The genome sequence of the northern goshawk, Accipiter gentilis (Linnaeus, 1758)

We present a genome assembly from an individual female Accipiter gentilis (the northern goshawk; Chordata; Aves; Accipitriformes; Accipitridae). The genome sequence is 1,398 megabases in span. The majority of the assembly (99.98%) is scaffolded into 40 chromosomal pseudomolecules, with the W and Z chromosomes assembled. The complete mitochondrial genome was also assembled and is 16.6 kilobases in length.


Background
The northern goshawk, Accipiter gentilis, is a medium-sized, forest specialist, bird of prey inhabiting large parts of the Holarctic. The considerable morphological variation particularly within A. gentilis has resulted in the acknowledgement of 10 subspecies (Del Hoyo & Collar, 2015;Dickson & Remsen, 2013), with the nominate European subspecies, A. gentilis gentilis found across Europe, except for the Iberian Peninsula, southern Italy, and Greece, and extending eastwards to the Carpathians and part of Russia. However, mitochondrial phylogenetic analyses suggest two monophyletic groups within the species, a Neartcic clade and a Palearctic clade (Kunz et al., 2019).
In the UK, loss of woodland habitat and persecution drove the species to extinction by the end of the 19 th century, before it was reintroduced during the 1960s. Despite being legally protected, A. gentilis are still persecuted throughout Europe (Rutz et al., 2006) and their nests are frequently robbed. For instance, in Scotland, illegal killing of birds of prey in general, and northern goshawks in particular, has not declined during the last 20 years (RSPB, 2015), leading to the hypothesis that this might have contributed to the slow recovery of the population in the UK despite repeated reintroductions (Kenward, 2006).

Genome sequence report
The genome was sequenced from a single female A. gentilis ( Figure 1) collected from Northumberland, UK. A total of 34-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 31-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 113 missing/misjoins and removed 1 haplotypic duplication, reducing the assembly size by 0.04% and the scaffold number by 17.30%, and increasing the scaffold N50 by 9.56%.
The final assembly has a total length of 1,398 Mb in 454 sequence scaffolds with a scaffold N50 of 34.0 Mb (Table 1). The majority, 90.67%, of the assembly sequence was assigned to 40 chromosomal-level scaffolds, representing 38 autosomes (numbered by sequence length) and the W and Z chromosomes (Figure 2- Figure 5; Table 2). Microchromosomes 35, 36, 37, and 38 were curated based on homology to microchromosomes found in Gallus gallus, Taeniopygia guttata, and Cuculus canorus.
The assembly has a BUSCO v5.1.2 (Manni et al., 2021) completeness of 97.5% (single 96.7%, duplicated 0.8%) using the aves_odb10 reference set (n=8338). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A single female A. gentilis specimen (bAccGen1) was found dead in coniferous woodland in Northumberland, UK by Katherine August (University of Aberdeen) and Martin Davison (Northumbria Ringing Group). The specimen was identified by Martin Davison and frozen at -20°C. Dissection of tissue samples occurred while the specimen was frozen, with the samples then stored at -80°C prior to sending to the Wellcome Sanger Institute on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute. The bAccGen1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C and RNA sequencing. Heart tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. Fragment size analysis of 0.01-0.5 ng of DNA was then performed using an Agilent FemtoPulse. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 200-ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. HMW DNA was sheared into an average fragment size between 12-20 kb in a Megaruptor 3 system with speed setting 30. Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics Chromium 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 II (HiFi) and Illumina NovaSeq 6000 instruments. Hi-C data were generated in the Tree of Life laboratory from remaining heart tissue of bAccGen1 using the Arima v2 kit and sequenced on a NovaSeq 6000 instrument.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021); haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes (Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using yahs. The assembly was checked for contamination as described previously (Howe et al., 2021). Manual curation was performed using HiGlass (Kerpedjievet al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2021), which performs annotation using MitoFinder (Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

Ethics/compliance issues
The materials that have contributed to this genome note have been supplied by a Tree of Life collaborator. The Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use. The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.