A microarray platform and novel SNP calling algorithm to evaluate Plasmodium falciparum field samples of low DNA quantity

Jacob, Christopher G; Tan, John C.; Miller, Becky A.; Tan, Aiping; Takala-Harrison, Shannon; Ferdig, Michael T.; Plowe, Christopher V.

doi:10.1186/1471-2164-15-719

Cited by 15 publications

(21 citation statements)

References 16 publications

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“…Genotyping chips are both expensive and restricted to mutations present in the reference genome used at its creation (Hugerth and Andersson, 2017;Read and Massey, 2014). As a result, only a few organisms like Neisseria meningitidis (Bille et al, 2005(Bille et al, , 2008, Mycobacterium tuberculosis (Troesch et al, 1999) and P. falciparum (Jacob et al, 2014) with highly conserved genomes, very low rates of mutation (Dutilh et al, 2013) and that are of high global health significance were genotyped. These bottlenecks have primarily been resolved by the advent of next-generation sequencing (NGS) that offers a relatively cheap and fast solution to produce whole genomes at an unprecedented rate (Mardis, 2008;Schuster, 2008), paving the way for novel biomarker discoveries.…”

Section: Introductionmentioning

confidence: 99%

Current Affairs of Microbial Genome-Wide Association Studies: Approaches, Bottlenecks and Analytical Pitfalls

et al. 2020

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Microbial genome-wide association studies (mGWAS) are a new and exciting research field that is adapting human GWAS methods to understand how variations in microbial genomes affect host or pathogen phenotypes, such as drug resistance, virulence, host specificity and prognosis. Several computational tools and methods have been developed or adapted from human GWAS to facilitate the discovery of novel mutations and structural variations that are associated with the phenotypes of interest. However, no comprehensive, end-to-end, user-friendly tool is currently available. The development of a broadly applicable pipeline presents a real opportunity among computational biologists. Here, (i) we review the prominent and promising tools, (ii) discuss analytical pitfalls and bottlenecks in mGWAS, (iii) provide insights into the selection of appropriate tools, (iv) highlight the gaps that still need to be filled and how users and developers can work together to overcome these bottlenecks. Use of mGWAS research can inform drug repositioning decisions as well as accelerate the discovery and development of more effective vaccines and antimicrobials for pressing infectious diseases of global health significance, such as HIV, TB, influenza, and malaria.

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Section: Introductionmentioning

confidence: 99%

Current Affairs of Microbial Genome-Wide Association Studies: Approaches, Bottlenecks and Analytical Pitfalls

et al. 2020

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“…A key area of methods development is quantification of within-host diversity 7,[41][42][43][44][45][46] , estimation of inbreeding 7,47 , and deconvolution of mixed infections into individual strains. 48,49 The data have also been used to develop and test methods for estimating identity by descent 50,51 , imputation 52 , typing structural variants 53 , designing other SNP genotyping platforms 54 and data visualisation 8,55 . In a companion study we performed whole genome sequencing of experimental genetic crosses of P. falciparum, and this provided a benchmark to test the accuracy of our genotyping methods, and to conduct an in-depth analysis of indels, structural variants and recombination events which are complicated to ascertain in these population genetic samples 56 .…”

Section: Introductionmentioning

confidence: 99%

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples

Pearson

Amato

Kwiatkowski

2019

Preprint

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MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination. MethodsAll samples in this study were derived from blood samples obtained from patients with P. falciparum malaria, collected with informed consent from the patient or a parent or guardian. At each location, sample collection was approved by the appropriate local and institutional ethics committees. The following local and institutional committees gave ethical approval for the partner studies:Standard laboratory protocols were used to determine DNA quantity and proportion of human DNA in each sample as previously described 7,56 .

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“…The former situation is a larger 577 issue for clinical parasite isolates due to the abundance of white blood cells that contribute to extracellular DNA when they decay outside of the host [92]. Indeed, we observed more human of a leukodepletion step that is routinely employed to limit host cell contamination [93][94][95] and 583 2) lower overall sequencing output of that particular run (Table S10).…”

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confidence: 90%

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Liu

Huckaby

Brown

et al. 2020

Preprint

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24Single cell genomics is a rapidly advancing field; however, most techniques are designed for 25 mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, 26 Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed 27 base content. Through optimization of a quasi-linear genome amplification method, we achieve 28 more even genome coverage and better targeting of the parasite genome over contaminants. 29 These improvements are particularly important for expanding the accessibility of single cell 30 approaches to new organisms and cell types and for improving the study of genetic mechanisms 31 of adaptation. 32 33 Keywords: whole-genome amplification, AT-skewed genome, malaria, single cell 34 sequencing, MALBAC 35 36 Background 37 Malaria is a life-threatening disease caused by protozoan Plasmodium parasites. P. falciparum 38 causes the greatest number of human malaria deaths [1]. The clinical symptoms of malaria occur 39 when parasites invade human erythrocytes and undergo rounds of asexual reproduction by 40 maturing from early forms into late stage parasites and bursting from erythrocytes to begin the 41 cycle again [2]. In this asexual cycle, parasites possess a single haploid genome during the early 42 stages; rapid genome replication in the later stages leads to an average of 16 genome copies [2].43 44Due to a lack of an effective vaccine, antimalarial drugs are required to treat malaria. However, 45 drug efficacy is threatened by the frequent emergence of resistant populations [3]. Copy number variations (CNVs), or the amplification and deletion of a genomic element, is one of the major 47 sources of genomic variation in P. falciparum that contribute to antimalarial resistance [4][5][6][7][8][9][10][11][12][13][14][15]. 48Similar to bacteria and viruses [16][17][18], a high rate of CNVs may initiate genomic changes that 49 contribute to the rapid adaptation of this organism [7,19]. Despite the importance of CNVs, their 50 dynamics in evolving populations are not well understood. 52The majority of CNVs in P. falciparum have been identified by analyzing bulk DNA in which 53 the CNVs are present in a substantial fraction of individual parasites in the population due to 54 positive selection [8,10,11,20,21]. However, many CNVs likely remain undetected because 55 they are presumably either deleterious or offer no advantages for parasite growth or transmission 56 [22] and are therefore present in low frequency [20,22]. Currently, most CNVs are identified 57 using read-depth analysis of short read sequencing data, which derives an average signal across 58 the population. For this reason, genetic variants must be present in a high frequency (i.e. ~50%) 59 in the population to be detected [23][24][25]. Sequencing at very high depth improves the detection 60 of low frequency CNVs, but the sensitivity is limited to large-scale CNVs present in > 5% cells 61 [26][27][28]. Analysis methods that rely on the detection of reads that span CNV junctions h...

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A microarray platform and novel SNP calling algorithm to evaluate Plasmodium falciparum field samples of low DNA quantity

Cited by 15 publications

References 16 publications

Current Affairs of Microbial Genome-Wide Association Studies: Approaches, Bottlenecks and Analytical Pitfalls

Current Affairs of Microbial Genome-Wide Association Studies: Approaches, Bottlenecks and Analytical Pitfalls

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples

Single cell sequencing of the small and AT-skewed genome of malaria parasites

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