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 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 five species—Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Strongyloides stercoralis, and Trichuris muris—with an optimal protocol. A sixth species—Dracunculus medinensis—was included for comparison. 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 both between species and between different life stages from some species described. These results demonstrate the feasibility of whole-genome sequencing of individual parasites, and highlight a new avenue toward generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

Genome-wide analysis ofSchistosoma mansonireveals population structure and praziquantel drug selection pressure within Ugandan hot-spot communities

“…Despite the falling cost and rising throughput of nucleic acid sequencing, we were limited in the number of miracidia that we could sequence in this study. An additional limitation is the labour-intensive process of hatching and washing miracidia necessary to obtain high-quality data due to the nonselective nature of the whole-genome sequencing approach [67].…”

“…DNA was extracted using a modified CGP buffer protocol as previously described [67,68]. The individual spots containing miracidia were punched from the FTA cards using a 2 mm Harris micro-punch and placed in 96-well plates.…”

Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes

“…The three types of DNA extraction tested in this study were chelating, precipitation, and silica binding. DNA extraction method comparisons have previously been conducted on individual nematodes of other species [ 16 ], however, they did not include the traditional phenol-chloroform/CTAB method, or a chelating method as has been done in this study. The variety of DNA extraction methods available highlights the difficulty in comparing and standardizing methods between studies and organisms.…”

“…Studies focusing on several DNA extraction methods are uncommon. SR Doyle et al [ 16 ] compared 5 methods to extract individual nematode DNA from 8 different species and found a Cancer Genome Project method was ideal. This method utilised Whatman® FTA® cards for sample collection which did not limit the DNA extraction and whole genome sequencing of parasite samples.…”

Assessment of three DNA extraction kits for the absolute quantification of strongyle nematode eggs in faecal samples

“…As is the case with all polymerase chain reaction (PCR) based diagnostics, the key step is the acquisition of a sufficient amount and quality of template genomic DNA (gDNA). To date, few studies have systematically explored the suitability of various commercially available DNA extraction kits in order to obtain genomic DNA from parasites of veterinary importance for subsequent analyses [ 11 , 14 , 15 ]. Nevertheless, although a variety of commercial kits for DNA extraction are available on the market, comparatively little is known about the efficacy of each on the quantitative molecular detection of H. contortus in faecal samples containing the parasite’s eggs.…”