The Draft Genome Assembly of Dermatophagoides pteronyssinus Supports Identification of Novel Allergen Isoforms in Dermatophagoides Species

Abstract

Background: Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF) are highly similar disease-associated mites with frequently overlapping geographic distributions. A draft genome of DP was assembled to identify candidate allergens in DP homologous to those in DF, investigate allergen isoforms, and facilitate comparisons with related Acari.

Methods: PacBio and Illumina whole genome sequencing was performed on DP. Assembly and reconstruction of the genomes were optimized for isoform identification in a heterogeneous population. Bioinformatic analyses of Acari genomes were peformed.

Results: The predicted size of the DP nuclear genome is 52.5 Mb. A predicted protein set of 19,368 proteins was identified, including all 19 currently recognized allergens from this species. Orthologs for 12 allergens established for DF were found. The population of DP mites showed a high level of heterozygosity that allowed the identification of 43 new isoforms for both established and candidate allergens in DP, including a new isoform for the major allergen Der p 23. Reanalyzing the previous DF data assuming heterozygosity, 14 new allergen isoforms could be indentified. Some new isoforms were observed in both species suggesting that these isoforms pre-dated speciation. The high quality of both genomes allowed an examination of synteny which showed many allergen orthologs are physically clustered but with species specific exon/intron structures. Comparative genomic analyses with other Acariformes mites showed that most of the allergen homologs are widely conserved within this Superorder.

Conclusions: Candidate allergens in DP were identified to facilitate future serological studies. While DP and DF are highly similar genetically, species-specific allergen isoforms exist to facilitate molecular differentiation.

Keywords: Dermatophagoides farinae; Dermatophagoides pteronyssinus; Allergens; Genome; House dust mite.

Figure 1. A multiple sequence alignment of the isoforms of groups 23 and 3

A) All known and newly identified isoforms of group 23 from both D. pteronyssinus and D. farinae are shown aligned. B) Both known and newly identified isoforms of group 3 from both species are included. The three residues boxed in red are examples of amino acid variation conserved between species. As L17, D127, and A138 are observed in both species, the most parsimonious assumption is that these are the ancestral amino acids at these three position and the other is derived after speciation. A list of conserved amino acid substitutions in all allergen isoforms is listed in Table 2.

Figure 2. Conserved genomic organization of allergens

A) clusters of unrelated allergens that are conserved between D. pteronyssinus and D. farinae. B) Cartoon depiction of the organization of allergens that are part of gene families. In both, orientation of the genes is indicated by an arrow, and the approximate distance between linked genes is shown, not to scale. In parenthesis beside the gene name is the number of exons in a given gene. All chitin binding domain and serpin genes are single exon genes. Orthologous relationships, as defined by the orthoparahomlist.pl script, are highlighted in red. The percent identity between members of a gene family within a species is on the right of a given gene pair. The orthologous relationships between the members of the serpin gene family is inconclusive aside from the Der 27 allergens.

Figure 3. Conserved orthologs in mite and tick genomes

Predicted proteomes from five publicly available mites and ticks, and D. pteronyssinus, were used. A Venn diagram showing the distribution of conserved ortholog clusters. A cluster is defined as a group of related proteins having a BLAST similarity of at least 10⁻⁰⁵. The central overlap of all six species (3224 clusters) represents those proteins that have one or more orthologs in each of the six species at a BLAST cutoff of 10⁻⁰⁵, whereas the various other overlapping groups contain clusters representing orthologs conserved between two or more species. The outer spikes (i.e., the D. pteronyssinus spike containing 334 clusters) represent clusters (gene families) of two or more proteins unique to a given species only. The area of the spikes are not proportional to the numbers of genes they contain.