scholarly journals Approaches to estimating inbreeding coefficients in clinical isolates of Plasmodium falciparum from genomic sequence data

2015 ◽  
Author(s):  
Lucas Amenga-Etego ◽  
Ruiqi Li ◽  
John D. O’Brien
Keyword(s):  
Plasmodium Falciparum ◽  
Genetic Epidemiology ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Infected Individual ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Extensive Literature ◽  
Data Set ◽  
Inbreeding Coefficients

AbstractThe advent of whole-genome sequencing has generated increased interest in modeling the structure of strain mixture within clinicial infections of Plasmodium falciparum (Pf). The life cycle of the parasite implies that the mixture of multiple strains within an infected individual is related to the out-crossing rate across populations, making methods for measuring this process in situ central to understanding the genetic epidemiology of the disease. In this paper, we show how to estimate inbreeding coefficients using genomic data from Pf clinical samples, providing a simple metric for assessing within-sample mixture that connects to an extensive literature in population genetics and conservation ecology. Features of the P. falciparum genome mean that some standard methods for inbreeding coefficients and related F-statistics cannot be used directly. Here, we review an initial effort to estimate the inbreeding coefficient within clinical isolates of P. falciparum and provide several generalizations using both frequentist and Bayesian approaches. The Bayesian approach connects these estimates to the Balding-Nichols model, a mainstay within genetic epidemiology. We provide simulation results on the performance of the estimators and show their use on ~ 1500 samples from the PF3K data set. We also compare the results to output from a recent mixture model for within-sample strain mixture, showing that inbreeding coefficients provide a strong proxy for the results of these more complex models. We provide the methods described within an open-source R package pfmix.

scholarly journals Long read assemblies of geographically dispersed Plasmodium falciparum isolates reveal highly structured subtelomeres

2018 ◽  
Vol 3 ◽  
pp. 52 ◽  
Author(s):  
Thomas D. Otto ◽  
Ulrike Böhme ◽  
Mandy Sanders ◽  
Adam J. Reid ◽  
Ellen I. Bruske ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Sequence Data ◽  
Gene Families ◽  
Complete Sequence ◽  
Clinical Isolates ◽  
Current Standard ◽  
Entire Genome ◽  
Variable Regions ◽  
Long Read ◽  
Subtelomeric Regions

Background: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters.  Results: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates.  We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.

scholarly journals Sustained Ex Vivo Susceptibility of Plasmodium falciparum to Artemisinin Derivatives but Increasing Tolerance to Artemisinin Combination Therapy Partner Quinolines in The Gambia

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Alfred Amambua-Ngwa ◽  
Joseph Okebe ◽  
Haddijatou Mbye ◽  
Sukai Ceesay ◽  
Fatima El-Fatouri ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Combination Therapy ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Ex Vivo ◽  
Artemisinin Combination Therapy ◽  
Artemisinin Resistance ◽  
The Gambia ◽  
Microsatellite Variation

ABSTRACT Antimalarial interventions have yielded a significant decline in malaria prevalence in The Gambia, where artemether-lumefantrine (AL) has been used as a first-line antimalarial for a decade. Clinical Plasmodium falciparum isolates collected from 2012 to 2015 were analyzed ex vivo for antimalarial susceptibility and genotyped for drug resistance markers (pfcrt K76T, pfmdr1 codons 86, 184, and 1246, and pfk13) and microsatellite variation. Additionally, allele frequencies of single nucleotide polymorphisms (SNPs) from other drug resistance-associated genes were compared from genomic sequence data sets from 2008 (n = 79) and 2014 (n = 168). No artemisinin resistance-associated pfk13 mutation was found, and only 4% of the isolates tested in 2015 showed significant growth after exposure to dihydroartemisinin. Conversely, the 50% inhibitory concentrations (IC50s) of amodiaquine and lumefantrine increased within this period. pfcrt 76T and pfmdr1 184F mutants remained at a prevalence above 80%. pfcrt 76T was positively associated with higher IC50s to chloroquine. pfmdr1 NYD increased in frequency between 2012 and 2015 due to lumefantrine selection. The TNYD (pfcrt 76T and pfmdr1 NYD wild-type haplotype) also increased in frequency following AL implementation in 2008. These results suggest selection for pfcrt and pfmdr1 genotypes that enable tolerance to lumefantrine. Increased tolerance to lumefantrine calls for sustained chemotherapeutic monitoring in The Gambia to minimize complete artemisinin combination therapy (ACT) failure in the future.

scholarly journals Inferring Strain Mixture within Clinical Plasmodium falciparum Isolates from Genomic Sequence Data

2016 ◽  
Vol 12 (6) ◽  
pp. e1004824 ◽  
Author(s):  
John D. O’Brien ◽  
Zamin Iqbal ◽  
Jason Wendler ◽  
Lucas Amenga-Etego
Keyword(s):  
Plasmodium Falciparum ◽  
Genomic Sequence ◽  
Sequence Data

scholarly journals Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification

2016 ◽  
Author(s):  
Samuel O. Oyola ◽  
Cristina V. Ariani ◽  
William L. Hamilton ◽  
Mihir Kekre ◽  
Lucas N. Amenga-Etego ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Genome Sequencing ◽  
Whole Genome Amplification ◽  
Sequence Data ◽  
White Blood Cells ◽  
Clinical Samples ◽  
Limiting Factor ◽  
Whole Genome ◽  
Genome Amplification ◽  
Field Samples

ABSTRACTTranslating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum 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, we evaluate the use of selective whole genome amplification (sWGA) to sequence the P. falciparum genome from clinical DBS samples, and compare the results to current methods using leucodepleted VB. 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 (P. falciparum DNA) and contaminating (human) genomes. Using sWGA method, we sequenced clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB. Greater than 18-fold enrichment of P. falciparum DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5x 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. The sWGA methods described here provide a reliable and scalable way of generating P. falciparum genome sequence data from DBS samples. Our data indicate that it will be possible to get good quality sequence data on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in P. falciparum genome sequencing from field samples, and paves the way for large-scale epidemiological applications.

scholarly journals Getting close to nature – Plasmodium knowlesi reference genome sequences from contemporary clinical isolates

2021 ◽  
Author(s):  
Damilola R Oresegun ◽  
Peter Thorpe ◽  
Ernest Diez Benavente ◽  
Susana Campino ◽  
Muh Fauzi ◽  
...  
Keyword(s):  
Plasmodium Knowlesi ◽  
Sequence Data ◽  
Family Members ◽  
Gene Families ◽  
Core Gene ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Cell Agglutination ◽  
Sequencing Platform ◽  
Kir Genes

Plasmodium knowlesi, a malaria parasite of old-world macaque monkeys, is used extensively to model Plasmodium biology. Recently P. knowlesi was found in the human population of Southeast Asia, particularly Malaysia. P. knowlesi causes un-complicated to severe and fatal malaria in the human host with features in common with the more prevalent and virulent malaria caused by Plasmodium falciparum. As such P. knowlesi presents a unique opportunity to inform an experimental model for malaria with clinical data from same-species human infections. Experimental lines of P. knowlesi represent well characterised genetically static parasites and to maximise their utility as a backdrop for understanding malaria pathophysiology, genetically diverse contemporary clinical isolates, essentially wild-type, require comparable characterization. The Oxford Nanopore PCR-free long-read sequencing platform was used to sequence P. knowlesi parasites from archived clinical samples. The sequencing platform and assembly pipeline was designed to facilitate capturing data on important multiple gene families, including the P. knowlesi schizont-infected cell agglutination (SICA) var genes and the Knowlesi-Interspersed Repeats (KIR) genes. The SICAvar and KIR gene families code for antigenically variant proteins that have been difficult to resolve and characterise. Analyses presented here suggest that the family members have arisen through a process of gene duplication, selection pressure and variation. Highly evolving genes tend to be located proximal to genetic elements that drive change rather than regions that support core gene conservation. For example, the virulence-associated P. falciparum erythrocyte membrane protein (PfEMP1) gene family members are restricted to relatively unstable sub-telomeric regions. In contrast the SICAvar nd KIR genes are located throughout the genome but as the study presented here shows, they occupy otherwise gene-sparse chromosomal locations. The novel methods presented here offer the malaria research community new tools to generate comprehensive genome sequence data from small clinical samples and renewed insight into these complex real-world parasites.

scholarly journals An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates

Genomics Data ◽  
2014 ◽  
Vol 2 ◽  
pp. 393-395 ◽  
Author(s):  
Amit Kumar Subudhi ◽  
P.A. Boopathi ◽  
Shilpi Garg ◽  
Sheetal Middha ◽  
Jyoti Acharya ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Clinical Isolates ◽  
Transcriptome Data ◽  
Antisense Transcripts ◽  
Data Set ◽  
Natural Antisense Transcripts

scholarly journals A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India

2016 ◽  
Vol 54 (6) ◽  
pp. 1500-1511 ◽  
Author(s):  
Pavitra N. Rao ◽  
Swapna Uplekar ◽  
Sriti Kayal ◽  
Prashant K. Mallick ◽  
Nabamita Bandyopadhyay ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Artemisinin Combination Therapy ◽  
Amplicon Sequencing ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Ion Torrent ◽  
Content Type ◽  
Ion Torrent Pgm ◽  
Reference Strains

A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of sixPlasmodium falciparumgenes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverseP. falciparumreference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

Integrated Quantitative Analysis of the Phosphoproteome and Transcriptome in Tamoxifen-resistant Breast Cancer*

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Breast Cancer ◽  
High Rate ◽  
System Level ◽  
Clinical Samples ◽  
Transcriptome Data ◽  
Data Set ◽  
Reporter Assays ◽  
Treated Breast ◽  
Mcf 7 ◽  
Resistant Cells

Quantitative phosphoproteome and transcriptome analysisof ligand-stimulated MCF-7 human breast cancer cells wasperformed to understand the mechanisms of tamoxifen resistanceat a system level. Phosphoproteome data revealed thatWT cells were more enriched with phospho-proteins thantamoxifen-resistant cells after stimulation with ligands.Surprisingly, decreased phosphorylation after ligand perturbationwas more common than increased phosphorylation.In particular, 17?-estradiol induced down-regulation inWT cells at a very high rate. 17?-Estradiol and the ErbBligand heregulin induced almost equal numbers of up-regulatedphospho-proteins in WT cells. Pathway and motifactivity analyses using transcriptome data additionallysuggested that deregulated activation of GSK3? (glycogensynthasekinase 3?) and MAPK1/3 signaling might be associatedwith altered activation of cAMP-responsive elementbindingprotein and AP-1 transcription factors intamoxifen-resistant cells, and this hypothesis was validatedby reporter assays. An examination of clinical samples revealedthat inhibitory phosphorylation of GSK3? at serine 9was significantly lower in tamoxifen-treated breast cancerpatients that eventually had relapses, implying that activationof GSK3? may be associated with the tamoxifen-resistantphenotype. Thus, the combined phosphoproteomeand transcriptome data set analyses revealed distinct signal

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