Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum

ABSTRACTMolecular markers for surveillance ofPlasmodium falciparumresistance to current antimalarials are sorely needed. A 28-day efficacy study of artemether-lumefantrine in eastern Sudan identified 5 treatment failures among 100 evaluable patients; 9 further individuals were parasite positive by PCR during follow-up. Polymorphisms inpfatpase6andpfmdr1were evaluated by DNA sequencing. One individual carried parasites with a novelpfmdr1polymorphism (F1044L).pfmdr1gene amplification in parasites prior to treatment occurred in three individuals who had recurrent infection during follow-up.

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Extra-binomial variation approach for analysis of pooled DNA sequencing data

Bioinformatics ◽

10.1093/bioinformatics/bts553 ◽

2012 ◽

Vol 28 (22) ◽

pp. 2898-2904 ◽

Cited By ~ 3

Author(s):

Xin Yang ◽

John A. Todd ◽

David Clayton ◽

Chris Wallace

Keyword(s):

Dna Sequencing ◽

Sequencing Data ◽

Variation Approach ◽

Pooled Dna

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Sensitive, highly multiplexed sequencing of microhaplotypes from the Plasmodium falciparum heterozygome

10.1101/2020.02.25.964536 ◽

2020 ◽

Cited By ~ 2

Author(s):

Sofonias K Tessema ◽

Nicholas J Hathaway ◽

Noam B Teyssier ◽

Maxwell Murphy ◽

Anna Chen ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Low Cost ◽

Flexible Tool ◽

High Coverage ◽

Laboratory Methods ◽

Targeted Next Generation Sequencing ◽

Sequencing Method ◽

Sample Frequency ◽

Genomic Regions ◽

High Diversity

AbstractBackgroundTargeted next generation sequencing offers the potential for consistent, deep coverage of information rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited.MethodsA bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, WGS data were generated for the same controls.ResultsUsing publicly available genomes, 4465 high diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median HE = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%) and sensitivity (90% for haplotypes with 5% within sample frequency in DBS with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic SNP barcodes.DiscussionThe bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

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Reconstructing mutational lineages in breast cancer by multi-patient-targeted single cell DNA sequencing

10.1101/2021.11.16.468877 ◽

2021 ◽

Author(s):

Nicholas Navin ◽

Jake Leighton ◽

Min Hu ◽

Emi Sei ◽

Funda Meric-Bernstam

Keyword(s):

Breast Cancer ◽

Dna Sequencing ◽

Single Cell ◽

Copy Number ◽

Somatic Mutations ◽

Single Cells ◽

Tumor Evolution ◽

Sequencing Method ◽

Genomic Regions ◽

Microfluidics Platform

Single cell DNA sequencing (scDNA-seq) methods are powerful tools for profiling mutations in cancer cells, however most genomic regions characterized in single cells are non-informative. To overcome this issue, we developed a Multi-Patient-Targeted (MPT) scDNA-seq sequencing method. MPT involves first performing bulk exome sequencing across a cohort of cancer patients to identify somatic mutations, which are then pooled together to develop a single custom targeted panel for high-throughput scDNA-seq using a microfluidics platform. We applied MPT to profile 330 mutations across 23,500 cells from 5 TNBC patients, which showed that 3 tumors were monoclonal and 2 tumors were polyclonal. From this data, we reconstructed mutational lineages and identified early mutational and copy number events, including early TP53 mutations that occurred in all five patients. Collectively, our data suggests that MPT can overcome technical obstacles for studying tumor evolution using scDNA-seq by profiling information-rich mutation sites.

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Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa527 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sofonias K Tessema ◽

Nicholas J Hathaway ◽

Noam B Teyssier ◽

Maxwell Murphy ◽

Anna Chen ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Low Cost ◽

Dried Blood Spots ◽

Whole Genome Sequencing Data ◽

Sequencing Data ◽

Flexible Tool ◽

High Coverage ◽

Targeted Next Generation Sequencing ◽

Genomic Regions ◽

High Diversity

Abstract Background Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. Methods A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls. Results Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes. Conclusions The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

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SNV identification from single-cell RNA sequencing data

Human Molecular Genetics ◽

10.1093/hmg/ddz207 ◽

2019 ◽

Vol 28 (21) ◽

pp. 3569-3583 ◽

Cited By ~ 3

Author(s):

Patricia M Schnepp ◽

Mengjie Chen ◽

Evan T Keller ◽

Xiang Zhou

Keyword(s):

Dna Sequencing ◽

Single Cell ◽

Rna Sequencing ◽

Single Cells ◽

Specific Gene ◽

Sequencing Data ◽

Single Nucleotide Variants ◽

Single Cell Rna Sequencing ◽

Sequencing Studies ◽

Genomic Regions

Abstract Integrating single-cell RNA sequencing (scRNA-seq) data with genotypes obtained from DNA sequencing studies facilitates the detection of functional genetic variants underlying cell type-specific gene expression variation. Unfortunately, most existing scRNA-seq studies do not come with DNA sequencing data; thus, being able to call single nucleotide variants (SNVs) from scRNA-seq data alone can provide crucial and complementary information, detection of functional SNVs, maximizing the potential of existing scRNA-seq studies. Here, we perform extensive analyses to evaluate the utility of two SNV calling pipelines (GATK and Monovar), originally designed for SNV calling in either bulk or single-cell DNA sequencing data. In both pipelines, we examined various parameter settings to determine the accuracy of the final SNV call set and provide practical recommendations for applied analysts. We found that combining all reads from the single cells and following GATK Best Practices resulted in the highest number of SNVs identified with a high concordance. In individual single cells, Monovar resulted in better quality SNVs even though none of the pipelines analyzed is capable of calling a reasonable number of SNVs with high accuracy. In addition, we found that SNV calling quality varies across different functional genomic regions. Our results open doors for novel ways to leverage the use of scRNA-seq for the future investigation of SNV function.

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