Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples
MetadataShow full item record
AbstractBACKGROUND: Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitaemia samples. In this study, enzymatic digestion with MspJI prior to sWGA and whole genome sequencing was evaluated to determine whether this approach improved genome coverage compared to sWGA alone. The potential of sWGA to cause amplification bias in polyclonal infections was also examined. METHODS: DNA extracted from laboratory-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA. RESULTS: MspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI?+?sWGA and sWGA alone, particularly for low parasitaemia samples. The optimized sWGA (filtration?+?sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. CONCLUSION: This optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitaemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies.
Selective whole genome amplification
Whole genome sequencing
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85082790753&doi=10.1186%2fs12936-020-03195-8&partnerID=40&md5=b42acae35263869e71d75dbbf663d053; http://hdl.handle.net/10713/12543
- Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification.
- Authors: Oyola SO, Ariani CV, Hamilton WL, Kekre M, Amenga-Etego LN, Ghansah A, Rutledge GG, Redmond S, Manske M, Jyothi D, Jacob CG, Otto TD, Rockett K, Newbold CI, Berriman M, Kwiatkowski DP
- Issue date: 2016 Dec 20
- Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples.
- Authors: Cowell AN, Loy DE, Sundararaman SA, Valdivia H, Fisch K, Lescano AG, Baldeviano GC, Durand S, Gerbasi V, Sutherland CJ, Nolder D, Vinetz JM, Hahn BH, Winzeler EA
- Issue date: 2017 Feb 7
- Selective whole genome amplification of Plasmodium malariae DNA from clinical samples reveals insights into population structure.
- Authors: Ibrahim A, Diez Benavente E, Nolder D, Proux S, Higgins M, Muwanguzi J, Gomez Gonzalez PJ, Fuehrer HP, Roper C, Nosten F, Sutherland C, Clark TG, Campino S
- Issue date: 2020 Jul 2
- Direct whole-genome sequencing of Plasmodium falciparum specimens from dried erythrocyte spots.
- Authors: Nag S, Kofoed PE, Ursing J, Lemvigh CK, Allesøe RL, Rodrigues A, Svendsen CA, Jensen JD, Alifrangis M, Lund O, Aarestrup FM
- Issue date: 2018 Feb 23
- Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples.
- Authors: Teyssier NB, Chen A, Duarte EM, Sit R, Greenhouse B, Tessema SK
- Issue date: 2021 Feb 26