Evaluating the utility of deep genome skimming for phylogenomic analyses: A case study in the species-rich genus Rhododendron

Abstract

Deep genome skimming (DGS) has emerged as a promising approach to recover orthologous nuclear genes for large-scale phylogenomic analyses. However, its reliability with low DNA quality and quantity typical of archival specimens, such as herbarium material, remains largely unexplored. We used Rhododendron as a case study to evaluate best practices for DGS in phylogenetic analyses at both deep and shallow scales. We first investigated locus recovery variation with sequencing depth, before evaluating the phylogenetic utility of different sets of loci, including Angiosperms353, target nuclear exons, and extended exon-flanking regions. We found DGS effectively recovered nuclear genes from herbarium specimens, with ∼15× coverage performing similarly to deeper sequencing. The recovery of target exon and flanking regions was improved by using supercontigs as a reference, offering a potential solution to limited sequencing depth. The high-integrity nuclear sequences recovered robust phylogenetic relationships within Rhododendron. Notably, exon-flanking regions showed significant potential for resolving relationships at shallow scales. Genes recovered with taxon-specific references had less missing data than those recovered by Angiosperms353 and generated higher-resolution phylogenetic trees. This study demonstrates the utility of DGS data for obtaining numerous nuclear genes from herbarium specimens for phylogenetic studies, and makes recommendations for best practices regarding sequencing coverage, locus selection, and bioinformatic approaches.

Keywords: Deep genome skimming; Degraded DNA; Herbarium specimens; Non-targeted exon-flanking regions; Phylogenomics; Target enrichment.

Fig. 1

Sequence recovery efficiency for 5358 genes using HybPiper. E-exon (A) and E-flank (B) represent the exons and flanking regions recovered using exon as reference, respectively. S-exon (C) and S-flank (D) represent the exons and flanking regions recovered using intron-containing sequences (supercontigs) as reference, respectively. Each column corresponds to a gene, and each row corresponds to a sample. The shading in each cell indicates the proportion of the recovered sequence length relative to the reference length, with 1.0 representing complete recovery. sgdm represents silica-gel dried material and hbsp represents herbarium specimen. The labels 10×, 15× and 20× represent the sequencing depths. Full data are provided in Tables S3–S6.

Fig. 2

Number of recovered exons (A) and exon-flanking regions (B) of 5358 genes. sgdm represents silica-gel dried material and hbsp represents herbarium specimen. The labels 10×, 15× and 20× represent the sequencing depths. E/S-exon/flank indicates the exons/flanking regions recovered using exons/supercontigs as reference for read sorting.

Fig. 3

Relative lengths of sequences recovered from silica-gel dried materials and herbarium specimens at sequencing depths of 10×, 15× and 20×. sgdm represents silica-gel dried material and hbsp represents herbarium specimen. E/S-exon/flank represents the exons/flanking regions recovered using exons/supercontigs as reference for read sorting.

Fig. 4

Relative lengths of sequences recovered from all silica-gel dried materials (A) and herbarium specimen subsets (B). E/S-exon/flank represents the exons/flanking regions recovered using exons/supercontigs as reference for read sorting. Combinations of E/S and 10/15/20× represent the recovered sequence from herbarium specimens with sequencing depths of 10×, 15× and 20×, using exons/supercontigs as reference for read sorting.

Fig. 5

Phylogenetic trees inferred using ASTRAL and IQ-TREE from exon datasets E10×-exon, S10×-exon, E15×-exon, S15×-exon, E20×-exon and S20×-exon (A–L). Numbers in parentheses represent the gene count in the dataset. Comparisons of phylogenetic trees constructed from datasets using exons or supercontigs as reference for read sorting were made. Accessions with conflicting placements between the trees are connected with dashed lines, and accessions or clades with conflicts in the primary topology are highlighted in red. Branch support values are shown near the nodes, where values are omitted when fully supported (LPP = 1.0 and UFBS = 100%).