Evaluation of DNA extraction methods on individual helminth egg and larval stages for whole genome sequencing

Abstract

Whole genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative, however, these samples are typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole genome sequencing of individual immature helminth samples. These approaches do not use whole genome amplification, a common but costly approach to increase the yield of low input samples. We first tested individual parasites from two species spotted onto FTA cards - egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni - before further testing on an additional six species - Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Dracunculus medinensis, Strongyloides stercoralis, and Trichuris muris - with an optimal protocol. Whole genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation between species, and within species but between life stages, described. These results demonstrate the feasibility of whole genome sequencing of individual parasites, and highlight a new avenue towards generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

Parasite Population Genetic Contributions to the Schistosomiasis Consortium for Operational Research and Evaluation within Sub-Saharan Africa

“… 33 , 34 Additional refinements developed through SCORE studies and beyond now allow repeat analyses on unamplified genomic DNA, cost-effectively and without genome amplification bias, thereby improving data quality and giving the potential for greater analytical depth. 35 , 36 Such new methodologies and refinements allow an enormous reduction in the logistical effort required in assaying parasite populations. Furthermore, these methodologies can be applied to natural schistosome populations across continents, allowing wide-scale genetic and genomic analyses of schistosome populations over space and time, and encompasses all life stages from adult worms to free-living miracidia and cercariae (e.g., refs.…”

Hatching of parasitic nematode eggs: a crucial step determining infection

“…Under controlled conditions, it would therefore be possible to access genetic material that is otherwise inaccessible in the unembryonated eggs present in patient or environmental samples. Indeed, we have shown that, while DNA can be recovered successfully from larval stages, this is not the case for eggs of certain species, which have a lower DNA content that is difficult to access through the eggshell [82]. Thus, by obtaining embryonated eggs and larvae, better parasite detection and diagnosis…”

“…OPEN ACCESS using molecular techniques, and studies on genome variation in parasite populations via new high-throughput sequencing approaches, will be possible [82,83]. Surveillance of parasite populations using their genomic data has the potential to reveal signatures of demographic change or of recent selection due to anthelmintic drug use, and even detect the emergence of anthelmintic resistance [83].…”

Challenges and opportunities for the adoption of molecular diagnostics for anthelmintic resistance

“…96% ethanol; Ayana et al, 2019 ) or on a solid substrate (e.g. FTA cards; Doyle et al, 2019a ), allowing greater flexibility of sampling, transport, and storage. Use across multiple drug classes : the FECRT can inform on drug resistance across all drug classes using a similar experimental format.…”

“…96% ethanol; Ayana et al, 2019 ) or on a solid substrate (e.g. FTA cards; Doyle et al, 2019a ), allowing greater flexibility of sampling, transport, and storage.…”