Chiropteran types I and II interferon genes inferred from genome sequencing traces by a statistical gene-family assembler

Abstract

Background: The rate of emergence of human pathogens is steadily increasing; most of these novel agents originate in wildlife. Bats, remarkably, are the natural reservoirs of many of the most pathogenic viruses in humans. There are two bat genome projects currently underway, a circumstance that promises to speed the discovery host factors important in the coevolution of bats with their viruses. These genomes, however, are not yet assembled and one of them will provide only low coverage, making the inference of most genes of immunological interest error-prone. Many more wildlife genome projects are underway and intend to provide only shallow coverage.

Results: We have developed a statistical method for the assembly of gene families from partial genomes. The method takes full advantage of the quality scores generated by base-calling software, incorporating them into a complete probabilistic error model, to overcome the limitation inherent in the inference of gene family members from partial sequence information. We validated the method by inferring the human IFNA genes from the genome trace archives, and used it to infer 61 type-I interferon genes, and single type-II interferon genes in the bats Pteropus vampyrus and Myotis lucifugus. We confirmed our inferences by direct cloning and sequencing of IFNA, IFNB, IFND, and IFNK in P. vampyrus, and by demonstrating transcription of some of the inferred genes by known interferon-inducing stimuli.

Conclusion: The statistical trace assembler described here provides a reliable method for extracting information from the many available and forthcoming partial or shallow genome sequencing projects, thereby facilitating the study of a wider variety of organisms with ecological and biomedical significance to humans than would otherwise be possible.

Figure 1

Schematic of the statistical model. The observed sequencing traces X_i result from the observation of genes ξ. These genes are related by descent from the common ancestor α.

Figure 2

Diagram indicating the labeling of traces and assemblies for the discussion of progressive alignment in the text.

Figure 3

Phylogenetic tree of the type-I interferons in Pteropus vampyrus (Pva), Myotis lucifigus (Mlu), Sus scrofa (Ssc), Homo sapiens (Hsa), and Ornithorhynchus anatinus (Oan). The tree was thinned for clarity by omitting one member of any pair of sequences differing by fewer than 3 amino acids. The branch length is proportional to the evolutionary distance.

Figure 4

The posterior probability mass function on the number of genes in the IFNA and IFND families as estimated by cloning and sequencing from peripheral blood cells from Pteropus vampyrus.

Figure 5

Gene expression time course by qRT-PCR in fresh P.vampyrus PBMCs stimulated in culture by the indicated compound. Expression levels are relative to the housekeeping gene PPIA. The error bars represent standard errors of the mean over biological duplicates.