Approaches to genotyping individual miracidia of Schistosoma japonicum

Xiao, Ning; Remais, Justin V.; Brindley, Paul J.; Qiu, Dongchuan; Carlton, Elizabeth J.; Li, Rongzhi; Liu, Yang; Blair, David

doi:10.1007/s00436-013-3587-9

Cited by 9 publications

(9 citation statements)

References 38 publications

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“…Furthermore, we designed PCR primers for 1,609 new microsatellite loci that could be used alone or in conjunction with high-throughput sequencing experiments—this resource provides a considerable increase from the 17 microsatellite loci used previously to genotype S . japonicum samples [ 31 ]. Importantly, this study demonstrates the ability to obtain many orders of magnitude more data than was previously possible, even from archived miracidia samples collected six years in the past.…”

Section: Discussionmentioning

confidence: 99%

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Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia

Shortt¹,

Card²,

Schield³

et al. 2017

PLoS Negl Trop Dis

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BackgroundIn areas where schistosomiasis control programs have been implemented, morbidity and prevalence have been greatly reduced. However, to sustain these reductions and move towards interruption of transmission, new tools for disease surveillance are needed. Genomic methods have the potential to help trace the sources of new infections, and allow us to monitor drug resistance. Large-scale genotyping efforts for schistosome species have been hindered by cost, limited numbers of established target loci, and the small amount of DNA obtained from miracidia, the life stage most readily acquired from humans. Here, we present a method using next generation sequencing to provide high-resolution genomic data from S. japonicum for population-based studies.Methodology/Principal FindingsWe applied whole genome amplification followed by double digest restriction site associated DNA sequencing (ddRADseq) to individual S. japonicum miracidia preserved on Whatman FTA cards. We found that we could effectively and consistently survey hundreds of thousands of variants from 10,000 to 30,000 loci from archived miracidia as old as six years. An analysis of variation from eight miracidia obtained from three hosts in two villages in Sichuan showed clear population structuring by village and host even within this limited sample.Conclusions/SignificanceThis high-resolution sequencing approach yields three orders of magnitude more information than microsatellite genotyping methods that have been employed over the last decade, creating the potential to answer detailed questions about the sources of human infections and to monitor drug resistance. Costs per sample range from $50-$200, depending on the amount of sequence information desired, and we expect these costs can be reduced further given continued reductions in sequencing costs, improvement of protocols, and parallelization. This approach provides new promise for using modern genome-scale sampling to S. japonicum surveillance, and could be applied to other schistosome species and other parasitic helminthes.

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Section: Discussionmentioning

confidence: 99%

“…Archived field-collected samples of S . japonicum miracidia were collected from infected humans in Sichuan Province, China in 2010 as described elsewhere [ 9 , 31 ]. Briefly, participants were tested for infection using the miracidia hatching test and miracidia were collected from positive hatching tests.…”

Section: Methodsmentioning

confidence: 99%

Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia

Shortt¹,

Card²,

Schield³

et al. 2017

PLoS Negl Trop Dis

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“…All participants testing positive for S. japonicum in this study were promptly treated and county health officials implemented several mass and targeted chemotherapy campaigns during the study period. While there is no conclusive evidence that praziquantel resistance is occurring in China, we have previously found the same individuals repeatedly test positive for S. japonicum and it is possible these individuals harbor uncured infections [ 36 , 37 ]. We do not know to what extent treatment failure is occurring, or the extent to which it is caused by praziquantel resistance vs. non-compliance due to populations tiring of repeated treatment campaigns.…”

Section: Discussionmentioning

confidence: 99%

Associations between Schistosomiasis and the Use of Human Waste as an Agricultural Fertilizer in China

et al. 2015

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BackgroundHuman waste is used as an agricultural fertilizer in China and elsewhere. Because the eggs of many helminth species can survive in environmental media, reuse of untreated or partially treated human waste, commonly called night soil, may promote transmission of human helminthiases.Methodology/Principal FindingsWe conducted an open cohort study in 36 villages to evaluate the association between night soil use and schistosomiasis in a region of China where schistosomiasis has reemerged and persisted despite control activities. We tested 2,005 residents for Schistosoma japonicum infection in 2007 and 1,365 residents in 2010 and interviewed heads of household about agricultural practices each study year. We used an intervention attributable ratio framework to estimate the association between night soil use and S. japonicum infection. Night soil use was reported by half of households (56% in 2007 and 46% in 2010). Village night soil use was strongly associated with human S. japonicum infection in 2007. We estimate cessation of night soil use would lead to a 49% reduction in infection prevalence in 2007 (95% CI: 12%, 71%). However, no association between night soil and schistosomiasis was observed in 2010. These inconsistent findings may be due to unmeasured confounding or temporal shifts in the importance of different sources of S. japonicum eggs on the margins of disease elimination.Conclusions/SignificanceThe use of untreated or partially treated human waste as an agricultural fertilizer may be a barrier to permanent reductions in human helminthiases. This practice warrants further attention by the public health community.

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“…Furthermore, s amples may need to be transported efficiently to a laboratory setting without a significant loss of this already limited material. A number of approaches have been tested to preserve macromolecules from individual parasites for transport and storage, including ethanol, RNAlater and Whatman® FTA® cards, from which robust PCR and microsatellite data could be profiled (Boué et al, 2017;Campbell et al, 2017;Gower et al, 2007;Marek et al, 2014;Webster, 2009;Webster et al, 2012;Xiao et al, 2013) . Although under ideal conditions the detection of a single DNA molecule is possible, the limited material available per parasite has to date largely restricted assaying to a small number of loci, limiting the amount of information obtained from any individual parasite.…”

Section: Introductionmentioning

confidence: 99%

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

Doyle

Sankaranarayan

Allen

et al. 2019

Preprint

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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.

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Approaches to genotyping individual miracidia of Schistosoma japonicum

Cited by 9 publications

References 38 publications

Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia

Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia

Associations between Schistosomiasis and the Use of Human Waste as an Agricultural Fertilizer in China

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

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