Long-read RNA sequencing of human and animal filarial parasites improves gene models and discovers operons

Abstract

Filarial nematodes (Filarioidea) cause substantial disease burden to humans and animals around the world. Recently there has been a coordinated global effort to generate and curate genomic data from nematode species of medical and veterinary importance. This has resulted in two chromosome-level assemblies ( Brugia malayi and Onchocerca volvulus ) and 10 additional draft genomes from Filarioidea. These reference assemblies facilitate comparative genomics to explore basic helminth biology and prioritize new drug and vaccine targets. While the continual improvement of genome contiguity and completeness advances these goals, experimental functional annotation of genes is often hindered by poor gene models. Short-read RNA sequencing data and expressed sequence tags, in cooperation with ab initio prediction algorithms, are employed for gene prediction, but these can result in missing clade-specific genes, fragmented models, imperfect mapping of gene ends, and lack of isoform resolution.Long-read RNA sequencing can overcome these drawbacks and greatly improve gene model quality. Here, we present Iso-Seq data for B. malayi and Dirofilaria immitis , etiological agents of lymphatic filariasis and canine heartworm disease, respectively. These data cover approximately half of the known coding genomes and substantially improve gene models by extending untranslated regions, cataloging novel splice junctions from novel isoforms, and correcting mispredicted junctions. Furthermore, we validated computationally predicted operons, identified new operons, and merged fragmented gene models. We carried out analyses of poly(A) tails in both species, leading to the identification of non-canonical poly(A) signals.Finally, we prioritized and assessed known and putative anthelmintic targets, correcting or validating gene models for molecular cloning and target-based antiparasitic screening efforts.Overall, these data significantly improve the catalog of gene models for two important parasites, and they demonstrate how long-read RNA sequencing should be prioritized for future improvement of parasitic nematode genome assemblies. Author SummaryReference genomes for parasitic nematodes are important resources that enable the study of nematode evolution and molecular biology, and they also hold promise for hastening the development of chemotherapeutics to treat parasitic diseases. Recent years have seen an explosion in the availability of reference genomes for filarial worms, which cause diseases in both humans and animals, but much work remains to be done in order to fully potentiate the true utility of these resources. We carried out long-read RNA sequencing of Brugia malayi and Dirofilaria immitis , two important filarial worms that cause lymphatic filariasis and canine heartworm disease, respectively. We used these RNA sequencing data to correct many errors in the gene models of the reference genomes of these two species, and we also carried out novel analyses of poly(A) tails and operons. These datasets will greatly improve the B. malayi a...

Advances in our understanding of nematode ion channels as potential anthelmintic targets

“…Recent studies have identified a third splice variant that contains both the exons that show alternative usage in the a and the f isoforms. This suggests existence of a complex in vivo genomic regulatory mechanisms that can combat the insult caused by anthelmintics like emodepside ( Wheeler et al, 2020a ).…”

Pharmacological profiling of a Brugia malayi muscarinic acetylcholine receptor as a putative antiparasitic target

“…The open-reading frame (ORF) for the longest predicted isoform of Bma-gar-3 (isoform a, WormBase ParaSite version 17) was selected for study based on manual assessment and alignment with homologous GARs. This isoform is supported by long-read RNA sequencing data [115] that extends the model upstream of the 5’ end of isoform b. The ORF was synthesized (GenScript) and cloned into pPD133.48, L4663 (a gift from Andrew Fire, Addgene plasmid #1665) using BamHI/KpnI sites to create pMZ0005 (myo3p::Bma-gar-3::unc-54-3’UTR).…”

Improving helminth genome resources in the post-genomic era

“…o Examples: S. mansoni [45], Schistosoma japonicum [51], Schistosoma haematobium [52], Hymenolepis microstoma [46], H. contortus [14], B. malayi/Dirofilaria immitis [53], Heterodera glycines [54], Clonorchis sinensis [55], Ascaris suum [56], Oscheius tipulae [57], P. pacificus [58], Nector americanus [59]. • Community annotation o Engaging school-age/undergraduate students [60].…”