Computational cloning of drug target genes of a parasitic nematode, Oesophagostomum dentatum

Abstract

BackgroundGene identification and sequence determination are critical requirements for many biological, genomic, and bioinformatic studies. With the advent of next generation sequencing (NGS) technologies, such determinations are predominantly accomplished in silico for organisms for which the genome is known or for which there exists substantial gene sequence information. Without detailed genomic/gene information, in silico sequence determination is not straightforward, and full coding sequence determination typically involves time- and labor-intensive PCR-based amplification and cloning methods.ResultsAn improved method was developed with which to determine full length gene coding sequences in silico using de novo assembly of RNA-Seq data. The scheme improves upon initial contigs through contig-to-gene identification by BLAST nearest–neighbor comparison, and through single-contig refinement by iterative-binning and -assembly of reads. Application of the iterative method produced the gene identification and full coding sequence for 9 of 12 genes and improved the sequence of 3 of the 12 genes targeted by benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs in the swine nodular parasite Oesophagostomum dentatum. The approach improved upon the initial optimized assembly with Velvet that only identified full coding sequences for 2 genes.ConclusionsOur reiterative methodology represents a simplified pipeline with which to determine longer gene sequences in silico from next generation sequence data for any nematode for which detailed genetic/gene information is lacking. The method significantly improved upon an initial Velvet assembly of RNA-Seq data that yielded only 2 full length sequences. The identified coding sequences for the 11 target genes enables further future examinations including: (i) the use of recombinant target protein in functional assays seeking a better understanding of the mechanism of drug resistance, and (ii) seeking comparative genomic and transcriptomic assessments between parasite isolates that exhibit varied drug sensitivities.

Molecular Pathology: An Advanced Approach for Rapid Diagnosis of Diseases

“…NGS technology is playing a key role in the study of etiology, genomics and epidemiology of various animal infectious diseases.NGS systems have played the central role in the completion of large animal genomes and also in the studies of genomic variations.Hence, the whole genomic sequences of bovines, pig, sheep equines and avian species are now available (Bai et al,2012).NGS have been helpful in the documentation of intra-host genetic diversity in the Cryptosporidium parvumby targeting specific polymeric genes in its genomic sequence (Grinberg et al,2013).NGS techniques have facilitated the studies on the transcriptomes of different parasitic species and their 1118 developmental stages (Cantacessiet al,2012), e.g. characterization of the transcriptome of Eimeria species from chicken (Matsubayashiet al,2013)and Taenia species from sheep (Wu et al,2012).The RNA sequence data has also been helpful in predicting potential drug targets (Romine et al,2013)and the genes responsible for anti-helmintic resistance(Cwiklinskiet al,2013). The evolution of FMDV intra-sample sequence diversity (Morelli et al,2013)was studied by using DNA sequencing techniques, under the experimental conditions of serial transmission in the ovine hosts, which resulted in the documentation of fine-scale evolution of FMDV.…”

Perusal of parasitic nematode ‘omics in the post-genomic era

“…Members of the genus Strongylus and genera within the subfamily Cyathostominae are also important pathogens of horses [12]. O. dentatum transcripts encode proteins associated with the targets of benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs [95]. Proteomic work identified several proteins down-regulated during hydrolase inhibitor-mediated developmental arrest [96], as well as 11 proteins hypothesized to be involved in exsheathment [97].…”

The Conqueror Worm: recent advances with cholinergic anthelmintics and techniques excite research for better therapeutic drugs

“…Knowledge has advanced of genomes of B. malayi ( Ghedin et al , 2007 ), A. suum ( Jex et al , 2011 ) and transcriptomes of O. dentatum ( Romine et al , 2013 ). With new sequencing technology, knowledge of the genomes of representative parasitic nematodes has advanced rapidly with B. malayi and A. suum .…”