DOI: 10.5061/dryad.v9s4mw6vs
title: Evaluation of four methods to identify the homozygotic sex chromosome in small populations

description: Whole genomes are commonly assembled into a collection of scaffolds and
 often lack annotations of autosomes, sex chromosomes and, and organelle
 genomes (i.e., mitochondrial and chloroplast). As these chromosome types
 can have highly disparate evolutionary histories, it is imperative to take
 this information into account when analyzing genomic variation. Here we
 assessed the accuracy of four methods for identifying the homogametic sex
 chromosome using two whole genome sequenced (WGS) and 133 RAD sequenced
 white-tailed eagles (Haliaeetus albicilla): i) difference in read depth
 per scaffold, ii) heterozygosity per scaffold in a male and female bird,
 iii) mapping to a reference genome of a related species (chicken) with
 identified sex chromosomes, and iv) an analysis of SNP-loadings from a
 principal components analysis (PCA), based on low-depth RADseq data from
 133 individuals. In i and ii, the WGS were mapped to a reference genome
 consisting of 1142 assembled scaffolds from the golden eagle (Aquila
 chrysaetos) with no identified chromosomes. The read depth per scaffold
 identified 86.41% of the homogametic sex chromosome (Z) with few false
 positives. The SNP-loading scores found 78.6% of the Z-chromosome but had
 a false positive discovery rate of more than 10%. The heterozygosity per
 scaffold did not provide clear results due to a lack of diversity in both
 the Z and autosomal chromosomes, and potential interference from the
 heterogametic sex chromosome (W).
description: Blood samples were collected
 from white-tailed eagle chicks as a part of an ongoing monitoring program
 in Iceland since 2001 by the Natural History Institute of Iceland. The sex
 of the chicks was determined in the field based on morphology. Three to
 ten mL of blood was extracted from each chick. The blood was stored in
 EDTA buffer at -20 degrees until DNA extraction. DNA from blood
 samples from 133 chicks were extracted using the ThermoFisher GeneJET
 Whole Blood Genomics DNA Purification Mini Kit following the standard
 protocol (Thermo Fisher, 2016). DNA concentration was estimated using the
 NanoDrop 1000 and run on 0.7% agarose gels to evaluate the fragment size.
 Samples with concentration higher than 60 ng/µl were selected for library
 preparation and sequencing. The 133 samples
 were prepared for double digest restriction-site associated DNA sequencing
 (ddRADseq) using modified protocols from Elshire et al. (2011) and
 Peterson et al. (2012). Total genomic DNA (100-500 ng) was sequentially
 digested using the restriction endonucleases Sau3AI (1U) and ApeKI (2U),
 respectively, each for four hours at manufacturer (NEB) recommended
 temperatures in NEB Buffer 4. Digested DNA (100 ng) was ligated to
 adapters (sequences in Elshire et al. (2011)) containing unique
 combinatorial barcodes (16 unique 5 bp barcodes for ApeKI adapters and
 five unique 6 bp barcodes for Sau3AI adapters) for each individual
 (barcode and adapter sequences in Supplementary Information S1) using T4
 DNA ligase (NEB) in supplied buffer at 21°C for four hours. Ligation
 reactions contained a 6:1 molar excess of adapter to fragmented DNA,
 calculated using the mean fragment size determined from an agarose gel.
 Ligated DNA was pooled and purified using magnetic beads (Macherey-Nagel
 NGS clean-up and size selection) following the manufacturers protocol.
 Size selection of ligated DNA fragments was performed on a Pippin Prep
 (Sage Science) with 2% ethidium-free agarose gels and external size
 standard. The narrow range setting included a mean fragment size of 350 bp
 ± 18 bp. The eluate was split among eight PCR reactions and amplified
 using the primers and PCR conditions as in Elshire et al. (2011). Each PCR
 reaction had a total volume of 25 μL containing; 1x OneTaq Master Mix with
 Standard Buffer (NEB), 0.5 mM each primer, and 8 μL template DNA. PCR
 products were pooled and purified using magnetic beads before
 quantification using a SYBR Gold fluorometric assay (protocol in
 Supplementary Information S2). The library was prepared for sequencing
 following manufacturer’s instructions with a final concentration of 38 nM.
 The library was sequenced on an Illumina HiSeq2500 using the Illumina
 TruSeq kit (2x125bp) following the manufacturer’s instructions. The
 sequencing was done on one lane and obtained 303 million unambiguous PE
 reads. Two individuals
 of white-tailed eagle, a male and a female, were selected for high-depth
 whole genome shotgun sequencing with two lanes each on an Illumina
 HiSeqX.  Library preparation and sequencing was done at deCODE genetics,
 using the TruSeq Nano sample preparation method. Two reference
 assemblies from male golden eagles (ZZ), one in 1142 scaffolds and one
 assembled to chromosome level (GenBank Assembly Accession numbers:
 GCA_000766835.1 and GCA_900496995.2, respectively) and female chicken (ZW)
 (GenBank Assembly Accession: GCA_0000023315.3) were downloaded from NCBI
 and used in the analysis (Doyle et al., 2014; Hillier et al.,
 2004). The
 white-tailed eagle RADseq data was demultiplexed, sorting sequence reads
 into individual files, both for forward and reverse sequences using the
 command process_radtags in Stacks (Catchen et al., 2013; Catchen et al.,
 2011). Standard setting was used for the RADseq data, applying the option
 “r”, to rescue barcodes and RAD-Tags. After
 demultiplexing, FastQC (Babraham Bioinformatics, 2010) was run for quality
 control. For the RADseq data, we found an excess of specific sequences
 (kmers) which were removed using AdapterRemoval v2 (version 2.2.2)
 (Schubert, Lindgreen & Orlando, 2016). The high depth shotgun
 sequenced individuals were tested in the same way but found no excess of
 kmers.  The
 Burrows-Wheeler Aligner (BWA) and SAMtools (Li & Durbin, 2009; Li
 et al., 2009) were used to process RADseq and high depth shotgun data and
 map reads to the golden eagle scaffold assembly of 1142 scaffolds with no
 identified chromosomes (GCA_000766835.1) (Doyle et al., 2014).