DOI: 10.5061/dryad.c59zw3r87
title: A high-quality genome assembly and annotation of the dark-eyed junco Junco hyemalis, a recently diversified songbird

description: The dark-eyed junco (Junco hyemalis) is one of the most common passerines
 of North America, and has served as a model organism in studies related to
 ecophysiology, behavior and evolutionary biology for over a century. It is
 composed by at least six distinct, geographically structured forms of
 recent evolutionary origin presenting remarkable variation in phenotypic
 traits, migratory behavior and habitat. Here we report a high-quality
 genome assembly and annotation of the dark-eyed junco generated using a
 combination of shotgun libraries and proximity ligation ChicagoTM and
 Dovetail HiCTM libraries. The final assembly is 1,031,523,571 bp long,
 with 98.3% of the sequence located in 30 full or nearly full chromosome
 scaffolds, and with a N50/L50 of 71,3 Mb/5 scaffolds. We identified 19,026
 functional genes combining gene prediction and similarity approaches, of
 which 15,967 were associated to GO terms. Genome assembly and annotated
 set of genes yielded 95.4% and 96.2% completeness scores, respectively,
 when compared with the BUSCO avian dataset. This new assembly for J.
 hyemalis provides a valuable resource for genome evolution analysis, as
 well as for identifying functional genes involved in adaptive processes
 and speciation.
description: <em>Genome sequencing
 and assembly</em><br>A high-quality genome was produced
 combining newly generated shotgun reads and sequence data from proximity
 ligation libraries. Preparation of proximity ligation libraries
 Chicago<sup>TM</sup> and HiC<sup>TM</sup>, as well
 as scaffolding with the software pipeline HiRise<sup>
 </sup>(Putnam et al. 2016; https://dovetailgenomics.com) was
 conducted at Dovetail Genomics, LLC. The sequenced sample consisted of
 muscle tissue obtained from a female <em>J. hyemalis
 carolinensis</em>, collected at Mountain Lake Biological Station in
 Pembroke, Virginia, USA (37.3751°N, 80.5228°W), currently deposited at the
 Moore Laboratory of Zoology, Occidental College, Los Angeles, California
 (voucher number: MLZ:bird: 69236). Briefly, a de novo draft assembly was
 first built using shotgun, paired-end libraries (mean insert size ~350 bp)
 and the Meraculous pipeline (Chapman et al. 2011). For the
 Chicago<sup>TM</sup> and the Dovetail
 HiC<sup>TM</sup> library preparation, chromatin was fixed with
 formaldehyde. Fixed chromatin was then digested with DpnII and free blunt
 ends were ligated. Crosslinks were reversed and the DNA purified from
 protein. Resulting nucleic material was then sheared to ~350 bp mean
 fragment size and sequencing libraries were generated using NEBNext Ultra
 enzymes and Illumina-compatible adapters. Sequencing of the libraries was
 carried out on an Illumina HiSeq X platform. The shotgun reads, Chicago
 library reads, and Dovetail Hi-C library reads were then used as input
 data for HiRise, a software pipeline designed specifically for using
 proximity ligation data to scaffold genome assemblies (Putnam et al.
 2016). The mitochondrial genome was assembled using
 NOVOplasty2.7.2 (Dierckxsens et al. 2017) and the shotgun data. The NADH
 dehydrogenase subunit 2 (ND2) mitochondrial gene sequenced for a previous
 study (Friis et al. 2016) and available in NCBI (GenBank accession no.
 KX461682.1) was used for the input seed sequence.
 <em>Identification of repetitive regions
 </em><br>We first created a repeat library for the junco
 genome by modelling <em>ab initio</em> repeats using Repeat
 Modeler 1.0.11 (Smit and Hubley 2019) in scaffolds longer than 100 Kb with
 default options. The resulting repeat library was merged with known bird
 repeat libraries from the RepBase database (RepBase-20181026) (Bao et al.
 2015), Dfam_Consensus-20181026 and repeats from the zebra finch. Then, we
 used Repeat Masker 4.0.7 (Smit et al. 2015) to identify and mask repeat
 regions in the whole-genome assembly, and classified the repeat
 distribution by chromosome. <em>Gene prediction
 and functional annotation</em><br>Gene prediction was
 conducted using BRAKER v2.1.5 (Hoff et al. 2019) and GeMoMa v1.7.1
 (Keilwagen et al. 2019). We used the repeat soft-masked genome assembly
 and we first trained Augustus with the conserved orthologous genes from
 BUSCO Aves_odb10 as proteins from short evolutionary distance (Stanke et
 al. 2006; Gremme et al. 2005; see Figure 3B from Hoff et al. 2019). The
 predicted proteins resulting from Augustus training were combined with
 homology-based annotations using the zebra finch (GCF_008822105.2; Warren
 et al. 2010) (GCF_008822105.2; Warren et al. 2010) and chicken
 (GCF_000002315.6; International Chicken Genome Sequencing Consortium 2004)
 annotated genes with the GeMoMa pipeline, obtaining the final reported
 gene models. We applied a similarity-based search
 approach to conduct the functional annotation of the junco predicted
 proteins. We first used BLASTP against the UniProt SwissProt database and
 the annotated proteins from the zebra finch genome (Warren et al. 2010;
 UniProt Consortium 2019) (E-value 10<sup>-5</sup>). We only
 considered as positives those hits covering at least 2/3 of the query
 sequence length or 80% of the total subject sequence. We also used
 InterProScan v5.31 (Jones et al. 2014) in order to identify specific
 protein-domain signatures in the predicted genes. The functional
 annotation, including Gene Ontology terms, was integrated from all
 searches providing a curated set of junco coding genes (Fig. S2, Supp.
 Inf.). We used GenomeTools (Gremme et al. 2013) to calculate the number
 and mean length of genes, exons, introns and CDS (Coding Sequence) from
 the annotation file in general feature format (GFF).
 <em>Gene completeness assessment and genome
 synteny</em><br>We assessed gene completeness and gene
 annotation in the genome assembly using BUSCO (Benchmarking Universal
 Single-Copy Orthologs) v4.0.5 (--auto-lineage-euk option; Waterhouse et
 al. 2018). BUSCO evaluations were conducted using the 255 and 8,338
 single-copy orthologous genes in Eukaryota_odb10 and Aves_odb10 datasets,
 respectively. In addition, we used MUMmer (Delcher et al. 2003) to explore
 synteny with the zebra finch (<em>Taeniopygia guttata</em>)
 genome v87 available in Ensembl (Yates et al. 2016).
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description: The genome assembly here provided has also been deposited at
 GenBank along with raw sequence data from Chicago and Hi-C libraries under
 the accession QZWM00000000.2, Bioproject (accession: PRJNA493001;
 Biosample accession: SAMN10120167).