About: Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples

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About: Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples Goto Sponge NotDistinct Permalink

An Entity of Type : schema:ScholarlyArticle, within Data Space : covidontheweb.inria.fr associated with source document(s)

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type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
title	Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples
Creator	Adams, Matthew Laufer, Miriam Silva, Joana Takala-Harrison, Shannon Moser, Kara Serre, David Shah, Zalak Shrestha, Biraj Stucke, Emily
source	PMC
abstract	BACKGROUND: 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.
has issue date	2020-03-30(xsd:dateTime)
bibo:doi	10.1186/s12936-020-03195-8
bibo:pmid	32228559
has license	cc-by
sha1sum (hex)	6db3a7777b634b1c5d242259783c6850e1d1a51f
schema:url	https://doi.org/10.1186/s12936-020-03195-8
resource representing a document's title	Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples
has PubMed Central identifier	PMC7106660
has PubMed identifier	32228559
schema:publication	Malar J
resource representing a document's body	covid:6db3a7777b634b1c5d242259783c6850e1d1a51f#body_text
is schema:about of	named entity 'leukocyte' named entity 'sequencing' named entity 'amplification' named entity 'parasite' named entity 'EVALUATED' named entity 'APPROACHES' named entity 'DIFFICULT' named entity 'ENRICH' named entity 'DATA' named entity 'DEPLETION' named entity 'SELECTIVE' named entity 'SAMPLES' named entity 'BEING' named entity 'LEUKOCYTE' named entity 'CAUSE' named entity 'selective' named entity 'large' named entity 'limits' named entity 'data' named entity 'compared' named entity 'determine' named entity 'genome sequencing' named entity 'WGS' named entity 'enzymatic digestion' named entity 'high-quality' named entity 'parasite' named entity 'leukocyte' named entity 'genome sequencing' named entity 'Plasmodium falciparum' named entity 'parasitaemia' named entity 'genome sequencing data' named entity 'sequencing data' named entity 'enzymatic digestion' named entity 'Quantitative PCR' named entity 'P. falciparum' named entity 'P. vivax' named entity 'genome' named entity 'genome sequencing' named entity 'enzymatic digestion' named entity 'genome' named entity 'Nashville' named entity 'genome' named entity 'Ipswich, MA' named entity 'Next-generation sequencing' named entity 'directly sequenced' named entity 'genomes' named entity 'genome sequence data' named entity 'clones' named entity 'next-generation sequencing' named entity 'enzyme' named entity 'parasites' named entity 'parasites' named entity 'human DNA' named entity 'human genome' named entity 'experimental design' named entity 'parasites' named entity 'PCR' named entity 'malaria' named entity 'SNPs' named entity 'restriction endonuclease' named entity 'genome sequencing' named entity 'polymerase' named entity 'Plasmodium vivax' named entity 'P. falciparum' named entity 'digestion' named entity 'R package' named entity 'reaction mixture' named entity 'parasites' named entity 'red blood cells' named entity 'parasitaemia' named entity 'human genome' named entity 'genetically similar' named entity 'microsatellites'

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