Annotation of Two Large Contiguous Regions from the Haemonchus contortus Genome Using RNA-seq and Comparative Analysis with Caenorhabditis elegans

Roz Laing,Michael Quail,Anna V Protasio,Karen Mungall,Steven Laing,John S Gilleard,Martin Hunt,Frank Jackson,Robin Beech,Gary Saunders,Matthew Berriman,Clotilde K S Carlow

doi:10.1371/journal.pone.0023216

Abstract

The genomes of numerous parasitic nematodes are currently being sequenced, but their complexity and size, together with high levels of intra-specific sequence variation and a lack of reference genomes, makes their assembly and annotation a challenging task. Haemonchus contortus is an economically significant parasite of livestock that is widely used for basic research as well as for vaccine development and drug discovery. It is one of many medically and economically important parasites within the strongylid nematode group. This group of parasites has the closest phylogenetic relationship with the model organism Caenorhabditis elegans, making comparative analysis a potentially powerful tool for genome annotation and functional studies. To investigate this hypothesis, we sequenced two contiguous fragments from the H. contortus genome and undertook detailed annotation and comparative analysis with C. elegans. The adult H. contortus transcriptome was sequenced using an Illumina platform and RNA-seq was used to annotate a 409 kb overlapping BAC tiling path relating to the X chromosome and a 181 kb BAC insert relating to chromosome I. In total, 40 genes and 12 putative transposable elements were identified. 97.5% of the annotated genes had detectable homologues in C. elegans of which 60% had putative orthologues, significantly higher than previous analyses based on EST analysis. Gene density appears to be less in H. contortus than in C. elegans, with annotated H. contortus genes being an average of two-to-three times larger than their putative C. elegans orthologues due to a greater intron number and size. Synteny appears high but gene order is generally poorly conserved, although areas of conserved microsynteny are apparent. C. elegans operons appear to be partially conserved in H. contortus. Our findings suggest that a combination of RNA-seq and comparative analysis with C. elegans is a powerful approach for the annotation and analysis of strongylid nematode genomes.

Highlights

H. contortus is a parasitic nematode of small ruminants of major economic importance
Gene structure and content H. contortus is a member of the closest phylogenetic clade of parasitic nematodes to C. elegans and the last common ancestor is estimated to have existed 400 million years ago [22]
We have used a generation transcriptomic approach to annotate two large contigs and these represent the largest contiguous sections of genomic sequence yet annotated for a strongylid nematode

Summary

Introduction

H. contortus is a parasitic nematode of small ruminants of major economic importance. The major challenge facing parasitic nematode genomics is not genome sequencing per se, but assembly and annotation. The production of a high quality finished genome sequence for H. contortus is an important aim as it will provide a reference genome for many parasitic nematodes in the strongylid nematode group. These include some of the most important ‘‘neglected tropical diseases’’ of humans and economically important parasites of livestock, many of which are currently being sequenced to varying levels of completion (e.g. http://www.sanger.ac.uk/resources/downloads/ helminths/ and http://www.nematode.net/). We present the results of this annotation, along with a comparative analysis of H. contortus and C. elegans gene structure and genome organisation

Methods

Results

Conclusion