We present a genome assembly from an individual male Lutra lutra (the Eurasian river otter; Vertebrata; Mammalia; Eutheria; Carnivora; Mustelidae). The genome sequence is 2.44 gigabases in span. The majority of the assembly is scaffolded into 20 chromosomal pseudomolecules, with both X and Y sex chromosomes assembled

Betteridge, Emma

Blaxter, Mark

Chadwick, Elisabeth

Challis, Richard

Corton, Craig

Doulcan, Jale Doulcan

Dudchenko, Olga

Durbin, Richard

Hailer, Frank

Howe, Kerstin

Lieberman Aiden, Erez

McCarthy, Shane

Mead, Dan

Oliver, Karen

Omer, Arina

Portela Miguez, Roberto

Sims, Ying

Skelton, Jason

Smith, Michelle

Torrance, James

Tracey, Alan

Weisz, David

English

Online Research @ Cardiff

 Open Peer ReviewAny reports and responses or comments on thearticle can be found at the end of the article.DATA NOTEThe genome sequence of the Eurasian river otter, Lutra lutra Linnaeus 1758 [version 1; peer review: awaiting peer review]Dan Mead ,       Frank Hailer , Elisabeth Chadwick , Roberto Portela Miguez ,         Michelle Smith , Craig Corton , Karen Oliver , Jason Skelton , Emma Betteridge ,       Jale Doulcan Doulcan , Olga Dudchenko , Arina Omer , David Weisz ,       Erez Lieberman Aiden , Shane McCarthy , Kerstin Howe , Ying Sims ,       James Torrance , Alan Tracey , Richard Challis , Richard Durbin ,Mark Blaxter 1Wellcome Genome Campus, Wellcome Sanger Institute,, Hinxton, CB10 1SA, UKCardiff Otter Project, Cardiff University School of Biosciences, Cardiff, CF10 3AX, UKDepartment of Life Sciences, Natural History Museum, London, SW7 5BD, UKBaylor College of Medicine, Houston, Texas, TX 77030 USA, USAAbstractWe present a genome assembly from an individual male   (theLutra lutraEurasian river otter; Vertebrata; Mammalia; Eutheria; Carnivora;Mustelidae). The genome sequence is 2.44 gigabases in span. Themajority of the assembly is scaffolded into 20 chromosomalpseudomolecules, with both X and Y sex chromosomes assembled.KeywordsLutra lutra river otter genome sequence chromosomal1 2 2 31 1 1 1 11 4 4 44 1 1 11 1 1 11234 Reviewer Status AWAITING PEER REVIEW 19 Feb 2020,  :33 (First published: 5)https://doi.org/10.12688/wellcomeopenres.15722.1 19 Feb 2020,  :33 (Latest published: 5)https://doi.org/10.12688/wellcomeopenres.15722.1v1Page 1 of 7Wellcome Open Research 2020, 5:33 Last updated: 19 FEB 2020  Mark Blaxter ( )Corresponding author: mark.blaxter@sanger.ac.uk  : Conceptualization, Investigation, Writing – Review & Editing;  : Formal Analysis, Investigation, Resources, Writing –Author roles: Mead D Hailer FReview & Editing;  : Writing – Review & Editing;  : Data Curation, Formal Analysis, Investigation, Resources, Writing –Chadwick E Portela Miguez RReview & Editing;  : Formal Analysis, Investigation, Methodology, Writing – Review & Editing;  : Formal Analysis, Investigation,Smith M Corton CMethodology, Writing – Review & Editing;  : Formal Analysis, Investigation, Methodology, Supervision, Writing – Review & Editing; Oliver K Skelton: Writing – Review & Editing;  : Formal Analysis, Investigation, Methodology, Writing – Review & Editing;  : FormalJ Betteridge E Doulcan JDAnalysis, Investigation, Methodology, Writing – Review & Editing;  : Formal Analysis, Investigation, Methodology, Visualization,Dudchenko OWriting – Review & Editing;  : Formal Analysis, Investigation, Methodology, Software, Writing – Review & Editing;  : Writing –Omer A Weisz DReview & Editing;  : Software, Supervision, Writing – Review & Editing;  : Data Curation, Formal Analysis,Lieberman Aiden E McCarthy SInvestigation, Software, Writing – Review & Editing;  : Data Curation, Formal Analysis, Investigation, Software, Supervision, Validation,Howe KWriting – Review & Editing;  : Data Curation, Formal Analysis, Investigation, Software, Writing – Review & Editing;  : DataSims Y Torrance JCuration, Formal Analysis, Investigation, Software, Supervision, Validation, Writing – Review & Editing;  : Data Curation, Formal Analysis,Tracey AInvestigation, Software, Validation, Writing – Review & Editing;  : Data Curation, Formal Analysis, Investigation, Software, Validation,Challis RWriting – Review & Editing;  : Conceptualization, Data Curation, Formal Analysis, Investigation, Software, Validation, Visualization, WritingDurbin R– Review & Editing;  : Conceptualization, Data Curation, Formal Analysis, Investigation, Software, Validation, Visualization, Writing –Blaxter MReview & Editing No competing interests were disclosed.Competing interests: This work was supported by the Wellcome Trust through core funding to the Wellcome Sanger Institute (WT206194). SMcCGrant information:and RD were supported by Wellcome grant WT207492. MB was supported by Wellcome grant WT218328. ELA was supported by an NSF PhysicsFrontiers Center Award (PHY1427654), the Welch Foundation (Q-1866), a USDA Agriculture and Food Research Initiative Grant (2017-05741),and an NIH Encyclopedia of DNA Elements Mapping Center Award (UM1HG009375). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. © 2020 Mead D  . This is an open access article distributed under the terms of the  , whichCopyright: et al Creative Commons Attribution Licensepermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mead D, Hailer F, Chadwick E   How to cite this article: et al. The genome sequence of the Eurasian river otter, Lutra lutra Linnaeus 1758 Wellcome Open Research 2020,  :33 ( )[version 1; peer review: awaiting peer review] 5 https://doi.org/10.12688/wellcomeopenres.15722.1 19 Feb 2020,  :33 ( ) First published: 5 https://doi.org/10.12688/wellcomeopenres.15722.1Page 2 of 7Wellcome Open Research 2020, 5:33 Last updated: 19 FEB 2020Species taxonomyEukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Carnivora; Caniformia; Mustelidae; Lutrinae; Lutra; Lutra lutra Linnaeus 1758 (NCBI txid 9657).BackgroundThe Eurasian river otter, Lutra lutra, is found along the coasts and inland waters of Europe, Asia, China, Japan, Java, Sri lanka, the Middle East and North Africa. Eurasia. Throughout Europe, populations of L. lutra declined precipitously through the latter half of the 20th century, and the species is of active conservation concern. In Ireland, L. lutra populations have remained relatively stable1, and in Britain river restoration and active intervention have resulted in increased populations, and recolonisation of watersheds from which otters had been eliminated2. There is active research of the continuing impacts of pollutants on otters (Pountney et al., 2015), and on the population genetic patterns that have resulted from their near-extinction and subsequent recovery in Britain (Stanton et al., 2014). Here we present a chromosomally assembled genome sequence for L. lutra, based on a male specimen from Britain.Genome sequence reportThe genome was sequenced from a naturally deceased single male L. lutra collected by the Cardiff Otter Project from Wincanton, Somerset. A total of 63-fold coverage in Pacific Biosciences single-molecule long reads (N50 24 kb) and 58-fold coverage in 10X Genomics read clouds (from molecules with an estimated N50 of 57 kb) were generated. Primary assembly contigs were scaffolded with chromosome conformation HiC data (17-fold coverage). The final assembly has a total length of 2.44 Gb in 43 sequence scaffolds with a scaffold N50 of 149.0 Mb (Table 1). The majority, 92.7%, of the assembly sequence was assigned to 20 chromosomal-level scaffolds representing 18 autosomes (numbered by sequence length), and the X and Y sex chromosomes (Figure 1–Figure 4; Table 2). The assembly has a BUSCO (Simão et al., 2015) completeness Table 1. Genome data for Lutra lutra mLutLut1.Project accession dataAssembly identifier mLutLut1Species Lutra lutra Specimen NHMUK ZD 2019.215NCBI taxonomy ID 9657BioProject PRJEB35340Biosample ID SAMEA994731Isolate information Wild casualty; maleRaw data accessionsPacificBiosciences SEQUEL I ERR3313238, ERR3313239-ERR3313241, ERR3313246, ERR3313327, ERR3313330, ERR3313333-ERR331334110X Genomics Illumina ERR3316145-ERR3316148, ERR3316169-ERR3316171Hi-C Illumina SRR10119468Genome assemblyAssembly accession GCA_902655055.1Accession of alternate haplotype GCA_902653095.1Span (Mb) 2,438.00Number of contigs 228Contig N50 length (Mb) 30.40Number of scaffolds 43Scaffold N50 length (Mb) 149.00Longest scaffold (Mb) 223.45BUSCO* genome score C:95.8%[S:94.3%,D:1.5%],F:1.9%,M:2.3%,n:4104*BUSCO scores based on the mammalia_odb9 BUSCO set using v3.0.2. C= complete [S= single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/mLutLut1_1/dataset/mLutLut1_1/busco.1 Vincent Wildlife Trust https://www.vincentwildlife.ie/species/otter2 National Biodiversity network Atlas https://species.nbnatlas.org/species/NBNSYS0000005133#overviewPage 3 of 7Wellcome Open Research 2020, 5:33 Last updated: 19 FEB 2020Figure 1. Genome assembly of Lutra lutra mLutLut1: BlobToolKit Snailplot. The plot shows N50 metrics for L. lutra assembly mLutLut1 and BUSCO scores for the Euarchontoglires set of orthologues. The interactive version of this figure is hosted here.Figure 2. Genome assembly of Lutra lutra mLutLut1: BlobToolKit GC-coverage plot. The interactive version of this figure is hosted here.Page 4 of 7Wellcome Open Research 2020, 5:33 Last updated: 19 FEB 2020Figure 3. Genome assembly of Lutra lutra mLutLut1: BlobToolKit Cumulative sequence plot. The interactive version of this figure is hosted here.Figure 4. Genome assembly of Lutra lutra mLutLut1: Hi-C contact map. Hi-C contact map of the L. lutra mLutLut1 assembly, visualized in Juicebox (Durand et al., 2016). An interactive version of the map hosted here, powered by Juicebox.js (Robinson et al., 2018).Page 5 of 7Wellcome Open Research 2020, 5:33 Last updated: 19 FEB 2020Table 3. Software tools used.Software tool Version SourceFalcon-unzip falcon-kit 1.2.2 (Chin et al., 2016)purge_dups 1.0.0 (Guan et al., 2020) 3D-DNA 180419 (Dudchenko et al., 2018)scaff10x 4.2 https://github.com/wtsi-hpag/Scaff10Xarrow GenomicConsensus 2.3.3 https://github.com/PacificBiosciences/GenomicConsensuslongranger align 2.2.2 https://support.10xgenomics.com/genome-exome/software/pipelines/latest/advanced/other-pipelinesfreebayes v1.1.0-3-g961e5f3 (Garrison & Marth, 2012)bcftools consensus 1.9 http://samtools.github.io/bcftools/bcftools.htmlgEVAL 2016 (Chow et al., 2016)BlobToolKit 1 (Challis et al., 2019)Table 2. Chromosomal pseudomolecules in the genome assembly of Lutra lutra mLutLut1.ENA accession Chromosome Size (Mb) GC% LR738403.1 1 223.45 41LR738404.1 2 210.65 39LR738405.1 3 201.32 39.5LR738406.1 4 197.71 41.7LR738407.1 5 165.81 40.3LR738408.1 6 154.43 40.1LR738409.1 7 149.01 41.9LR738410.1 8 144.75 41.3LR738411.1 9 144.09 42.9LR738412.1 10 114.66 42.7LR738413.1 11 108.79 40.6LR738414.1 12 96.45 43LR738415.1 13 95.73 42.7LR738416.1 14 89.08 43.1LR738417.1 15 69.99 42.8LR738418.1 16 61.48 46.9LR738419.1 17 60.35 46.2LR738420.1 18 40.43 48.2LR738421.1 X 99.69 41.2LR738422.1 Y 2.25 38.8MethodsThe river otter specimen was collected from Wincanton, Somerset by the Cardiff Otter Project. A full tissue dissec-tion and preservation in 80% ethanol was undertaken and the specimen accessioned by the Natural History Museum, London.DNA was extracted using an agarose plug extraction from spleen tissue following the Bionano Prep Animal Tissue DNA Isolation Soft Tissue Protocol. Pacific Biosciences CLR long read and 10X Genomics read cloud sequencing libraries were constructed according to the manufacturers’ instructions. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL I and Illumina HiSeq X instruments. Hi-C data were generated by the Aiden lab using an optimised version of their protocols (Dudchenko et al., 2017).Assembly was carried out using Falcon-unzip (Chin et al., 2016), haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020) and a first round of scaffolding carried out with 10X Genomics read clouds using scaff10x (https://github.com/wtsi-hpag/Scaff10X). Scaf-folding with Hi-C data (Rao et al., 2014) was carried out with 3D-DNA (Dudchenko et al., 2017), followed by manual curation with Juicebox Assembly Tools (Dudchenko et al., 2018; Durand et al., 2016; Robinson et al., 2018) and visualisa-tion in HiGlass (Kerpedjiev et al., 2018). The Hi-C scaffolded assembly was polished with arrow using the PacBio data, then polished with the 10X Genomics Illumina data by aligning to the assembly with longranger align, calling variants with freebayes (Garrison & Marth, 2012) and applying homozygous non-reference edits using bcftools consensus (https://github.com/VGP/vgp-assembly/tree/master/pipeline/freebayes-polish). Two rounds of the Illumina polishing were applied. The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016). We removed two of 95.8% using the mammalia_odb9 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.Page 6 of 7Wellcome Open Research 2020, 5:33 Last updated: 19 FEB 2020low-coverage scaffolds that were likely to have derived from the ribosomal DNA cistron of a Sarcocystis species (most simi-lar to Sarcocystis lutrae). The genome was analysed within the BlobToolKit environment (Challis et al., 2019).Data availabilityEuropean Nucleotide Archive: Lutra lutra (Eurasian otter) genome assembly, mLutLut1. BioProject accession number PRJEB35340; https://www.ebi.ac.uk/ena/data/view/PRJEB35340.The genome sequence is released openly for reuse. The L. lutra genome sequencing initiative is part of the Wellcome Sanger Institute’s “25 genomes for 25 years” project3. It is also part of the Vertebrate Genome Project (VGP)4 ordinal references programme, the DNA Zoo Project5 and the Darwin Tree of Life (DToL) project6. The specimen has been preserved in ethanol and deposited with the Natural History Museum, London under registration number NHMUK ZD 2019.215 where it will remain accessible to the research community for posterity. All raw data and the assembly have been deposited in the ENA. The genome will be annotated and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.AcknowledgementsWe thank Mike Stratton and Julia Wilson for their continuing support for the 25 genomes for 25 years project.3 https://www.sanger.ac.uk/science/collaboration/25-genomes-25-years4 https://vertebrategenomesproject.org/5 https://www.dnazoo.org/6 https://www.darwintreeoflife.org/References	 Challis R, Richards E, Rajan J, et al.: BlobToolKit – Interactive Quality Assessment of Genome Assemblies. bioRxiv. 2019.  Publisher Full Text  Chin CS, Peluso P, Sedlazeck FJ, et al.: Phased diploid genome assembly with single-molecule real-time sequencing. Nat Methods. 2016; 13(12): 1050–54. PubMed Abstract | Publisher Full Text | Free Full Text  Chow W, Brugger K, Caccamo M, et al.: gEVAL - a web-based browser for evaluating genome assemblies. Bioinformatics. 2016; 32(16): 2508–10.  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The genome sequence of the Eurasian river otter, Lutra lutra Linnaeus 1758 [version 1; peer review: 2 approved]

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The genome sequence of the Eurasian river otter, Lutra lutra Linnaeus 1758 [version 1; peer review: 2 approved]

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