Evaluation of DNA extraction methods on individual Helminth egg and larval stages for whole-genome sequencing
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...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
Frontiers Media
2019
|
| _version_ | 1826279888313647104 |
|---|---|
| author | Doyle, SR Sankaranarayanan, G Allan, F Berger, D Jimenez Castro, PD Bryant Collins, JB Crellen, T Duque-Correa, MA Ellis, P Jaleta, TG Laing, R Maitland, K McCarthy, C Moundai, T Softley, B Thiele, E Ouakou, PT Tushabe, JV Webster, JP Weiss, AJ Lok, J Devaney, E Kaplan, RM Cotton, JA Berriman. M Holroyd, N |
| author_facet | Doyle, SR Sankaranarayanan, G Allan, F Berger, D Jimenez Castro, PD Bryant Collins, JB Crellen, T Duque-Correa, MA Ellis, P Jaleta, TG Laing, R Maitland, K McCarthy, C Moundai, T Softley, B Thiele, E Ouakou, PT Tushabe, JV Webster, JP Weiss, AJ Lok, J Devaney, E Kaplan, RM Cotton, JA Berriman. M Holroyd, N |
| author_sort | Doyle, SR |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T00:05:31Z |
| format | Journal article |
| id | oxford-uuid:77676a1a-c6f4-40f2-a023-02642abb3d16 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:05:31Z |
| publishDate | 2019 |
| publisher | Frontiers Media |
| record_format | dspace |
| spelling | oxford-uuid:77676a1a-c6f4-40f2-a023-02642abb3d162022-03-26T20:23:51ZEvaluation of DNA extraction methods on individual Helminth egg and larval stages for whole-genome sequencingJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:77676a1a-c6f4-40f2-a023-02642abb3d16EnglishSymplectic ElementsFrontiers Media2019Doyle, SRSankaranarayanan, GAllan, FBerger, DJimenez Castro, PDBryant Collins, JBCrellen, TDuque-Correa, MAEllis, PJaleta, TGLaing, RMaitland, KMcCarthy, CMoundai, TSoftley, BThiele, EOuakou, PTTushabe, JVWebster, JPWeiss, AJLok, JDevaney, EKaplan, RMCotton, JABerriman. MHolroyd, NWhole-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. |
| spellingShingle | Doyle, SR Sankaranarayanan, G Allan, F Berger, D Jimenez Castro, PD Bryant Collins, JB Crellen, T Duque-Correa, MA Ellis, P Jaleta, TG Laing, R Maitland, K McCarthy, C Moundai, T Softley, B Thiele, E Ouakou, PT Tushabe, JV Webster, JP Weiss, AJ Lok, J Devaney, E Kaplan, RM Cotton, JA Berriman. M Holroyd, N Evaluation of DNA extraction methods on individual Helminth egg and larval stages for whole-genome sequencing |
| title | Evaluation of DNA extraction methods on individual Helminth egg and larval stages for whole-genome sequencing |
| title_full | Evaluation of DNA extraction methods on individual Helminth egg and larval stages for whole-genome sequencing |
| title_fullStr | Evaluation of DNA extraction methods on individual Helminth egg and larval stages for whole-genome sequencing |
| title_full_unstemmed | Evaluation of DNA extraction methods on individual Helminth egg and larval stages for whole-genome sequencing |
| title_short | Evaluation of DNA extraction methods on individual Helminth egg and larval stages for whole-genome sequencing |
| title_sort | evaluation of dna extraction methods on individual helminth egg and larval stages for whole genome sequencing |
| work_keys_str_mv | AT doylesr evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT sankaranarayanang evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT allanf evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT bergerd evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT jimenezcastropd evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT bryantcollinsjb evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT crellent evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT duquecorreama evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT ellisp evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT jaletatg evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT laingr evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT maitlandk evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT mccarthyc evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT moundait evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT softleyb evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT thielee evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT ouakoupt evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT tushabejv evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT websterjp evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT weissaj evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT lokj evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT devaneye evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT kaplanrm evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT cottonja evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT berrimanm evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing AT holroydn evaluationofdnaextractionmethodsonindividualhelmintheggandlarvalstagesforwholegenomesequencing |