Direct whole-genome sequencing of Plasmodium falciparum specimens from dried erythrocyte spots

Sidsel Nag*, Poul Erik Kofoed, Johan Ursing, Camilla Koldbæk Lemvigh, Rosa Lundbye Allesøe, Amabelia Rodrigues, Christina Aaby Svendsen, Jacob Dyring Jensen, Michael Alifrangis, Ole Lund, Frank Møller Aarestrup

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Background: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individuals living in rural areas, away from main infrastructure and the electrical grid. The aim of this study was to describe a low-tech procedure to sample P. falciparum specimens for direct whole genome sequencing (WGS), without use of electricity and cold-chain. Methods: Venous blood samples were collected from malaria patients in Bandim, Guinea-Bissau and leukocyte-depleted using Plasmodipur filters, the enriched parasite sample was spotted on Whatman paper and dried. The samples were stored at ambient temperatures and subsequently used for DNA-extraction. Ratios of parasite:human content of the extracted DNA was assessed by qPCR, and five samples with varying parasitaemia, were sequenced. Sequencing data were used to analyse the sample content, as well as sample coverage and depth as compared to the 3d7 reference genome. Results: qPCR revealed that 73% of the 199 samples were applicable for WGS, as defined by a minimum ratio of parasite:human DNA of 2:1. WGS revealed an even distribution of sequence data across the 3d7 reference genome, regardless of parasitaemia. The acquired read depths varied from 16 to 99×, and coverage varied from 87.5 to 98.9% of the 3d7 reference genome. SNP-analysis of six genes, for which amplicon sequencing has been performed previously, confirmed the reliability of the WGS-data. Conclusion: This study describes a simple filter paper based protocol for sampling P. falciparum from malaria patients for subsequent direct WGS, enabling acquisition of samples in remote settings with no access to electricity.

Original languageEnglish
Article number91
JournalMalaria Journal
Volume17
Issue number1
Number of pages8
ISSN1475-2875
DOIs
Publication statusPublished - 23 Feb 2018

Keywords

  • Dried blood spots
  • Dried erythrocyte spots
  • Leukocyte depletion
  • Malaria
  • Plasmodium falciparum
  • Sub-Saharan Africa
  • Whole-genome sequencing

Cite this

@article{d5c999efbaa04b178b5ead8a3643a733,
title = "Direct whole-genome sequencing of Plasmodium falciparum specimens from dried erythrocyte spots",
abstract = "Background: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individuals living in rural areas, away from main infrastructure and the electrical grid. The aim of this study was to describe a low-tech procedure to sample P. falciparum specimens for direct whole genome sequencing (WGS), without use of electricity and cold-chain. Methods: Venous blood samples were collected from malaria patients in Bandim, Guinea-Bissau and leukocyte-depleted using Plasmodipur filters, the enriched parasite sample was spotted on Whatman paper and dried. The samples were stored at ambient temperatures and subsequently used for DNA-extraction. Ratios of parasite:human content of the extracted DNA was assessed by qPCR, and five samples with varying parasitaemia, were sequenced. Sequencing data were used to analyse the sample content, as well as sample coverage and depth as compared to the 3d7 reference genome. Results: qPCR revealed that 73{\%} of the 199 samples were applicable for WGS, as defined by a minimum ratio of parasite:human DNA of 2:1. WGS revealed an even distribution of sequence data across the 3d7 reference genome, regardless of parasitaemia. The acquired read depths varied from 16 to 99×, and coverage varied from 87.5 to 98.9{\%} of the 3d7 reference genome. SNP-analysis of six genes, for which amplicon sequencing has been performed previously, confirmed the reliability of the WGS-data. Conclusion: This study describes a simple filter paper based protocol for sampling P. falciparum from malaria patients for subsequent direct WGS, enabling acquisition of samples in remote settings with no access to electricity.",
keywords = "Dried blood spots, Dried erythrocyte spots, Leukocyte depletion, Malaria, Plasmodium falciparum, Sub-Saharan Africa, Whole-genome sequencing",
author = "Sidsel Nag and Kofoed, {Poul Erik} and Johan Ursing and Lemvigh, {Camilla Koldb{\ae}k} and Alles{\o}e, {Rosa Lundbye} and Amabelia Rodrigues and Svendsen, {Christina Aaby} and Jensen, {Jacob Dyring} and Michael Alifrangis and Ole Lund and {M{\o}ller Aarestrup}, Frank",
year = "2018",
month = "2",
day = "23",
doi = "10.1186/s12936-018-2232-6",
language = "English",
volume = "17",
journal = "Malaria Journal",
issn = "1475-2875",
publisher = "BioMed Central",
number = "1",

}

Direct whole-genome sequencing of Plasmodium falciparum specimens from dried erythrocyte spots. / Nag, Sidsel; Kofoed, Poul Erik; Ursing, Johan; Lemvigh, Camilla Koldbæk; Allesøe, Rosa Lundbye; Rodrigues, Amabelia; Svendsen, Christina Aaby; Jensen, Jacob Dyring; Alifrangis, Michael; Lund, Ole; Møller Aarestrup, Frank .

In: Malaria Journal, Vol. 17, No. 1, 91, 23.02.2018.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Direct whole-genome sequencing of Plasmodium falciparum specimens from dried erythrocyte spots

AU - Nag, Sidsel

AU - Kofoed, Poul Erik

AU - Ursing, Johan

AU - Lemvigh, Camilla Koldbæk

AU - Allesøe, Rosa Lundbye

AU - Rodrigues, Amabelia

AU - Svendsen, Christina Aaby

AU - Jensen, Jacob Dyring

AU - Alifrangis, Michael

AU - Lund, Ole

AU - Møller Aarestrup, Frank

PY - 2018/2/23

Y1 - 2018/2/23

N2 - Background: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individuals living in rural areas, away from main infrastructure and the electrical grid. The aim of this study was to describe a low-tech procedure to sample P. falciparum specimens for direct whole genome sequencing (WGS), without use of electricity and cold-chain. Methods: Venous blood samples were collected from malaria patients in Bandim, Guinea-Bissau and leukocyte-depleted using Plasmodipur filters, the enriched parasite sample was spotted on Whatman paper and dried. The samples were stored at ambient temperatures and subsequently used for DNA-extraction. Ratios of parasite:human content of the extracted DNA was assessed by qPCR, and five samples with varying parasitaemia, were sequenced. Sequencing data were used to analyse the sample content, as well as sample coverage and depth as compared to the 3d7 reference genome. Results: qPCR revealed that 73% of the 199 samples were applicable for WGS, as defined by a minimum ratio of parasite:human DNA of 2:1. WGS revealed an even distribution of sequence data across the 3d7 reference genome, regardless of parasitaemia. The acquired read depths varied from 16 to 99×, and coverage varied from 87.5 to 98.9% of the 3d7 reference genome. SNP-analysis of six genes, for which amplicon sequencing has been performed previously, confirmed the reliability of the WGS-data. Conclusion: This study describes a simple filter paper based protocol for sampling P. falciparum from malaria patients for subsequent direct WGS, enabling acquisition of samples in remote settings with no access to electricity.

AB - Background: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individuals living in rural areas, away from main infrastructure and the electrical grid. The aim of this study was to describe a low-tech procedure to sample P. falciparum specimens for direct whole genome sequencing (WGS), without use of electricity and cold-chain. Methods: Venous blood samples were collected from malaria patients in Bandim, Guinea-Bissau and leukocyte-depleted using Plasmodipur filters, the enriched parasite sample was spotted on Whatman paper and dried. The samples were stored at ambient temperatures and subsequently used for DNA-extraction. Ratios of parasite:human content of the extracted DNA was assessed by qPCR, and five samples with varying parasitaemia, were sequenced. Sequencing data were used to analyse the sample content, as well as sample coverage and depth as compared to the 3d7 reference genome. Results: qPCR revealed that 73% of the 199 samples were applicable for WGS, as defined by a minimum ratio of parasite:human DNA of 2:1. WGS revealed an even distribution of sequence data across the 3d7 reference genome, regardless of parasitaemia. The acquired read depths varied from 16 to 99×, and coverage varied from 87.5 to 98.9% of the 3d7 reference genome. SNP-analysis of six genes, for which amplicon sequencing has been performed previously, confirmed the reliability of the WGS-data. Conclusion: This study describes a simple filter paper based protocol for sampling P. falciparum from malaria patients for subsequent direct WGS, enabling acquisition of samples in remote settings with no access to electricity.

KW - Dried blood spots

KW - Dried erythrocyte spots

KW - Leukocyte depletion

KW - Malaria

KW - Plasmodium falciparum

KW - Sub-Saharan Africa

KW - Whole-genome sequencing

U2 - 10.1186/s12936-018-2232-6

DO - 10.1186/s12936-018-2232-6

M3 - Journal article

VL - 17

JO - Malaria Journal

JF - Malaria Journal

SN - 1475-2875

IS - 1

M1 - 91

ER -