GigaScience, 2022, 11, 1–7 DOI: 10.1093/gigascience/giac026 Data Note A high-quality, long-read genome assembly of the endangered ring-tailed lemur (Lemur catta) 1, ,† 1,2,† 3 3 4,5 Marc Palmada-Flores * , Joseph D. Orkin , Bettina Haase , Jacquelyn Mountcastle , Mads F. Bertelsen , Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giac026/6562532 by guest on 19 April 2022 3 1,‡ 3,5,6,7 1,8,9,10 Olivier Fedrigo , Lukas F. K. Kuderna , Erich D. Jarvis and Tomas Marques-Bonet 1 Department of Medicine and Life Sciences (MELIS), Institut de Biologia Evolutiva, Universitat Pompeu Fabra-CSIC, Barcelona 08003, Spain 2 Département d’anthropologie, Université de Montréal, Montréal, QC H3T 1N8, Canada 3 The Vertebrate Genomes Lab, The Rockefeller University, New York, NY 10065, USA 4 Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C 1870, Denmark 5 Center for Zoo and Wild Animal Health, Copenhagen Zoo, Frederiksber 1870, Denmark 6 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA 7 Laboratory of Neurogenetics of Language, The Rockefeller University, NY 10065, USA 8 Catalan Institution of Research and Advanced Studies (ICREA), Barcelona 08010, Spain 9 CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelon 08028a, Spain 10 Institut Català de Paleontologia Miquel Crusafont, Universitat Autònoma de Barcelona, Cerdanyola del Vallès 08193, Spain ∗ Correspondence address. Tomas Marques-Bonet, Institut de Biologia Evolutiva, Universitat Pompeu Fabra-CSIC, 08003 Barcelona, Spain. E-mail: tomas.marques@upf.edu † These authors contributed equally. ‡ Current affiliation: Illumina Artificial Intelligence Laboratory, Illumina Inc.; San Diego, California, USA Abstract Background: The ring-tailed lemur (Lemur catta) is a charismatic strepsirrhine primate endemic to Madagascar. These lemurs are of particular interest, given their status as a flagship species and widespread publicity in the popular media. Unfortunately, a recent population decline has resulted in the census population decreasing to <2,500 individuals in the wild, and the species’s classification as an endangered species by the IUCN. As is the case for most strepsirrhine primates, only a limited amount of genomic research has been conducted on L. catta, in part owing to the lack of genomic resources. Results: We generated a new high-quality reference genome assembly for L. catta (mLemCat1) that conforms to the standards of the Vertebrate Genomes Project. This new long-read assembly is composed of Pacific Biosciences continuous long reads (CLR data), Optical Mapping Bionano reads, Arima HiC data, and 10X linked reads. The contiguity and completeness of the assembly are extremely high, with scaffold and contig N50 values of 90.982 and 10.570 Mb, respectively. Additionally, when compared to other high-quality primate assemblies, L. catta has the lowest reported number of Alu elements, which results predominantly from a lack of AluS and AluY elements. Conclusions: mLemCat1 is an excellent genomic resource not only for the ring-tailed lemur community, but also for other members of the Lemuridae family, and is the first very long read assembly for a strepsirrhine. Keywords: Strepsirrhine, primate, long-read assembly, ALUs, repeats, lemuridae, mitogenome, scaffolding Context leaves and engage in a multi-male multi-female social structure The strepsirrhines are a remarkably diverse radiation of primates with a polygynandrous mating system [7]. Ring-tailed lemurs are that includes more than one-quarter of all recognized primate under severe conservation pressure; they are classified as "en- species [1]. The vast majority of strepsirrhines (103 species) are dangered" by the International Union for Conservation of Nature members of the Lemuroidea, colloquially known as “lemurs,” and (IUCN) [8], resulting primarily from deforestation, hunting, and endemic to Madagascar. Despite their geographic isolation, the capture for the pet trade. A recent population census has revealed lemur radiation is exceptionally diverse, including both the small- a dramatic population decline, with as few as 2,200 individuals est living primate (Microcebus berthae) and one of the largest (the remaining in the wild [9]. Of further concern, the species is dis- recently extinct subfossil lemur, Archaeoindris fontoynontyii) [2, 3]. tributed across a highly fragmented range with only 8 populations Although lemurs are highly diverse, they are comparatively under- of ≥100 individuals remaining [9]. Despite this near-term popula- studied relative to other primates, and ∼87% of species are threat- tion decline, a recent microsatellite analysis indicates that the ge- ened with extinction, raising major conservation challenges [1]. netic diversity of L. catta populations could be exceptionally high, Of particular interest, both ecologically and in the public imag- with evidence of genetic isolation by distance throughout their ination, are ring-tailed lemurs (Lemur catta, NCBI:txid9447; Fig. 1). geographic range [6]. Ring-tailed lemurs are medium-bodied, ecologically flexible mem- From a genomic perspective, relatively little is known about bers of the Lemuridae family and the sole member of the genus ring-tailed lemurs (and strepsirrhines more broadly). Genome as- Lemur [ 4–6]. In contrast to most other Lemuridae, L. catta predom- semblies have been published for 18 strepsirrhine species, but inantly inhabit the dry and seasonal forests of southern Mada- none of these assemblies has a contig N50 value >1 Mb, and only gascar [7]. They consume an omnivorous diet mostly of fruit and 3 of them are >100 kb [10]. Recently, an L. catta genome (Lem- Received: October 18, 2021. Revised: January 14, 2022. Accepted: February 19, 2022 C The Author(s) 2022. Published by Oxford University Press GigaScience. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 | GigaScience, 2022, Vol. 11, No. 1 Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giac026/6562532 by guest on 19 April 2022 Figure 1: Ring-tailed lemur (L. catta); photo courtesy of Copenhagen Zoo. Cat_v1_BIUU) was assembled by the Zoonomia consortium [11]; insert PacBio library was prepared using the Pacific Biosciences given that it is derived from Illumina short reads, its metrics and Express Template Prep Kit v2.0 (No. 100–938-900) following the application are still limited compared to the genome quality of manufacturer protocol. The library was then size selected (>20 recent highly contiguous assemblies [12]. This general lack of ge- kb) using the Sage Science BluePippin Size-Selection System. A to- nomic resources remains a limitation for the comparative and tal of 23 PacBio 1M v3 (#101–531-000) smrtcells were sequenced on population genomics of lemurs. the Sequel instrument (PacBio Sequel System, RRID:SCR_017989) Here, we present a new high-quality genome assembly of L. (sequencing kit 3.0 No. 101–597-800) with a 10-hour movie and 2 catta (mLemCat1) that conforms to the standards of the Verte- hours pre-extension time. Unfragmented uHMW DNA was used to brate Genomes Project (VGP). mLemCat1 was assembled with a generate a linked-reads library on the 10X Genomics Chromium combination of Pacific Biosciences (PacBio) continuous long reads (Genome Library Kit & Gel Bead Kit v2 PN-120258, Genome Chip (CLR data), Optical Mapping Bionano reads, Arima HiC data, and Kit v2 PN-120257, i7 Multiplex Kit PN-120262). This 10X library was 10X linked-reads. Our new assembly will allow for a deep assess- sequenced on an Illumina Novaseq (Illumina NovaSeq 6000 Se- ment of the genome biology and conservation genomics of endan- quencing System, RRID:SCR_016387) S4 150-bp PE lane. uHMW gered ring-tailed lemurs. Additionally, given the paucity of high- DNA was labeled for Bionano Genomics optical mapping (Bio- contiguity strepsirrhine assemblies, it will allow major advances Nano Irys system, RRID:SCR_016754) using the Bionano Prep Di- in the genomics across the Lemuridae family. rect Label and Stain (DLS) Protocol (30206E) and run on 1 Saphyr (Saphyr, RRID:SCR_017992) instrument chip flow cell. Hi-C prepa- ration was performed by Arima Genomics using the Arima-HiC Data Description kit (P/N: A510008) and an Illumina-compatible library was gen- erated using the KAPA Hyper Prep kit (P/N: KK8504). This library Bionano: San Diego/CA/USA; Qubit: Thermo Fisher Scientific: was then sequenced on an Illumina HiSeq X Ten (Illumina HiSeq Waltham/MA/USA; PacBio: Menlo Park/CA/USA; Sage Science: X Ten, RRID:SCR_016385) (150 bp PE) at ∼60× coverage following Beverly/MA/USA; 10xgenomics: Pleasanton/CA/USA; Illumina: the manufacturer’s protocols. Assuming a genome size of 3.21 Gb San Diego/CA/USA; Arima Genomics: San Diego/CA/USA; KAPA from the GoaT database [13], the present genome (mLemCat1) has (Roche) Basel Switzerland. been produced with 86.43× of 10X linked-reads data, 66.68× of Arima data, 154.57× of Bionano data, and 62.88× of PacBio data. Library preparation and sequencing A sample of spleen tissue was collected post-mortem from a male at the Copenhagen Zoo (Denmark) in 2015 and immediately flash- De novo assembly frozen (ZIMS Global Accession No. GAN: DKL15-03 323). We iso- The genome was assembled following the VGP standard pipeline lated 30 μg of ultra-high molecular weight DNA (uHMW) from v1.6 [12], and the specific parameter settings are available on 35 mg of flash-frozen spleen tissue using the agarose plug Bio- the VGP GitHub repository (Additional File 1). Specifically, contigs nano Genomics protocol for animal tissue (DNA isolation fibrous were generated using FALCON (FALCON, RRID:SCR_018804) [14] tissue protocol No. 30071C). uHMW DNA quality was assessed by a and FALCON-Unzip [15], producing primary and alternate assem- Pulsed-Field Gel assay and quantified with a Qubit 2 Fluorometer blies. We used purge_dups (purge dups, RRID:SCR_021173) [16] to (Supplementary Fig. S1). identify false duplications caused by regions of high heterozygos- A 10 μg quantity of uHMW DNA was sheared using a 26G blunt- ity. Purged contigs were removed from the primary assembly and end needle (PacBio protocol PN 101–181-000 Version 05). A large- added to the alternate assembly. We then scaffolded the primary Genome assembly of ring-tailed lemur | 3 Table 1: Genome quality metrics for the mLemCat1 genome assembly compared to previous assembly and standards Quality category Quality metric VGP standard mLemCat1 LemCat_v1_BIUU Continuity No. scaffolds 141 575,427 Scaffold N50 (Mb) 23–480 90.982 0.216 Largest scaffold (Mb) 285.823 2.320 No. contigs 518 580,026 Contig N50 (Mb) 1–25 10.570 0.158 Largest contig (Mb) 40.360 1.312 Gaps/Gb 75–1,500 179.5 2,001.3 Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giac026/6562532 by guest on 19 April 2022 Span (Gb) 2.122 2.298 Structural accuracy False duplications (%) 0.2–5.0 0.39 Base accuracy Base pair QV 39–43 44.45 k-mer completeness (%) 87–98 91.45 Functional completeness Genes (BUSCOs [S]) (%) 82–98 93.8 84.6 Chromosome status Organellesa 1 Complete allele 1 Complete allele No.: Number of. S: single-copy genes. BUSCOs database is vertebrata_odb10 (n=3,354) and the BUSCO version is: V4.0.6. a For example, mitochondrial. Figure 2: BUSCO assessment results. Comparison between mLemCat1 and LemCat_v1_BIUU Lemur catta assemblies using the Primates_ODB10 database (n = 13,780). The new mLemCat1 assembly shows a 7.3% increase in complete single-copy orthologous genes. assembly using 10X linked-read data with Scaff10X V2.0 [17], Bio- genome assembly (hg38), the L95 and N95 statistics (L95 = 24; N95 nano optical maps with Bionano Solve V.2.1 [18], and Arima Hi- = 46.710 Mb for hg38; L95 = 28; N95 = 21.924 Mb for mLemCat1) C data with Salsa V2.2 [19]. We assembled the mitochondrial are similar, given the expected chromosomes for both (22 auto- genome separately using MitoVGP [20] with PacBio and 10X data. somes + 2 sexual chromosomes in human, and 27 autosomal + The primary scaffolds, alternate contigs, and mitochondrial as- 2 sex chromosomes for L. catta) [24]. Further comparison can be sembly were polished simultaneously. We first performed Polish- found in Supplementary Table S1. The overall GC content of this ing and gap filling with the original PacBio data using Arrow [14], assembly is 40.48%. followed by 2 rounds of short-read polishing using the 10X linked- The mLemCat1 assembly has a high level of accuracy and read data. Specifically, 10X data were mapped to the assembly completeness that conforms to the proposed standards of the using Longranger V2.1.3 [21] and polishing was done with Free- VGP [12]. We assessed the base and structural accuracies of the Bayes (FreeBayes, RRID:SCR_010761) [22]. All computing was per- assembly with Merqury V1.1, using a Meryl V1.7 database [25] formed on the DNAnexus (DNAnexus, RRID:SCR_011884) cloud based on 130.708 Gb (84× coverage) of 10X linked-reads. The platform. base pair QV of the primary assembly is 44.35, which exceeds the VGP standard. The k-mer completeness is 91.45%. We classi- Genome quality assessment fied the structural accuracy using the false duplications percent- Compared to the currently available short-read L. catta genome age calculated in the false_duplications.sh script from Merqury (LemCat_v1_BIUU) [11], the new mLemCat1 assembly has higher V1.1. The assembly is estimated to have 0.39% false duplications contiguity values, fewer scaffolds, and a slightly smaller assembly based on the percentage of k-mers found in unexpected copy size (Table 1). We generated basic continuity assembly metrics for numbers. both assemblies using QUAST V5.0.2 (QUAST, RRID:SCR_001228) To assess the functional completeness of the assembly, we [23], which are presented in Table 1. The assembly has a total recovered BUSCO genes from both mLemCat1 and the exist- scaffold size of 2.122 Gb within 141 scaffolds. The mLemCat1 con- ing Illumina-based assembly (LemCat_v1_BIUU) (Fig. 2). Specif- tig and scaffold N50 values are 10.570 and 90.982 Mb, represent- ically, we conducted a gene completeness assessment using ing 20.41- and 421.21-fold increases, respectively, compared with BUSCO (BUSCO, RRID:SCR_015008) V4.0.6 [26], setting human as the LemCat_v1_BIUU assembly. In comparison with the human the reference species in the –augustus_species parameter, and 4 | GigaScience, 2022, Vol. 11, No. 1 A B Macaque SINE −15.87% DNA −15.67% Marmoset Simple Repeat −1.34% Human Satellite −0.3% Chimpanzee Catarrhine Lemur Orangutan Platyrrhine LTR −19.39% Gorilla Mouse Lemur Ring-tailed Lemur LINE −47.44% 0 2 4 6 8 10 12 Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giac026/6562532 by guest on 19 April 2022 ALUs (%) C Ring-tailed Lemur Gray Mouse Lemur Common Marmoset Rhesus Macaque Alu Alu Alu Alu 100 100 100 100 75 75 75 75 AluJ FRAM AluJ FRAM AluJ FRAM AluJ FRAM 50 50 50 50 25 25 25 25 0 0 0 0 AluS FLAM AluS FLAM AluS FLAM AluS FLAM AluY FAM AluY FAM AluY FAM AluY FAM Sumatran Orangutan Western Gorilla Human Chimpanzee Alu Alu Alu Alu 100 100 100 100 75 75 75 75 AluJ FRAM AluJ FRAM AluJ FRAM AluJ FRAM 50 50 50 50 25 25 25 25 0 0 0 0 AluS FLAM AluS FLAM AluS FLAM AluS FLAM AluY FAM AluY FAM AluY FAM AluY FAM Figure 3: (A) Percentages of elements in the L. catta genome (mLemCat1) masked by RepeatMasker. (B) Percentage of Alus masked in primate long-read assemblies. (C) Spider plots of the total number of different Alu-like elements masked in each genome assembly. Lemurs have fewer AluS elements than anthropoid primates. Axis values represent 1,000× events: FAM (Fossil Alu Monomer); FLAM (Free Left Alu Monomers; FRAM (Free Right Alu Monomer; AluJ (oldest); AluS (intermediate); AluY (youngest); Alu (non-specified). LINE: long interspersed nuclear element; LTR: long terminal repeat; SINE: short interspersed nuclear element [31]. using the primates_OrthoDB10 database, which comprises a to- web server [30]. With the annotation results we plotted a map of tal of 13,780 genes. Of the 13,780 possible BUSCOs, we identified the mitochondrion with GenomeVx [31] (Supplementary Fig. S3). 12,138 single-copy (88.1%), 100 duplicates (0.7%), and 188 frag- Thirteen main protein-coding genes have been annotated in this mented genes (1.4%) in mLemCat1, leaving 9.8% of BUSCOs miss- new mitogenome including nad1, nad2, nad3, nad4, nad4L, nad5, ing. In contrast, we could only recover 11,132 single-copy BUSCOs nad6, cox1, cox2, cox3, atp6, atp8, and cob. (80.8%) from LemCat_v1_BIUU, with 15.3% of BUSCO genes miss- ing. We also ran the same analyses, but changed the database to vertebrata_odb10 and obtained even better results for mLem- Analysis of the repeatome Cat1 compared to LemCat_v1_BIUU, which we report in Table 1. To assess the structure and variety of repeat elements in the L. The present assembly (mLemCat1) can be useful to create syn- catta genome, we analyzed mLemCat1 with RepeatMasker (Re- teny plots between the present species and others, such as hu- peatMasker, RRID:SCR_012954) V4.1.2-p1. Non-default settings in- mans (Supplementary Fig. S2) because it has N50 statistics com- cluded the use of sensitive mode, the query assumed species parable to other high-quality primate genomes, like the pig-tailed set to primates, nhmmscan 3.3.2 (Nov 2020), and FamDB: HMM- macaque [27], olive baboon [28], and golden snub-nosed monkey Dfam_3.3, without the exclusion of simple repeats. In total, 50.32% [29] genome assemblies that have been recently published (Sup- of the bases in the L. catta genome (mLemCat1) are masked as plementary Table S2). interspersed repeats, including long and short interspersed nu- clear elements, long terminal repeats, and DNA elements (Fig. 3A, Supplementary Table S3). In general terms, the portion of the Mitogenome of L. catta genome that comprises repetitive elements is similar to that re- We assembled a gapless mitochondrial genome with a span of ported for other high-quality catarrhine genomes [32, 33], al- 17,086 bp using both PacBio CLR (long reads) and 10X data (short though there are fewer satellites (0.30%), simple repeats (0.68%), reads) using MitoVGP V2.2 with additional parameters “-f 18 000 and low-complexity elements (0.13%) (Supplementary Table S3). -v LENIENT”, as described in the Additional File 2: Table S3 of For- In comparison with the previous Illumina-only assembly (Lem- menti et al. [20], and annotated the assembly using the MITOS2 Cat_v1_BIUU) we observed minor differences in the structure and Genome assembly of ring-tailed lemur | 5 Total Interspersed Repeats LINEs LINE 1 LINE 2 DNA Elements MIRs LTR Elements Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giac026/6562532 by guest on 19 April 2022 hAT−Charlie ERVL−MaLRs Repeat Element Total Bases Masked SINEs TcMar−Tigger204211 ERVL L3/CR1 Unclassified Small RNA Satellites ERV Class I ERV Class II ALUs Low Complexity Simple Repeats −0.5 −0.3 −0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 Percent Difference (mLemCat1 vs. LemCat_V1_BIUU) Figure 4: Comparison of repeat variety and structure between mLemCat1 and LemCat_V1_BIUU assemblies. LINE: long interspersed nuclear element; LTR: long terminal repeat; SINE: short interspersed nuclear element. variety of repeat elements (Fig. 4). The new long-read–based as- rhines, with the fewest being found in the L. catta genome (3.66% sembly has 1.31% more interspersed repeats (50.32% vs 49.01%), of repeat elements) (Fig. 3B, Supplementary Table S5). In con- and a higher percentage of sequence in each repeat subtype, ex- trast to the other primates assessed, for which AluS elements are cept for satellites, simple repeats, low-complexity elements, and most abundant, AluJ is the most common element in mLemCat1 ERV classes I and II. We also observed both a lower percentage of (54.17% of Alu events). Both lemurs have fewer AluS events than sequence and a smaller number of Alu events in mLemCat1. Ad- the anthropoids and fewer AluY events than the catarrhines, con- ditionally, the total number of masked bases is lower in the new sistent with previous reports of the expansion of these 2 families assembly, but they represent a higher percentage of the sequence, after the Catarrhini-Strepsirrhini split [39]. The fact that the com- owing to mLemCat1 having a shorter span. mon marmoset has the highest number of AluS elements (Fig. 3C) Alus are the most abundant repeat elements in the human confirms that the burst that started before the Catarrhini and genome, and differences in their rates, distribution, and prolifera- Platyrrhini parvorders diverged continued with different activity tion could have led to distinct functional changes in multiple pri- in both lineages after their split. Recent Alu activity (AluY events) mate lineages [34]. Alu elements have been present since the ear- is most abundant in catarrhines, particularly the rhesus macaque, liest stages of primate evolution, are frequently located in gene- which, when compared to great apes (Fig. 3C), has a higher overall rich regions, and may play an important role in gene regulation percentage of Alus (Fig. 3B). [35–38]. To compare the Alu repeat landscape of L. catta with those of other highly contiguous primate assemblies, we ran the same version of RepeatMasker as above adding the -alu option. The Conclusion genomes used for the comparison were long-read–based assem- We have assembled a new high-quality genome reference for the blies, including human (hg38), chimpanzee (panTro6), western go- ring-tailed lemur (L. catta) that satisfies the VGP quality assembly rilla (gorGor6), Sumatran orangutan (ponAbe3), rhesus macaque standards. Compared to pre-existing genomic resources, the new (rheMac10), common marmoset (calJac4), and gray mouse lemur assembly has higher contiguity and completeness and contains (Mmur_3.0) (Supplementary Table S4). more single-copy complete BUSCO genes with fewer fragmented We identified substantially fewer Alu elements in the lemur or missing genes. Additionally, we analyzed the L. catta repeatome genomes (L. catta and Microcebus murinus) than those of the catar- and observed substantially fewer Alu events compared to other 6 | GigaScience, 2022, Vol. 11, No. 1 high-quality primate assemblies. This assembly illustrates how and LCF/BQ/DR20/11790032. T.M.B. is supported by funding long-reads and further scaffolding data such as HiC or optical from the European Research Council (ERC) under the European mappings can drastically improve the contiguity and complete- Union’s Horizon 2020 research and innovation programme (grant ness of an assembly, which also allows for improved analysis of agreement No. 864203), BFU2017-86471-P (MINECO/FEDER, UE), structural variation. We suggest that this new assembly will be an “Unidad de Excelencia María de Maeztu,” funded by the AEI excellent resource for the mammalian genomics community, with (CEX2018-000792-M), Howard Hughes International Early Career, particular value for the conservation genomics of lemurs (Fig. 1). NIH 1R01HG010898-01A1, and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixe- ment de la Generalitat de Catalunya (GRC 2017 SGR 880). Data Availability Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giac026/6562532 by guest on 19 April 2022 The raw sequencing data and assembly are available via NCBI Authors’ Contributions BioProject: PRJNA562215. mLemCat1 assembly and the raw reads used to generate it can be accessed at GenomeArk [41]. The M.P.F. and J.D.O. analyzed the data; J.M. and B.H. generated the complete mitogenome of mLemCat1 is available in Genomeark data; B.H. generated the draft assembly; M.F.B. collected the sam- as mLemCat1.MT.20190820.fasta.gz [41]. Specific command line ples. M.P.F. and J.D.O. wrote the manuscript with contributions parameters are available in Additional File 1. The supporting from all authors. T.M.B., E.D.J., O.F., and L.F.K.K. designed the datasets are available in the GigaScience database (GigaDB) [40]. research. Additional Files Additional File 1: Links to the websites with the assembly pipeline References specifics used to create Lemur catta (mLemCat1) genome assembly 1. Estrada, A, Garber, PA, Rylands, AB, et al. Impending extinction and command lines used to perform the different analyses. crisis of the world’s primates: why primates matter. Sci Adv Supplementary Figure S1: Pulse Field Gel assay (Sage Pippin 2017;3(1):e1600946. Pulse) used for quality control of the ultra-High Molecular Weight 2. Orkin, JD, Kuderna, LFK, Marques-Bonet, T. The diversity of pri- DNA mates: from biomedicine to conservation genomics. Annu Rev Supplementary Figure S2: A chromosomal overall synteny plot Anim Biosci 2021;9(1):103–24. between Lemur catta (mLemCat1 assembly; vertical axis) and 3. Fleagle, JG. Primate Adaptation and Evolution. Academic; 2013. Homo sapiens (hg38 assembly; horizontal axis) doi:10.1016/C2009-0-01979-5. Supplementary Figure S3: Representation of the Lemur catta mi- 4. Sussman, RW. Demography and social organization of free- togenome (mLemCat1.MT.20190820) ranging Lemur catta in the Beza Mahafaly Reserve, Madagascar. Supplementary Table S1: Continuity metrics of human (hg38) Am J Phys Anthropol 1991;84(1):43–58. and Lemur catta (mLemCat1) assemblies 5. Cameron, A, Gould, L. Fragment-adaptive behavioural strate- Supplementary Table S2: Comparison of scaffold N50 and assem- gies and intersite variation in the ring-tailed lemur (Lemur catta) bly size of the latest primate genomes published in GigaScience in south-central Madagascar. In: LK Marsh, C Chapman, eds. Supplementary Table S3: Repetitive elements identified by Re- Primates in Fragments: Complexity and Resilience. New York, NY: peatMasker in the L. catta genome assembly (mLemCat1) Springer. 2013:227–43. Supplementary Table S4: Primate assembly lengths and respec- 6. Chandrashekar, A, Knierim, JA, Khan, S, et al. Genetic population tive quantifications of their Alu content structure of endangered ring-tailed lemurs (Lemur catta) from Supplementary Table S5: Alu-monomers and Alu subfamilies nine sites in southern Madagascar. Ecol Evol 2020;10(15):8030–43. count in each assembly 7. Sauther, ML, Sussman, RW, Gould, L. The socioecology of the ringtailed lemur: Thirty-five years of research. 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Curr Opin Competing Interests Genet Dev 2020;62:65–71. “LFKK is currently an employee of Illumina Inc.” All other authors 11. Zoonomia Consortium. A comparative genomics mul- declare no competing interests. titool for scientific discovery and conservation. Nature 2020;587(7833):240–5. 12. Rhie, A, McCarthy, SA, Fedrigo, O, et al. Towards complete and Funding error-free genome assemblies of all vertebrate species. Nature The project that gave rise to these results received the support 2021;592(7856):737–46. of 2 fellowships from "la Caixa" Foundation (ID 100010434) and 13. Challis, RJ, Kumar, S, Stevens, L, et al. GenomeHubs: from the European Union’s Horizon 2020 research and innova- simple containerized setup of a custom Ensembl tion programme under the Marie Skłodowska-Curie grant agree- database and web server for any species. Database ment No. 847648. The fellowship codes are LCF/BQ/PI20/11760004 2017;2017:doi:10.1093/database/bax039. Genome assembly of ring-tailed lemur | 7 14. 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