Rezansoff, A. M. et al. (2019) The confounding effects of high genetic diversity on the determination and interpretation of differential gene expression analysis in the parasitic nematode Haemonchus contortus. International Journal for Parasitology, 49(11), pp. 847-858. (doi: 10.1016/j.ijpara.2019.05.012) (PMID:31525371)
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201176.pdf - Accepted Version 3MB |
Abstract
Differential expression analysis between parasitic nematode strains is commonly used to implicate candidate genes in anthelmintic resistance or other biological functions. We have tested the hypothesis that the high genetic diversity of an organism such as Haemonchus contortus could complicate such analyses. First, we investigated the extent to which sequence polymorphism affects the reliability of differential expression analysis between the genetically divergent H. contortus strains MHco3(ISE), MHco4(WRS) and MHco10(CAVR). Using triplicates of 20 adult female worms from each population isolated under parallel experimental conditions, we found that high rates of sequence polymorphism in RNAseq reads were associated with lower efficiency read mapping to gene models under default TopHat2 parameters, leading to biased estimates of inter-strain differential expression. We then showed it is possible to largely compensate for this bias by optimising the read mapping single nucleotide polymorphism (SNP) allowance and filtering out genes with particularly high single nucleotide polymorphism rates. Once the sequence polymorphism biases were removed, we then assessed the genuine transcriptional diversity between the strains, finding ≥824 differentially expressed genes across all three pairwise strain comparisons. This high level of inter-strain transcriptional diversity not only suggests substantive inter-strain phenotypic variation but also highlights the difficulty in reliably associating differential expression of specific genes with phenotypic differences. To provide a practical example, we analysed two gene families of potential relevance to ivermectin drug resistance; the ABC transporters and the ligand-gated ion channels (LGICs). Over half of genes identified as differentially expressed using default TopHat2 parameters were shown to be an artifact of sequence polymorphism differences. This work illustrates the need to account for sequence polymorphism in differential expression analysis. It also demonstrates that a large number of genuine transcriptional differences can occur between H. contortus strains and these must be considered before associating the differential expression of specific genes with phenotypic differences between strains.
| Item Type: | Articles |
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| Additional Information: | We are grateful for funding from Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (grant number RGPIN-2015-03976), NSERC-CREATE Host-Parasites Interactions (HPI) program and the Biotechnology and Biological Sciences Research Council (BBSRC), UK. The Moredun Research Institute, United Kingdom (DJB and AAM) receives funding from the Scottish Government. NS was funded by the Higher Education Funding Council of England (HEFCE), the Department for Environment, Food and Rural Affairs (DEFRA), UK and the Scottish Funding Council (SFC) Veterinary Training Research Initiative (VTRI) programme VT0102 and supported by Pfizer Animal Health. NH, SD and JAC contributions were supported by Wellcome Trust grant WT206194. |
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Devaney, Professor Eileen and Laing, Dr Roz and Cotton, Professor James |
| Authors: | Rezansoff, A. M., Laing, R., Martinelli, A., Stasiuk, S., Redman, E., Bartley, D., Holroyd, N., Devaney, E., Sargison, N. D., Doyle, S., Cotton, J. A., and Gilleard, J. S. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine |
| Journal Name: | International Journal for Parasitology |
| Publisher: | Elsevier |
| ISSN: | 0020-7519 |
| ISSN (Online): | 1879-0135 |
| Published Online: | 13 September 2019 |
| Copyright Holders: | Copyright © 2019 Australian Society for Parasitology |
| First Published: | First published in International Journal for Parasitology 49(11):847-858 |
| Publisher Policy: | Reproduced in accordance with the publisher copyright policy |
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