The genome sequence of the red-legged partridge, Alectoris rufa Linnaeus 1758

Sample collection

We randomly selected a wild female from Ciudad Real (central Iberian Peninsula) and a farm-raised male from Lleida (northeastern Iberian Peninsula). They were both anesthetized by inhaling isoflurane, before blood samples (0.25 ml) were drawn from the brachial vein of the wing using a sterile syringe with a 20G needle. After the procedure, the birds were allowed to recover and closely monitored for any signs of distress. The protocol was approved by the Ethics Committee on Animal Experimentation of the University of Lleida in 2016 (Ref. CEEA 09-06/16).

Sample extraction, library construction and sequencing

High molecular weight (HMW) DNA was extracted from that blood for library preparation with the genomic DNA sequencing kit of Oxford Nanopore technology (ONT) as described in² and then sequenced the libraries using a GridION platform.

Chromatin conformation capture sequencing (Hi-C) libraries were prepared using the Hi-C High-Coverage kit (Arima Genomics) in the Metazoa Phylogenomics Lab (Institute of Evolutionary Biology [CSIC-UPF]). Sample concentration was assessed by Qubit DNA HS Assay kit (Thermo Fisher Scientific), and library preparation was carried out using the ACCEL-NGS 2S PLUS DNA LIBRARY KIT (Swift Bioscience) and using the 2S Set A single indexes (Swift Bioscience). We carried out library amplification with the KAPA HiFi DNA polymerase (Roche). The amplified libraries were sequenced on the NovaSeq 6000 (Illumina) with a read length of 2x151bp and a ~60Gb coverage, resulting in ~400M reads per library.

Nuclear genome assembly

Nuclear genome assembly was performed using Nanopore raw data from one female and one male, as described in our previous work.² We implemented and used the Dorado base caller⁵ to improve per-base quality of the original noisy ONT raw reads.

Assembly was carried out with NextDenovo pipeline.⁶ The yielded contig-level assembly was polished using the NextPolish v1.4.0⁶ based on the ONT data. Haplotypic duplications were identified and removed using purge_haplotigs.⁷ The purge_haplotigs contigs were used to filter out mitochondrial genome form the nuclear contig, as described below.

Fastp⁸ was used to process the Hi-C removing low quality and duplication raw reads and HapHiC⁹ was used on the Hi-C data to scaffolds the purged contigs. HapHiC is an allele-aware tool that enables scaffolding the haplotype-phased genome assembly into chromosome-scale pseudomolecules without the need for a preexistent reference genome. Chromosome assignment results were validated based on their synteny with respect to the Gallus gallus reference genome GCA_024206055.2,¹⁰ using Ragtag v2.1.0.¹¹ Note that RagTag orders, orients, and joins sequences with gaps without altering the input query. We performed a final round of gap filling with Medaka¹² using the ONT raw reads. This was sufficient to generate the final gapless genome and capture the chromatin signal of both sex chromosomes of A. rufa. We independently assembled the male and female autosomes using sex-specific Hi-C data. We choose the autosome with the highest quality as the reference. However, due to the lower quality of the male Hi-C data, we used only female Hi-C data to assemble the sex chromosomes.

Mitochondrial genome assembly and annotation

The mitogenome was extracted from the polished and purged nuclear contigs using a combined approach. First, the get_organelle_from_assembly.py script from GetOrganelle¹³ was applied to the nuclear contigs, producing an uncircularized, gapped sequence. Next, Illumina short reads were used to circularize the mitogenome assembly. For annotation, MitoZ v3.6¹⁴ was employed. The genome assembly metrics were estimated using the Gfastats tool.¹⁵ The BUSCO completeness score¹⁶ was calculated within the BlobtoolKit2.¹⁷

Genome sequence report

We sequenced the genome of two A. rufa individuals, one male and one female. We summarize the statistics and deposition data in Table 2. We generated a total of 60-fold coverage data in Nanopore Ultra-long reads (N50 20 kb). We also generated 60-fold coverage data in Illumina NovaSeq6000 Hi-C sequencing. We scaffolded the primary assembly contigs from nanopore sequences with chromosome conformation HiC data. We also performed a final quality control step for the HiC scaffolds by validating them through synteny comparison to the Gallus gallus reference genome. The final assembly has a total length of 1.38Gb in 46 sequence scaffolds with scaffold/contig N50 of 92 Mb. 99.91% of the assembly sequence was assigned to 40 chromosomal-level scaffolds representing 38 autosomes plus the W and Z sex chromosomes ( Figures 1, 2, 3, and 4 and Table 2). The mitochondrial genome was 16.694 kbp long and contained 38 genes, including 22 tRNAs and 14 protein coding genes, with a GC percentage of 45.27 % ( Figure 5). The genome has a BUSCO completeness of 99.1% using the aves_odb10 reference dataset of orthologue single copy genes. The statistics for each chromosome, together with their ENA accession numbers are given in Table 3.

Figure 1. BlobToolKit Snailplot, representing the N50 metrics for A. rufa assembly and BUSCO scores for the Aves set of orthologues.

The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly shown in red.

Figure 2. Juicebox visualization of the manually curated Hi-C contact map for the A. rufa assembly.

Left-hand side: Original mapping. Right-hand side: Contact map after manual curation.

Figure 3. BlobToolKit GC-coverage plot.

Circles are sized in proportion to scaffold length. Histograms show the distribution of scaffold length sum along each axis. Y-axis ontogene covariance (Chordata).

Figure 4. Cumulative sequence length plot of the A. rufa assembly.

The grey line shows cumulative length for all sequences.

Figure 5. Mitochondrial genome gene map of A. rufa.

The outermost track displays the annotated genes features. The middle track shows the coverage depth from ONT sequencing in 100 bp windows. The innermost track illustrates the GC content across the mitogenome, also in 100 bp windows.