.uk/).

Sample collection

The production of high-quality genome assemblies required us to obtain high-quality cells or tissue that would yield high-molecular-weight (HMW) DNA for long-read sequencing technologies (CLR and ONT) and optical mapping (Bionano). Therefore, we obtained fresh-frozen samples of various tissues (Supplementary Table 8). All samples were obtained according to approved protocols of the respective animal care and use committees or permits obtained by the respective persons and institutions listed in Supplementary Table 8. Additional details of the samples are on their respective BioSample pages (https://www.ncbi.nlm.nih.gov/biosample; accession numbers in Supplementary Table 8). All tissue types tested yielded a sufficient quantity and quality of DNA for sequencing and assembly, but we found that blood worked best for species that have nucleated red blood cells (that is, bird and reptiles), and spleen or cultured cells worked best for mammals, as of to date. Analysis of different tissue types will be presented elsewhere (in preparation).

Isolation of high-molecular-weight DNA

Agarose plug DNA isolation

For tissue, HMW DNA was extracted using the Bionano animal tissue DNA isolation fibrous tissue protocol (cat no. RE-013-10; document number 30071), according to the manufacturer’s guidelines. A total of 25–30 mg was fixed in 2% formaldehyde and homogenized using the Qiagen TissueRuptor or manual tissue disruption. For nucleated blood, 27–54 μl was used with an adapted protocol (Bionano, personal communication) of the Bionano Prep Blood and Cell Culture DNA Isolation Kit (cat no. RE-130-10). Lysates were embedded into agarose plugs and treated with Proteinase K and RNase A. Plugs were then purified by drop dialysis with 1× TE. DNA quality was assessed using pulse field gel electrophoresis (PFGE) (Pippin Pulse, SAGE Science, Beverly, MA) or the Femto Pulse instrument (Agilent). PFGE revealed that we isolated ultra-high-molecular-weight DNA between ~100 and ~500 kb long.

Phenol–chloroform gDNA extraction

For some samples, we performed phenol–chloroform extractions for HMW gDNA. Snap-frozen tissue was pulverized into a fine powder with a mortar and pestle in liquid nitrogen. The powdered tissue was lysed overnight at 55 °C in high-salt tissue lysis buffer (400 mM NaCl, 20 mM Tris base (pH 8.0), 30 mM EDTA (pH 8.0), 0.5% SDS, 100 μg/ml Proteinase K), and powdered lung tissue was lysed overnight in Qiagen G2 lysis buffer (cat no. 1014636, Qiagen, Hilden, Germany) containing 100 μg/ml Proteinase K at 55 °C. RNA was removed by incubation in 50 μg/ml RNase A for 1 h at 37 °C. HMW gDNA was purified with two washes of phenol–chloroform-IAA equilibrated to pH 8.0, followed by two washes of chloroform-IAA, and precipitated in ice-cold 100% ethanol. Filamentous HMW gDNA was either spooled with shepherds hooks or collected by centrifugation. HMW gDNA was washed twice with 70% ethanol, dried for 20 min at room temperature and eluted in TE. For the flier cichlid muscle gDNA sample used for PacBio CLR and 10XG libraries, glycogen was precipitated by adding 1/10 (v/v) 0.3 M sodium acetate, pH 6.0 to the extracted genomic DNA, mixing carefully and spinning at room temperature at 10,000g. PFGE revealed thatDNA molecule length was between 50 and 300 kb—often lower in size than that obtained with the agarose plug but sufficient for long-range sequencing of CLR and linked read data types.

Others

We also used the Qiagen MagAttract HMW DNA kit (cat no. 67563) and the KingFisher Cell and Tissue DNA kit (Thermo Scientific; cat no. 97030196), following the manufacturers’ guidelines. These protocols yielded HMW DNA ranging from 30 to 50 kb. The Genomic Tip (Qiagen) kit was also used for tissue-based extraction of HMW DNA.

Libraries and sequencing

PacBio libraries and sequencing

DNA obtained from agarose plugs was sheared down to ~40 kb fragment size with a MegaRuptor device (Diagenode, Belgium) and fragmented using Covaris g-tubes (520079) or by needle shearing. PacBio large insert libraries were prepared with either the SMRTbell Template Prep Kit 1.0‐SPv3 (no.100‐991‐900) or the SMRTbell Express Template Prep Kit v1 (no. 101‐357‐000). Libraries were size-selected between 12 and 25 kb using Sage BluePippin (Sage Science, USA), depending on the DNA quality and extraction method. These libraries were sequenced on either RSII or Sequel I instruments, at least 60× coverage per species using Sequel Binding Kit and Sequencing Plate versions 2.0 and 2.1 with 10-h movie time (Supplementary Table 9).

10X Chromium libraries and sequencing

Unfragmented HMW DNA from the agarose plugs was used to generate linked read libraries on the 10X Genomics Chromium platform (Genome Library Kit & Gel Bead Kit v2 PN-120258, Genome Chip Kit v2 PN-120257, i7 Multiplex Kit PN-120262) following the manufacturer’s guidelines. We sequenced the 10X libraries at ~60× coverage per species on an Illumina NovaSeq S4 150-bp PE lane.

Bionano libraries and optical map imaging

Unfragmented ultra-HMW DNA from the agarose plugs was labelled using either two different nicking enzymes (BspQI and BssSI) or a direct labelling enzyme (DLE1) following the Bionano Prep Labelling NLRS (document number 30024) and DLS protocols, respectively (document number 30206). Labelled samples were then imaged on a Bionano Irys or on a Bionano Saphyr instrument. For all species, we aimed for at least 100× coverage per label (Supplementary Table 9).

Hi-C libraries and sequencing

Chromatin interaction (Hi-C) libraries were generated using either Arima Genomics, Dovetail Genomics, or Phase libraries on muscle, blood, or other tissue with in vivo cross-linking (Supplementary Table 9) and sequenced on Illumina instruments. Arima-HiC preparations were performed by Arima Genomics (https://arimagenomics.com/) using the Arima-HiC kit that uses two enzymes (P/N: A510008). The resulting Arima-HiC proximally ligated DNA was then sheared, size-selected around 200–600 bp using SPRI beads, and enriched for biotin-labelled proximity-ligated DNA using streptavidin beads. From these fragments, Illumina-compatible libraries were generated using the KAPA Hyper Prep kit (P/N: KK8504). The resulting libraries were PCR amplified and purified with SPRI beads. The quality of the final libraries was checked with qPCR and Bioanalyzer, and then sequenced on Illumina HiSeq X at ~60× coverage following the manufacturer’s protocols. Dovetail-HiC preparations were performed by Dovetail using a single-enzyme (DpnII) proximity ligation approach. Phase-HiC libraries were made by Phase Genomics using a Proximo Hi-C Library single-enzyme reaction.

Quality control

Before we performed any assembly, all genomic data of all data types from each sample were used to screen potential outlier libraries, outlier sequencing runs, or accidental species contamination with Mash73 by measuring sequence similarity (Supplementary Fig. 4). When running Mash, we used 21-mers to generate sketches with sketch size of 10,000 and compared among each sequencing run, and then differences assessed between sequencing sets.

Genome size, repeat content, and heterozygosity estimations

These estimations were made with k-mer-based methods applied to the Illumina short reads obtained from 10XG linked sequencing libraries. After trimming off barcodes during scaff10x74 preprocessing, canonical 31-mer counts were collected using Meryl23. With the resulting 31-mer histogram, GenomeScope71 was used to estimate the haploid genome length, repeat content, and heterozygosity. The thorny skate linked read data failed quality control, which we suspect was due to low complexity sequences from the high repeat content (54.1%) of the genome; so k-mers were collected later from Illumina whole-genome sequencing reads instead. The genome size and repeat content of the channel bull blenny were estimated from an alternative method that looks at the mode of long read overlap coverage and WindowMasker75, as the estimated genome size from GenomeScope was almost doubling the known haploid genome size (1.29 Gb versus 0.6 Gb) and repeat content (28.0% versus 58.0%), for reasons related to either the quality of the 10X data or species differences.

Benchmarking assembly steps with the Anna’s hummingbird

To develop the VGP standard pipeline, we compared various scaffolding, gap filling, and polishing tools. Default options were used unless otherwise noted. Detailed software versions are listed in Supplementary Table 2.

Contigging and scaffolding

FALCON76 and FALCON-Unzip17 (smrtanalysis 3.0.0) were used to generate contigs that used CLR. Canu77 1.5+67 was used to generate the combined PacBio CLR and Oxford Nanopore ONT assembly. To benchmark scaffolding with linked reads, we used scaff10x74 2.0. For the linked read-only assembly, Supernova 278 was used. For the optical maps, two-enzyme hybrid scaffolding was used in the Bionano Solve v3.2.1 software, using BspQI and BssSI initially, as well as DLE1 later when the technology was developed. For benchmarking Hi-C in scaffolding, Salsa 2.279 was used for scaffolding results in Fig. 1a, with Hi-C reads generated from Arima Genomics. Additional comparisons for the Hi-C libraries were performed using assemblies provided by Dovetail Genomics and Phase Genomics (Supplementary Table 3). We used Hi-C from Arima Genomics as it had the smallest number of PCR duplicates and better coverage for short and long interactions at the time of comparison (Supplementary Fig. 1). Assembly statistics from HiRise, Proximo HiC, 3D-DNA80 and Arima Hi-C are available in Supplementary Table 3. We concluded that all Hi-C scaffolding algorithms had similar performance. We decided to use Salsa, as HiRise and Proximo HiC were not open access, and 3D-DNA was computationally expensive on the DNAnexus platform. For short read assemblies, other than Supernova and the NRGene assembly, the assembly GCA_000699085.116 was used for benchmarking, which was generated with Illumina paired-end, multiple mate-pair libraries and the SoapDeNovo81 assembler. The NRGene assembly was provided by the company with DeNovo Magic.

Gap filling

We ran PBJelly with support –capturedOnly –spanOnly parameters, to avoid greedy gap closures with no spanning read support. For conservatively filling sequences, we compared different parameters in output stage with –minreads 1 and –minreads 4 in addition to no restrictions. We found that the number of gaps closed was similar to the gaps filled with Arrow76 (Supplementary Table 4) and chose not to run PBJelly82 for future assemblies.

Short-read polishing

Illumina polishing benchmarking was performed using Longranger83 2.1.3 and Pilon84 1.21 with –fix bases, local option (Supplementary Table 5). Later, for the VGP pipeline, we used FreeBayes85 as Pilon84 was not computationally scalable for large genomes with the updated Longranger 2.2.2.

Base-level accuracy estimate

Base-level accuracy was measured using a mapping-based approach and later using the k-mer-based approach23. To determine the number of rounds to polish, we used Illumina paired-end reads from the hummingbird16.

Mis-joins and missed-joins

The curated hummingbird assembly was mapped to the target assemblies with MashMap286 with –filter_mode one-to-one –pi 95 using 5 kb segments (-s 5000) for CLR assemblies and 1 kb (-s 1000) for SR assemblies to compensate for the shorter contig sizes, as contigs smaller than a segment size will be excluded from the alignment. The number of mis-joins and missed joins were identified using the assembly_comparison.pl used in the ‘Curation’ section below (Supplementary Methods, Supplementary Fig. 5).

VGP standard genome assembly pipeline 1.0 to 1.6

All 17 genomes were assembled with the VGP pipeline (Extended Data Fig. 2a) for benchmark purposes, with some uncurated. The pale spear-nosed bat, greater horseshoe bat, Canada lynx, platypus, male and female zebra finch, kākāpō, Anna’s hummingbird, Goode’s thornscrub tortoise, flier cichlid, and blunt-snouted clingfish assemblies were generated using the VGP pipeline 1.0 to 1.6 and curated for submission to NCBI and EBI public archives. The curated and submitted two-lined caecilian, zig-zag eel, climbing perch, channel bull blenny, eastern happy, and thorny skate assemblies were generated using a similar process developed in parallel (Supplementary Note 2). Two submitted curated versions of the female zebra finch were made, one using the standard VGP pipeline and the other using the VGP trio pipeline, so that comparative analyses could be performed by others.

Contigging

For PacBio data, contigs were generated from subreads using FALCON76 and FALCON-Unzip17, with one round of Arrow polishing (smrtanalysis 5.1.0.26412). A minimum read length of 2 kb or a cutoff at which reads longer than the cutoff include 50× coverage was used, whichever was longer. For calculating read coverage, we used estimated genome size from http://www.genomesize.com/ when available, or from the literature (Supplementary Table 11) while waiting for 10XG sequencing to estimate genome size using k-mers. FALCON and FALCON-Unzip were run with default parameters, except for computing the overlaps. Raw read overlaps were computed with DALIGNER parameters -k14 -e0.75 -s100 -l2500 -h240 -w8 to better reflect the higher error rate in early PacBio sequel I and II. Pread (preassembled read) overlaps were computed with DALIGNER parameters -k24 -e.90 -s100 -l1000 -h600 intending to collapse haplotypes for the FALCON step to better unzip genomes with high heterozygosity rate. FALCON-Unzip outputs both a pseudo-haplotype and a set of alternate haplotigs that represent the secondary alleles. We refer to these outputs as the primary contig set (c1) and alternate contig set (c2).

Purging false duplications

Heterotype false duplications occurred despite setting FALCON76 parameters to resolve up to 10% haplotype divergence. FALCON-Unzip17 also incorrectly retained some secondary alleles in the primary contig set, which appeared as false duplications. To reduce these false duplications, we ran Purge_Haplotigs13, first during curation (VGP v1.0 pipeline) and then later after contig formation (VGP v1.5 pipeline). To do the former, Purge_Haplotigs was run on the primary contigs (c1), and identified haplotigs were mapped to the scaffolded primary assembly with MashMap286 for removal. In the latter, identified haplotigs were moved from the primary contigs (c1) to the alternate haplotig set (p2). The remaining primary contigs were referred to as p1; p2 combined with c2 was referred to as q2. Later, in the VGP v1.6 pipeline, we replaced Purge_Haplotigs with Purge_Dups14, a new program developed by several of the authors in response to Purge_Haplotigs not removing partial false duplication at contig boundaries. Purging also removes excessive low-coverage (junk) and high-coverage (repeats) contigs. To calculate the presence and overall success of purging false duplications, we used a k-mer approach (Supplementary Methods, Supplementary Fig. 6).

Scaffolding with 10XG linked reads

The 10X Genomics linked reads were aligned to the primary contigs (p1), and an adjacency matrix was computed from the barcodes using scaff10x74 v2.0–2.1. Two rounds of scaffolding were performed. The first round was run with parameters -matrix 2000 -reads 12 -link 10, and the second round with parameters -matrix 2000 -reads 8 -link 10. A gap of 100 bp (represented with ‘N’s) was inserted between joined contigs. The resulting primary scaffold set was named s1.

Scaffolding with Bionano optical maps

Bionano cmaps were generated using the Bionano Pipeline in non-haplotype assembly mode and used to further scaffold the s1 assembly with Bionano Solve v3.2.187. We began with a one-enzyme nick map (BspQI), followed by a two-enzyme nick map (BspQI and BssSI), and then with a DLE-1 one-enzyme non-nicking approach when the later data type became available (Supplementary Table 9). Scaffold gaps were sized according to the software estimate. The resulting scaffold set was named s2.

Scaffolding with Hi-C reads

Hi-C reads were aligned to the s2 scaffolds using the Arima Genomics mapping pipeline88. In brief, both ends of a read pair were mapped independently using BWA-MEM89 with the parameter -B8, and filtered when mapping quality was <10. Chimeric reads containing a restriction enzyme site were trimmed from the restriction site onward, leaving only the 5′ end. The filtered single-read alignments were then rejoined as paired read alignments. The processed alignments were then used for scaffolding with Salsa279, which analyses the normalized frequency of Hi-C interactions between all pairs of contig ends to determine a likely ordering and orientation of each. We used parameters -m yes -i 5 -p yes to allow Salsa2 to break potentially mis-assembled contigs and perform five iterations of scaffolding. After feedback from curation, later versions of Salsa were developed, which more conservatively determine the number of iterations (v2.1) and actively break at mis-assemblies (v2.2), and run for the Canada lynx, Goode’s thornscrub tortoise, and two-lined caecilian. The restriction enzyme(s) used to generate each library were specified using parameters -e GATC,GANTC for Arima and -e GATC for Dovetail and Phase Genomics Hi-C data. The resulting Hi-C scaffolded assembly was named s3.

Consensus polishing

To polish bases in both haplotypes with minimal alignment bias, we concatenated the alternate haplotig set (c2 in v1.0 or q2 in v1.5–1.6) to the scaffolded primary set (s3) and the assembled mitochondrial genome (mitoVGP in v1.6). We then performed another round of polishing with Arrow (smrtanalysis 5.1.0.26412) using PacBio CLR reads, aligning with pbalign –minAccuracy=0.75 –minLength=50 –minAnchorSize=12 –maxDivergence=30 –concordant –algorithm=blasr –algorithmOptions=–useQuality –maxHits=1 –hitPolicy=random –seed=1 and consensus polishing with variantCaller –skipUnrecognizedContigs haploid -x 5 -q 20 -X120 –v –algorithm=arrow. While this round of polishing resulted in higher QV for all genomes herein considered, we noticed that it was particularly sensitive to the coverage cutoff parameter (-x). This is because Arrow generates a de novo consensus from the mapped reads without explicitly considering the reference sequence. Later, we found that the second round of Arrow polishing sometimes reduced the QV accuracy for some species. Upon investigation, this issue was traced back to option -x 5, which requires at least 5 reads to call consensus. Such low minimum requirements can lead to uneven polishing in low coverage regions. To avoid this behaviour, we suggest to increase the -x close to the half sequence coverage (for example, 30× when 60× was used for assembly) and check QV before moving forward.

For genomes with a combined assembly size larger than 4 Gb, we used Minimap290 with parameters -ax map-pb instead of Blasr91 to overcome reference index size limitations.

Two more rounds of base-pair polishing were performed with linked reads. The reads were aligned with Longranger align 2.2.2, which incorporates the Lauriat for barcode-aware alignment83. From the alignments, homozygous mismatches (variants) were called with FreeBayes83 v1.2.0 using default options. Consensus was called with bcftools consensus92 with -i’QUAL>1 && (GT=’’AA’’ || GT = ‘’Aa’’)’ -Hla.

VGP Trio Pipeline v1.0–v1.6

The trio pipeline is similarly designed to the standard pipeline, except for the use of parental data (Extended Data Fig. 3b). When parental genomes are available, the child’s CLR reads are binned to maternal and paternal haplotypes, and assembled separately as haplotype-specific contigs (haplotigs) using TrioCanu20. In brief, parental specific marker k-mers were collected using Meryl23 from the parental Illumina WGS reads of the parents. These markers were filtered and used to bin the child’s CLR read. A haplotype was assigned given the markers observed, normalized by the total markers in each haplotype. The subsequent purging, scaffolding, and polishing steps were similarly updated with the use of Purge_Dups14 (v1.6). We extended binning to linked reads and Hi-C reads, by excluding read pairs that had any parental-specific marker. The binned Hi-C reads were used to scaffold its haplotype assembly, and polished with the binned linked reads from the observation of haplotype switching using the standard polishing approach. During curation, one of the haplotype assemblies with the higher QV and/or contiguity was chosen as the representative haplotype. The heterogametic sex chromosome from the unchosen haplotype was added to the representative assembly. However, while curating several trios, we found that in regions of low divergence between shared parental homogametic sex chromosomes (that is, X or Z), a small fraction of offspring CLR data was mis-assigned to the wrong haplotype. This mis-alignment resulted in a duplicate, low-coverage offspring X or Z assembly in the paternal (for mammals) or maternal (for birds) haplotype, respectively, which required removal during curation. We are working on methods to improve the binning accuracy for resolution of this issue going forward.

For the female zebra finch in particular, contigs were generated before the binning was automated in the Canu assembler as TrioCanu1.7, and therefore a manual binning process was applied as described in the original Trio-binning paper20 (Supplementary Methods). Contigs were assembled for each haplotype using the binned reads, excluding unclassified reads. The contigs were polished with two rounds of Arrow polishing using the binned reads, and scaffolded following the v1.0 pipeline with no purging. Additional scaffolding rounds with Bionano (s4) and Hi-C were applied. Scaffolds were renamed according to the primary scaffold assembly of the same individual (s5), with sex chromosomes grouped as Z in the paternal assembly and W in the maternal assembly following synteny to the Z chromosome from the curated male zebra finch VGP assembly. Two rounds of SR polishing were applied using linked reads, by mapping on both haplotypes. After haplotype switches were discovered, additional rounds of polishing were applied using binned linked reads (Supplementary Methods).

Mitochondrial genome assembly

Similar to other recent methods93,94, we developed a reference-guided MT assembly pipeline. MT reads in the raw CLR data were identified by mapping the whole read set to an existing reference sequence of the specific species or of closely related species using Blasr. Filtered mtDNA CLRs were assembled into a single contig using Canu v1.8, polished with Arrow using CLR and then FreeBayes v1.0.2 together with bcftools v1.9 using short reads from the 10XG data (Extended Data Fig. 3c). The overlapping sequences at the ends of the contig were trimmed, and the remaining contig sequence circularized. The mitoVGP pipeline is made available at https://github.com/VGP/vgp-assembly/tree/master/mitoVGP. A more detailed protocol description of the assembly pipeline and new discoveries from the MT assemblies are published elsewhere33.

Curation

The VGP genome assembly pipeline produces high quality assemblies, yet no automated method to date is free from the production of errors, especially during the scaffolding stages. To minimize the impact of the remaining algorithmic shortcomings, we subjected all assemblies to rigorous manual curation. All data generated for a species in this study and other publicly available data (for example, genetic maps, gene sets and genome assemblies of the same or closely related species) were collated, aligned to the primary assembly and analysed in gEVAL95 (https://vgp-geval.sanger.ac.uk/index.html), visualizing discordances in a feature browser and issue lists. In parallel, Hi-C data were mapped to the primary assembly and visualized using Juicebox96 and/or HiGlass97. With these data, genome curators identified mis-joins, missed joins and other anomalies, and corrected the primary assembly accordingly. No change was made without unambiguous evidence from available data types; for example, a Hi-C suggested join would not be made unless supported by BioNano maps, long-read data, or gene alignments. When sequencing the heterogametic sex, we identified sex chromosomes based on half coverage, homology alignments to sex chromosomes in other species, and the presence of sex chromosome-specific genes.

Contamination removal

A succession of searches was used to identify potential contaminants in the generated assemblies.

1) A megaBLAST98 search against a database of common contaminants (ftp://ftp.ncbi.nlm.nih.gov/pub/kitts/contam_in_euks.fa.gz) requiring e ≤ 1 × 10−4, reporting matches with ≥98% sequence identity and match length 50–99 bp, ≥94% and match length 100–199 bp, or ≥90% and match length 200 bp or above.

2) A vecscreen (https://www.ncbi.nlm.nih.gov/tools/vecscreen/) search against a database of adaptor sequences (ftp://ftp.ncbi.nlm.nih.gov/pub/kitts/adaptors_for_screening_euks.fa)

3) After soft-masking repeats using Windowmasker75, a megaBLAST search against chromosome-level assemblies from RefSeq requiring e ≤ 1 × 10−4, match score ≥100, and sequence identity ≥98%; regions matching highly conserved rDNAs were ignored.

Manual inspection of the results was necessary to differentiate contamination from conservation and/or horizontal gene transfer. Adaptor sequences were masked; other contaminant sequences were removed. Assemblies were also checked for runs of Ns at the ends of scaffolds, created as artefacts of the iterative scaffolding process, and when found they were trimmed.

Organelle genomes

These were detected by a megaBLAST search against a database of known organelle genomes requiring e ≤ 1 × 10−4, sequence identity ≥90%, and match length ≥500; the databases are available at ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/mito.nt.gz and ftp://ftp.ncbi.nlm.nih.gov/refseq/release/plastid/*genomic.fna.gz. Only scaffolds consisting entirely of organelle sequences were assumed to be organelle genomes, and replaced by the genome from the separate organelle assembly pipeline. Organelle matches embedded in nuclear sequences that were found to be NuMTs were kept.

False duplication removal

Retained false duplications were identified using Purge_Haplotigs13 run either after scaffolding and polishing (Anna’s hummingbird, kākāpō, male zebra finch, female zebra finch, platypus, pale spear-nosed bat, and greater horseshoe bat) or on the c1 before scaffolding (two-lined caecilian, flier cichlid, Canada lynx, and Goode’s thornscrub tortoise). Subsequent manual curation identified additional haplotypic duplications for the listed assemblies and also those that were not treated with Purge_Haplotigs (Eastern happy, climbing perch, zig-zag eel). The evidence used included read coverage, sequence self-comparison, transcript alignments, Bionano map alignments and Hi-C 2D maps, all confirming the superfluous nature of one allele. The identified haplotype duplications were moved from the primary to the alternate assembly.

Chromosome assignment

For a scaffold to be annotated as a chromosome, we used evidence from Hi-C as well as genetic linkage or FISH karyotype mapping when available. For Hi-C evidence, we considered a scaffold as a complete chromosome (albeit with gaps) when there was a clear unbroken diagonal in the Juicebox or HiGlass plots for that scaffold and no other large scaffolds that could be joined to that same scaffold; if present and no unambiguous join was possible, we named it as an unlocalized scaffold for that chromosome. When we could not find evidence of a complete chromosome, we kept the scaffold number for its name. We named all evidence-validated scaffolds as chromosomes down to the smallest Hi-C box unit resolution allowed with these characteristics. When there was an established chromosome terminology for a given species or set of species, we use the established terminology except when our new assemblies revealed errors in the older assembly, such as scaffold/chromosome fusions, fissions, rearrangements, and non-chromosome names. For species without an established chromosome terminology, we named the scaffolds as chromosomes numbers 1, 2, 3 …, in descending order of scaffold size. For the sex chromosomes, we used the letters X and Y for mammals and Z and W for birds.

Using comparative genomics to assess assembly structure

In cases where a high-quality chromosome-level genome was available for a closely related species, comparative genome analysis was performed. The polished primary assembly (t3.p) was mapped to the related genome using MashMap286 with –pi 75 -s 300000. The number of chromosomal differences was identified using a custom script available at https://github.com/jdamas13/assembly_comparison. This resulted in the identification of ~60 to ~450 regions for each genome assembly flanking putative misassemblies or lineage-specific genome rearrangements. To identify which were real misassemblies, the identified discrepancies were communicated to the curation team for manual verification (see above).

To identify any possible remaining mis-joins, each curated avian and mammalian assembly was compared with the zebra finch (taeGut2) or human (hg38) genomes, respectively. Pairwise alignments between each of the VGP assemblies and the clade reference were generated with LastZ99 (version 1.04) using the following parameters: C = 0 E = 30 H = 2000 K = 3000 L = 2200 O = 400. The pairwise alignments were converted into the UCSC ‘chain’ and ‘net’ formats with axtChain (parameters: -minScore = 1000 -verbose = 0 -linearGap = medium) followed by chainAntiRepeat, chainSort, chainPreNet, chainNet and netSyntenic, all with default parameters100. Pairwise synteny blocks were defined using maf2synteny101 at 100-, 300-, and 500-kb resolutions. Evolutionary breakpoint regions were detected and classified using an ad hoc statistical approach102. This analysis identified 2 to 90 genomic regions per assembly that could be flanking misassemblies, lineage-specific chromosome rearrangements, or reference-specific chromosome rearrangements (116 in the human and 26 in the zebra finch). Determining the underlying cause for each of the flagged regions will need further verification. All alignments are available for visualization at the Evolution Highway comparative chromosome browser (http://eh-demo.ncsa.illinois.edu/vgp/).

Annotation

NCBI and Ensembl annotation pipeline used in this study are described in the Supplementary Methods.

Evaluation

Detailed methods for other types of evaluation, including BUSCO runs, mis-join and missed-join identification, reliable blocks, collapsed repeats, telomeres, RNA-seq and ATAC–seq mapping, and false gene duplications are in the Supplementary Methods. No statistical methods were used to predetermine sample size, the experiments were not randomized, and the investigators were not blinded to group during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.



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