Version Changes
Revised. Amendments from Version 1
Based on suggestions made by reviewers, we have made several revision and clarifications to improve the clarity and precision of our findings. We utilized RepeatMasker to analyze repetitive elements and have now included the findings in the result section. Additionally, we have specified the parameters used for each software in Table 3. We have rephrased the gene annotation section to clarify results for both the RefSeq and Ensemble annotation pipelines. We clarified that JupyterPlot is used for scaffold-level alignment and synteny plots in the syntenic analysis. Latly, QC metrics are specified in the abstract.
Abstract
We present a genome assembly of Caretta caretta (the Loggerhead sea turtle; Chordata, Testudines, Cheloniidae), generated from genomic data from two unrelated females. The genome sequence is 2.13 gigabases in size. The assembly has a busco completion score of 96.1% and N50 of 130.95 Mb. The majority of the assembly is scaffolded into 28 chromosomal representations with a remaining 2% of the assembly being excluded from these.
Keywords: Caretta caretta, Loggerhead sea turtle, genome sequence, chromosomal, reptile
Species taxonomy
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Archelosauria; Testudinata; Testudines; Cryptodira; Durocryptodira; Americhelydia; Chelonioidea; Cheloniidae; Caretta; Caretta caretta Linnaeus 1758 (NCBI txid 8467).
Introduction
The loggerhead sea turtle, Caretta caretta, is one of only seven extant marine turtle species and is globally distributed throughout the subtropical and temperate regions of the Mediterranean Sea and Pacific, Indian and Atlantic Oceans ( Wallace et al., 2010, Casale and Tucker, 2015). The species is divided in various Regional Management Units (RMUs) and management units (MUs) that vary greatly by population size, geographic range, and population trends ( Wallace et al., 2010, Casale and Tucker, 2015, Shamblin et al., 2014). Events such as fisheries bycatch ( Caracappa et al., 2018, Pulcinella et al., 2019), human intrusion and disturbance ( Mazaris et al., 2009), oceanic pollution ( Savoca et al., 2018), and climate change and severe weather ( Alduina et al., 2020) have caused the global population to continuously decline ( Casale and Tucker, 2015). Consequently, the highly migratory C. caretta requires the collaborative efforts of numerous international conservation and protection organizations ( Species at Risk Act, 2002), and is currently listed as Vulnerable by the International Union for the Conservation of Nature (IUCN) ( Casale and Tucker, 2015). The genome of C. caretta was sequenced as part of the Canadian BioGenome Project (CBP) and CanSeq150 initiatives. The C. caretta genome will provide insights into genomic diversity and architecture, and inform conservation genomics applications.
Methods
Sample collection
Blood samples from an adult female and a juvenile of unknown sex were collected from the Fondazione Cetacea (43.9940 N, 12.6745 E) by Nicola Ridolfi (veterinarian; Fondazione Cetacea). Animal husbandry and welfare were overseen by Fondazione Cetacea. The specimens were transferred to Canada with two CITES permits between institutions (IT002 and CA027).
Sample extraction, library construction and sequencing
High-molecular weight (HMW) DNA was extracted from nucleated blood using the MagAttract HMW DNA kit (QIAGEN, Germantown, MD, USA). Nanopore genome libraries were constructed according to manufacturer instructions and sequenced using the PromethION instrument (Oxford Nanopore Technologies). A PCR-free genome library was sequenced in a multiplexed pool of an Illumina NovaSeq 6000 instrument S4 flowcell with paired-end 150 bp (PE150) reads. A Hi-C library was constructed using the Arima-HiC kit 2.0 (Arima Genomics, San Diego, CA) and the Swift Biosciences Accel-NGS 2S Plus DNA Library Kit (Integrated DNA Technologies, Mississauga, ON, Canada) and subjected to PE150 sequencing on an Illumina NovaSeq 6000 instrument. All lab work were performed at Canada’s Michael Smith Genome Sciences Centre at BC Cancer.
Genome assembly
Assembly was carried out using Redbean ( Ruan and Li, 2019), followed by four rounds of racon ( Vaser et al., 2017) polishing and medaka (medaka, n.d.) polishing. Scaffolding with Hi-C data was carried out using nf-core/hic workflow ( Servant and Peltzer, 2019), Salsa ( Ghurye et al., 2019) and LongStitch ( Coombe et al., 2021). The Hi-C scaffolded assembly was polished using Illumina short-reads using Pilon ( Walker et al., 2014). Four rounds of manual assembly curation and re-scaffolding with nf-core/hic workflow ( Servant and Peltzer, 2019) and Salsa ( Ghurye et al., 2019) corrected 54 missing/misjoins. The changes were visualized with a Hi-C contact map using Juicer ( Durand et al., 2016b). JupiterPlots ( Chu, 2018) was used to perform scaffold-level alignment with Green turtle reference genome and generate synteny plot for synteny analysis. The final sequence was analyzed using BlobToolKit ( Challis et al., 2020) for quality assessment and RepeatMasker ( Tarailo‐Graovac & Chen, 2009) for annotation of repetitive regions. The parameter and version number of software tools are listed in Table 3.
Table 3. Software tools used.
| Software | Version | Parameters | Source |
|---|---|---|---|
| Racon | 1.4.13 | Default parameters | Vaser et al., 2017 |
| Medaka | 1.2.0 | Default parameters | https://github.com/nanoporetech/medaka |
| Pilon | 1.23 | Default parameters | Walker et al., 2014 |
| Salsa | 2.3 | -m CLEAN -e GATC,GANTC,CTNAG,TTAA | Ghurye et al., 2019 |
| BlobToolKit | 2.6.4 (BTK pipeline)
3.1.0 (Blobtoolkit) |
Default parameters | Challis et al., 2020 |
| nf-core/hic | 1.1.0 | --restriction_site ‘^GATC,G^ANTC,C^TNAG,T^TAA’ --ligation_site ‘GATCGATC,GANTGATC,GANTANTC,GATCANTC’ --skip_tads | Servant and Peltzer, 2019 |
| Juicer Tools | 2.13.06 | Default parameters | Durand et al., 2016b |
| Juice Box | 2.13.06 | Default parameters | Durand et al., 2016a |
| Redbean | 2.5 | Default parameters | Ruan and Li, 2019 |
| LongStitch | 1.0.1 | tigmint-ntLink-arks G=2e9 z=100 | Coombe et al., 2021 |
| Jupiter Plot | 1.0 | ng=98 | Chu, 2018 |
| Busco | 5.2.2 | -l sauropsida_odb10 | Manni et al., 2021 |
| Quast | 5.0.2 | Default parameters | Gurevich et al., 2013 |
| RepeatMasker | 4.1.5 | -species “Caretta caretta” | Tarailo‐Graovac & Chen, 2009 |
Results
Genome sequence report
The genomes of two unrelated loggerhead sea turtles were sequenced from the same population collected from the Fondazione Cetacea hospital, Riccione, Italy. A total of 39-fold coverage in Nanopore PromethION long reads were generated from a single adult female. Approximately 50-fold coverage in Illumina NovaSeq6000 150 bp paired-end (PE150) reads and 18-fold coverage in Illumina NovaSeq6000 Hi-C sequencing were generated from a second individual. Primary assembly contigs from Nanopore data were further polished with Illumina PE150 shotgun sequencing data and scaffolded with Hi-C data. The final assembly has a total length of 2.13 Gb in 2007 sequence scaffolds with a scaffold N50 of 130.95 Mb ( Table 1). The majority (98.0%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds representing the species’ known 28 autosomes ( Kamezaki, 1989, Machado et al., 2020) (numbered by sequence length; Figure 1– Figure 4; Table 2). Aligned reads from the second turtle to the final assembly had an estimated heterozygosity of 0.11% (2,449,606 heterozygous hits). Determining gene coverage using BUSCO, we estimated 96.1% gene completeness using the sauropsida_odb10 reference set ( Manni et al., 2021). The assembly was compared to a previous chromosome-scale assembly of the closely-related green sea turtle, Chelonia mydas ( Wang et al., 2013), which has been reported to hybridize with the loggerhead sea turtle ( James et al., 2004, Vilaça et al., 2012). The loggerhead sea turtle assembly showed strong synteny to the green sea turtle assembly, as shown in Figure 5. The primary haplotype (rCheMyd1.pri.v2) of the green sea turtle was downloaded from NCBI on July 16, 2022. The proportions of SINEs, LINEs, LTR elements, and DNA transposons within the genomic sequences were determined to be 1.55%, 8.75%, 0.13%, and 1.10%, respectively.
Table 1. Genome data for Caretta caretta, rCarCar2.
| Project accession data | |
|---|---|
| Assembly identifier | rCarCar2 |
| Species | Caretta caretta |
| Specimen | SJ_126, SJ_184 |
| NCBI Taxonomy ID | 8467 |
| BioProject | PRJNA826225 |
| BioSample ID | SAMN28968396, SAMN27958248 |
| Isolate Information | SJ_184/204:Loco2, SJ_126:Eziel1 |
| Raw data accessions | |
| Oxford Nanopore PromethION | SRX15677840, SRX15677841 |
| Hi-C Illumina | SRX15677843 |
| Illumina short-read | SRX15677842 |
| Genome assembly | |
| Assembly accession | GCA_023653815.1 |
| Assembly name | GSC_CCare_1.0 |
| Span (Mb) | 2,134 |
| Number of contigs | 2,753 |
| Contig N50 length (Mb) | 18,214 |
| Number of scaffolds | 2,008 |
| Scaffold N50 length (Mb) | 130,956 |
| Longest scaffold (Mb) | 345.7 |
| BUSCO * genome score | C:96.1%[S:95.2%,D:0.9%],F:0.4%,M:3.5%,n:7480 |
BUSCO scores based on the sauropsida_odb10 BUSCO set using v5.0.0. C=complete [S=single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison.
Figure 1. Genome assembly of Caretta caretta, rCarCar2: metrics.
Snail plot showing N50 metrics, base pair composition and BUSCO gene completeness for C. caretta (rCarCar2) generated from Blobtoolkit v.2.6.4 ( Challis et al., 2020). The plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 2,134,012,717 bp assembly. The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (345,741,823 bp) shown in red. Orange and pale-orange arcs show the N50 and N90 chromosome lengths (130,956,235 and 23,648,662 bp, respectively). The pale grey spiral shows the cumulative chromosome count on a log scale with white scale lines showing successive orders of magnitude. The blue and pale-blue area around the outside of the plot displays the distribution of GC (blue), AT (pale blue) and N (white) percentages using the same bins as the inner plot. A summary of complete (96.1%), fragmented (0.4%), duplicated (0.9%), and missing (3.5%) BUSCO genes in the sauropsida_odb10 set is show in the top right.
Figure 4. Genome assembly of Caretta caretta, rCarCar2: Hi-C contact map.
HiC contact map of rCarCar2 assembly visualized using JuiceBox v2.13.07 ( Durand et al., 2016a). Chromosomes are shown in order of size from left to right and top to bottom. As an additional confirmation for the quality of the assembly, the microchromosomes are visible as a cluster of spatially-associated contigs in the lower right, as reported in by Waters et al., 2021.
Table 2. Chromosomal pseudomolecules in the genome assembly of Caretta caretta, rCarCar2.
| RefSeq sequence | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| NC_064473.1 | 1 | 345.74 | 42.86 |
| NC_064474.1 | 2 | 265.32 | 42.62 |
| NC_064475.1 | 3 | 208.08 | 42.71 |
| NC_064476.1 | 4 | 135.63 | 42.34 |
| NC_064477.1 | 5 | 130.96 | 42.42 |
| NC_064478.1 | 6 | 128.66 | 43.74 |
| NC_064479.1 | 7 | 123.31 | 43.74 |
| NC_064480.1 | 8 | 108.54 | 43.66 |
| NC_064481.1 | 9 | 101.34 | 43.68 |
| NC_064482.1 | 10 | 85.28 | 44.40 |
| NC_064483.1 | 11 | 76.53 | 43.00 |
| NC_064484.1 | 12 | 43.19 | 43.81 |
| NC_064485.1 | 13 | 38.20 | 47.24 |
| NC_064486.1 | 14 | 35.79 | 45.97 |
| NC_064487.1 | 15 | 33.48 | 45.53 |
| NC_064488.1 | 16 | 25.69 | 46.28 |
| NC_064489.1 | 17 | 24.70 | 45.64 |
| NC_064490.1 | 18 | 23.65 | 46.93 |
| NC_064491.1 | 19 | 20.21 | 48.10 |
| NC_064492.1 | 20 | 19.04 | 47.85 |
| NC_064493.1 | 21 | 18.99 | 46.81 |
| NC_064494.1 | 22 | 17.93 | 52.48 |
| NC_064495.1 | 23 | 16.78 | 47.24 |
| NC_064496.1 | 24 | 16.65 | 49.92 |
| NC_064497.1 | 25 | 16.37 | 50.20 |
| NC_064498.1 | 26 | 13.31 | 54.27 |
| NC_064499.1 | 27 | 12.55 | 57.47 |
| NC_064500.1 | 28 | 5.34 | 57.00 |
Figure 5. Jupiter plot alignment of Caretta caretta with Chelonia mydas (green sea turtle).
Full genome alignment of Caretta caretta genome, rCarCar2 (right), and Chelonia mydas (green sea turtle) genome (primary haplotype v2), rCheMyd1 (left), generated using Jupiter Plot ( Chu, 2018). The left of the circle shows 28 green sea turtle chromosomes and the right of the circle shows 28 loggerhead sea turtle chromosomes. Coloured bands represent synteny between the genomes, and lines crossing the circle indicate genomic rearrangements, or break points in the scaffolds.
Figure 2. Genome assembly of Caretta caretta, rCarCar2: GC-content.
GC-coverage plot of C. caretta (rCarCar2) generated from Blobtoolkit v.2.6.4 ( Challis et al., 2020). Scaffolds are coloured by phylum with Chordata represented by blue and no-hit represented by pale blue. Circles are sized in proportion to scaffold length. Histograms show the distribution of scaffold length sum along each axis.
Figure 3. Genome assembly of Caretta caretta, rCarCar2: cumulative sequence length.
Cumulative sequence length of C. caretta (rCarCar2) generated from Blobtoolkit v.2.6.4 ( Challis et al., 2020). The grey line shows the cumulative length for all scaffolds. Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the BUSCO genes tax rule, with Chordata represented by blue and no-hit represented by pale blue.
Genome annotation
The loggerhead sea turtle genome assembly was annotated by both RefSeq annotation pipeline ( Li et al., 2020) and Ensembl gene annotation system ( Aken et al., 2016). The RefSeq annotation pipeline includes 24,923 genes and pseudogenes, and 54,583 mRNA transcripts ( NCBI Caretta caretta Annotation Release). The Ensembl annotation includes 19,633 coding genes, 4,161 non-coding genes and 42,302 mRNA transcripts ( Caretta caretta - Ensembl Rapid Release).
Funding Statement
Sequencing of the loggerhead sea turtle genome was supported through the Canadian BioGenome Project (Grant ID 18107, Genome Canada) and CanSeq150 program of Canada’s Genomics Enterprise (www.cgen.ca), as well as the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement 844756 (TurtleHyb).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 2; peer review: 2 approved]
Data availability
Underlying data
National Centre for Biotechnology Information BioProject: Loggerhead Sea turtle ( Caretta caretta) genome sequencing and assembly, rCarCar2. Accession number: PRJNA826225.
The genome sequence is released openly for reuse. The C. caretta genome sequencing initiative is part of the Canadian BioGenome Project and CanSeq150 Projects initiatives. All raw sequence data and the assembly have been deposited in INSDC databases. The genome is annotated through the Reference Sequence (RefSeq) database in BioProject accession number PRJNA853764. Raw data and assembly accession identifiers are reported in Table 1.
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