Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification
Identifieur interne : 000F59 ( Main/Merge ); précédent : 000F58; suivant : 000F60Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification
Auteurs : Samuel O. Oyola [Kenya] ; Cristina V. Ariani ; William L. Hamilton ; Mihir Kekre ; Lucas N. Amenga-Etego [Ghana] ; Anita Ghansah [Ghana] ; Gavin G. Rutledge ; Seth Redmond [États-Unis] ; Magnus Manske ; Dushyanth Jyothi ; Chris G. Jacob ; Thomas D. Otto ; Kirk Rockett [Royaume-Uni] ; Chris I. Newbold [Royaume-Uni] ; Matthew Berriman ; Dominic P. Kwiatkowski [Royaume-Uni]Source :
- Malaria Journal [ 1475-2875 ] ; 2016.
Descripteurs français
- KwdFr :
- ADN des protozoaires (), ADN des protozoaires (génétique), ADN des protozoaires (isolement et purification), Amorces ADN (génétique), Analyse de séquence d'ADN, Dessiccation, Génome de protozoaire, Humains, Plasmodium falciparum (génétique), Plasmodium falciparum (isolement et purification), Prélèvement biologique (), Sang (parasitologie), Techniques d'amplification d'acides nucléiques ().
- MESH :
- génétique : ADN des protozoaires, Amorces ADN, Plasmodium falciparum.
- isolement et purification : ADN des protozoaires, Plasmodium falciparum.
- parasitologie : Sang.
- ADN des protozoaires, Analyse de séquence d'ADN, Dessiccation, Génome de protozoaire, Humains, Prélèvement biologique, Techniques d'amplification d'acides nucléiques.
English descriptors
- KwdEn :
- Blood (parasitology), DNA Primers (genetics), DNA, Protozoan (chemistry), DNA, Protozoan (genetics), DNA, Protozoan (isolation & purification), Desiccation, Genome, Protozoan, Humans, Nucleic Acid Amplification Techniques (methods), Plasmodium falciparum (genetics), Plasmodium falciparum (isolation & purification), Sequence Analysis, DNA, Specimen Handling (methods).
- MESH :
- chemical , chemistry : DNA, Protozoan.
- chemical , genetics : DNA Primers, DNA, Protozoan.
- chemical , isolation & purification : DNA, Protozoan.
- genetics : Plasmodium falciparum.
- isolation & purification : Plasmodium falciparum.
- methods : Nucleic Acid Amplification Techniques, Specimen Handling.
- parasitology : Blood.
- Desiccation, Genome, Protozoan, Humans, Sequence Analysis, DNA.
Abstract
Translating genomic technologies into healthcare applications for the malaria parasite
Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8–12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired (
Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of
The sWGA methods described here provide a reliable and scalable way of generating
The online version of this article (doi:10.1186/s12936-016-1641-7) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/s12936-016-1641-7
PubMed: 27998271
PubMed Central: 5175302
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PMC:5175302Le document en format XML
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Whole genome sequencing of <italic>Plasmodium falciparum</italic>
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<author><name sortKey="Rutledge, Gavin G" sort="Rutledge, Gavin G" uniqKey="Rutledge G" first="Gavin G." last="Rutledge">Gavin G. Rutledge</name>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Blood (parasitology)</term>
<term>DNA Primers (genetics)</term>
<term>DNA, Protozoan (chemistry)</term>
<term>DNA, Protozoan (genetics)</term>
<term>DNA, Protozoan (isolation & purification)</term>
<term>Desiccation</term>
<term>Genome, Protozoan</term>
<term>Humans</term>
<term>Nucleic Acid Amplification Techniques (methods)</term>
<term>Plasmodium falciparum (genetics)</term>
<term>Plasmodium falciparum (isolation & purification)</term>
<term>Sequence Analysis, DNA</term>
<term>Specimen Handling (methods)</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>ADN des protozoaires ()</term>
<term>ADN des protozoaires (génétique)</term>
<term>ADN des protozoaires (isolement et purification)</term>
<term>Amorces ADN (génétique)</term>
<term>Analyse de séquence d'ADN</term>
<term>Dessiccation</term>
<term>Génome de protozoaire</term>
<term>Humains</term>
<term>Plasmodium falciparum (génétique)</term>
<term>Plasmodium falciparum (isolement et purification)</term>
<term>Prélèvement biologique ()</term>
<term>Sang (parasitologie)</term>
<term>Techniques d'amplification d'acides nucléiques ()</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA, Protozoan</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA Primers</term>
<term>DNA, Protozoan</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>DNA, Protozoan</term>
</keywords>
<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Plasmodium falciparum</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN des protozoaires</term>
<term>Amorces ADN</term>
<term>Plasmodium falciparum</term>
</keywords>
<keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Plasmodium falciparum</term>
</keywords>
<keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>ADN des protozoaires</term>
<term>Plasmodium falciparum</term>
</keywords>
<keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Nucleic Acid Amplification Techniques</term>
<term>Specimen Handling</term>
</keywords>
<keywords scheme="MESH" qualifier="parasitologie" xml:lang="fr"><term>Sang</term>
</keywords>
<keywords scheme="MESH" qualifier="parasitology" xml:lang="en"><term>Blood</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Desiccation</term>
<term>Genome, Protozoan</term>
<term>Humans</term>
<term>Sequence Analysis, DNA</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>ADN des protozoaires</term>
<term>Analyse de séquence d'ADN</term>
<term>Dessiccation</term>
<term>Génome de protozoaire</term>
<term>Humains</term>
<term>Prélèvement biologique</term>
<term>Techniques d'amplification d'acides nucléiques</term>
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>Translating genomic technologies into healthcare applications for the malaria parasite <italic>Plasmodium falciparum</italic>
has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and storage requirements compared with venous blood (VB). Here, the use of selective whole genome amplification (sWGA) to sequence the <italic>P. falciparum</italic>
genome from clinical DBS samples was evaluated, and the results compared with current methods that use leucodepleted VB.</p>
</sec>
<sec><title>Methods</title>
<p>Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8–12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired (<italic>P. falciparum</italic>
DNA) and contaminating (human) genomes.</p>
</sec>
<sec><title>Results</title>
<p>Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of <italic>P. falciparum</italic>
DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5× coverage for 50% of the genome was 0.03% (40 parasites per 200 white blood cells). Over 99% SNP concordance between VB and DBS samples was achieved after excluding missing calls.</p>
</sec>
<sec><title>Conclusion</title>
<p>The sWGA methods described here provide a reliable and scalable way of generating <italic>P. falciparum</italic>
genome sequence data from DBS samples. The current data indicate that it will be possible to get good quality sequence on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in <italic>P. falciparum</italic>
genome sequencing from field samples, and paves the way for large-scale epidemiological applications.</p>
</sec>
<sec><title>Electronic supplementary material</title>
<p>The online version of this article (doi:10.1186/s12936-016-1641-7) contains supplementary material, which is available to authorized users.</p>
</sec>
</div>
</front>
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