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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Genet. | doi: 10.3389/fgene.2019.00826

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

 Stephen R. Doyle1*,  Geetha Sankaranarayanan1, Duncan Berger1, Pablo D. Jimenez Castro2, 3, James B. Collins4, Thomas Crellen1, 5, Maria A. Duque-Correa1, Peter Ellis1, Tegegn G. Jaleta6, Roz Laing7,  Kirsty Maitland7, Catherine McCarthy1, Tchonfienet Moundai8, Ben Softley1,  Elizabeth Thiele9, Philippe T. Ouakou8,  John V. Tushabe1, 10, Joannne P. Webster11, Adam J. Weiss12,  James B. Lok6,  Eileen Devaney7,  Ray M. Kaplan4, James A. Cotton1, Matthew Berriman1 and  Nancy Holroyd1
  • 1Wellcome Trust Sanger Institute (WT), United Kingdom
  • 2Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, United States
  • 3Grupo de Parasitologia Veterinaria, Universidad Nacional de Colombia, Colombia
  • 4University of Georgia, United States
  • 5Nuffield Department of Medicine, Medical Sciences Division, University of Oxford, United Kingdom
  • 6University of Pennsylvania, United States
  • 7University of Glasgow, United Kingdom
  • 8Ministry of Public Health (Chad), Chad
  • 9Vassar College, United States
  • 10Uganda Virus Research Institute (UVRI), Uganda
  • 11Royal Veterinary College (RVC), United Kingdom
  • 12Guinea Worm Eradication Program, The Carter Center, United States

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 towards generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

Keywords: whole genome sequencing, diagnostics, DNA extraction, Low input, Genomics, Helminths

Received: 26 Apr 2019; Accepted: 12 Aug 2019.

Copyright: © 2019 Doyle, Sankaranarayanan, Berger, Jimenez Castro, Collins, Crellen, Duque-Correa, Ellis, Jaleta, Laing, Maitland, McCarthy, Moundai, Softley, Thiele, Ouakou, Tushabe, Webster, Weiss, Lok, Devaney, Kaplan, Cotton, Berriman and Holroyd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Stephen R. Doyle, Wellcome Trust Sanger Institute (WT), Saffron Walden, United Kingdom, sd21@sanger.ac.uk