Genomes of Leishmania parasites directly sequenced from patients with visceral leishmaniasis in the Indian subcontinent

AbstractWhole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in patient samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples.Author summaryVisceral leishmaniasis (VL) is caused by parasitic protozoa of the Leishmania donovani complex and is lethal in the absence of treatment. Whole Genome Sequencing (WGS) of L. donovani clinical isolates revealed hitherto cryptic population structure in the Indian Sub-Continent and provided insights into the epidemiology and potential mechanisms of drug resistance. However, several biases are likely introduced during the culture step. We report here the development of a method that allows determination of parasite genomes directly in clinical samples, and validate it on bone marrow and splenic aspirates of VL patients in Nepal. Our study sheds a new light on the biology of Leishmania in the human host: we found that intracellular parasites of the patients had very low levels of aneuploidy, in sharp contrast to the situation in cultivated isolates. Moreover, the observed differences in genomes between intracellular amastigotes of the patient and the derived cultured parasites suggests polyclonality of infections, with different clones dominating in clinical samples and in culture, likely due to fitness differences. We believe this method is most suitable for clinical studies and for molecular tracking in the context of elimination programs.

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0306 Exploring the feasibility of using copy number variants as genetic markers through large-scale whole genome sequencing experiments

Journal of Animal Science ◽

10.2527/jam2016-0306 ◽

2016 ◽

Vol 94 (suppl_5) ◽

pp. 146-146

Author(s):

D. M. Bickhart ◽

L. Xu ◽

J. L. Hutchison ◽

J. B. Cole ◽

D. J. Null ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genetic Markers ◽

Genome Sequencing ◽

Copy Number ◽

Large Scale ◽

Copy Number Variants ◽

Whole Genome

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Whole-genome sequencing from the New Zealand Saccharomyces cerevisiae population reveals the genomic impacts of novel microbial range expansion

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkaa027 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Peter Higgins ◽

Cooper A Grace ◽

Soon A Lee ◽

Matthew R Goddard

Keyword(s):

Saccharomyces Cerevisiae ◽

New Zealand ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Range Expansion ◽

Copy Number ◽

Small Scale ◽

Whole Genome ◽

Copy Number Changes ◽

The Impact

Abstract Saccharomyces cerevisiae is extensively utilized for commercial fermentation, and is also an important biological model; however, its ecology has only recently begun to be understood. Through the use of whole-genome sequencing, the species has been characterized into a number of distinct subpopulations, defined by geographical ranges and industrial uses. Here, the whole-genome sequences of 104 New Zealand (NZ) S. cerevisiae strains, including 52 novel genomes, are analyzed alongside 450 published sequences derived from various global locations. The impact of S. cerevisiae novel range expansion into NZ was investigated and these analyses reveal the positioning of NZ strains as a subgroup to the predominantly European/wine clade. A number of genomic differences with the European group correlate with range expansion into NZ, including 18 highly enriched single-nucleotide polymorphism (SNPs) and novel Ty1/2 insertions. While it is not possible to categorically determine if any genetic differences are due to stochastic process or the operations of natural selection, we suggest that the observation of NZ-specific copy number increases of four sugar transporter genes in the HXT family may reasonably represent an adaptation in the NZ S. cerevisiae subpopulation, and this correlates with the observations of copy number changes during adaptation in small-scale experimental evolution studies.

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Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples

BMC Veterinary Research ◽

10.1186/s12917-021-02945-3 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Jacqueline King ◽

Anne Pohlmann ◽

Kamila Dziadek ◽

Martin Beer ◽

Kerstin Wernike

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Economic Losses ◽

Clinical Samples ◽

Bovine Viral Diarrhea ◽

Sequence Information ◽

Whole Genome ◽

Diarrhea Virus ◽

Viral Diarrhea ◽

Viral Loads

Abstract Background As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5’ untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. Results To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19–32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. Conclusions Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

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Rare instances of non-random dropout with the monochrome multiplex qPCR assay for mitochondrial DNA copy number

10.1101/2021.10.11.463983 ◽

2021 ◽

Author(s):

Stephanie Y Yang ◽

Charles E Newcomb ◽

Stephanie L Battle ◽

Anthony YY Hsieh ◽

Hailey L Chapman ◽

...

Keyword(s):

Mitochondrial Dna ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Copy Number ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Dna Copy Number ◽

Loop Primer ◽

Mitochondrial Dna Copy Number ◽

D Loop

Mitochondrial DNA copy number (mtDNA-CN) is a proxy for mitochondrial function and has been of increasing interest to the mitochondrial research community. There are several ways to measure mtDNA-CN, ranging from whole genome sequencing to qPCR. A recent article from the Journal of Molecular Diagnostics described a novel method for measuring mtDNA-CN that is both inexpensive and reproducible. However, we show that certain individuals, particularly those with very low qPCR mtDNA measurements, show poor concordance between qPCR and whole genome sequencing measurements. After examining whole genome sequencing data, this seems to be due to polymorphisms within the D-loop primer region. Non-concordant mtDNA-CN was observed in all instances of polymorphisms at certain positions in the D-loop primer regions, however, not all positions are susceptible to this effect. In particular, these polymorphisms appear disproportionately in individuals with the L, T, and U mitochondrial haplogroups, indicating non-random dropout.

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Detection and characterization of copy number variants based on whole-genome sequencing by DNBSEQ platforms

10.1101/786962 ◽

2019 ◽

Author(s):

Junhua Rao ◽

Lihua Peng ◽

Fang Chen ◽

Hui Jiang ◽

Chunyu Geng ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Copy Number ◽

Copy Number Variants ◽

Copy Number Variant ◽

Whole Genome ◽

Genome Wide ◽

Wide Range ◽

Distribution Sensitivity ◽

Cnv Detection

AbstractBackgroundNext-generation sequence (NGS) has rapidly developed in past years which makes whole-genome sequencing (WGS) becoming a more cost- and time-efficient choice in wide range of biological researches. We usually focus on some variant detection via WGS data, such as detection of single nucleotide polymorphism (SNP), insertion and deletion (Indel) and copy number variant (CNV), which playing an important role in many human diseases. However, the feasibility of CNV detection based on WGS by DNBSEQ™ platforms was unclear. We systematically analysed the genome-wide CNV detection power of DNBSEQ™ platforms and Illumina platforms on NA12878 with five commonly used tools, respectively.ResultsDNBSEQ™ platforms showed stable ability to detect slighter more CNVs on genome-wide (average 1.24-fold than Illumina platforms). Then, CNVs based on DNBSEQ™ platforms and Illumina platforms were evaluated with two public benchmarks of NA12878, respectively. DNBSEQ™ and Illumina platforms showed similar sensitivities and precisions on both two benchmarks. Further, the difference between tools for CNV detection was analyzed, and indicated the selection of tool for CNV detection could affected the CNV performance, such as count, distribution, sensitivity and precision.ConclusionThe major contribution of this paper is providing a comprehensive guide for CNV detection based on WGS by DNBSEQ™ platforms for the first time.

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Whole genome sequencing of respiratory syncytial (RSV) virus from clinical samples with low viral load v1 (protocols.io.qsedwbe)

protocols.io ◽

10.17504/protocols.io.qsedwbe ◽

2018 ◽

Author(s):

Stephanie Goya ◽

Gabriel L ◽

Mercedes S ◽

Laura E ◽

Alicia S ◽

...

Keyword(s):

Viral Load ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Samples ◽

Whole Genome

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MBRS-59. SINGLE-CELL WHOLE-GENOME SEQUENCING DISSECTS INTRA-TUMOURAL GENOMIC HETEROGENEITY AND CLONAL EVOLUTION IN CHILDHOOD MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.563 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii408-iii408

Author(s):

Marina Danilenko ◽

Masood Zaka ◽

Claire Keeling ◽

Stephen Crosier ◽

Rafiqul Hussain ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Single Cell ◽

Genome Sequencing ◽

Copy Number ◽

Single Cell Analysis ◽

Mutational Analysis ◽

Single Cells ◽

Clonal Evolution ◽

Whole Genome ◽

Clinically Significant

Abstract Medulloblastomas harbor clinically-significant intra-tumoral heterogeneity for key biomarkers (e.g. MYC/MYCN, β-catenin). Recent studies have characterized transcriptional heterogeneity at the single-cell level, however the underlying genomic copy number and mutational architecture remains to be resolved. We therefore sought to establish the intra-tumoural genomic heterogeneity of medulloblastoma at single-cell resolution. Copy number patterns were dissected by whole-genome sequencing in 1024 single cells isolated from multiple distinct tumour regions within 16 snap-frozen medulloblastomas, representing the major molecular subgroups (WNT, SHH, Group3, Group4) and genotypes (i.e. MYC amplification, TP53 mutation). Common copy number driver and subclonal events were identified, providing clear evidence of copy number evolution in medulloblastoma development. Moreover, subclonal whole-arm and focal copy number alterations covering important genomic loci (e.g. on chr10 of SHH patients) were detected in single tumour cells, yet undetectable at the bulk-tumor level. Spatial copy number heterogeneity was also common, with differences between clonal and subclonal events detected in distinct regions of individual tumours. Mutational analysis of the cells allowed dissection of spatial and clonal heterogeneity patterns for key medulloblastoma mutations (e.g. CTNNB1, TP53, SMARCA4, PTCH1) within our cohort. Integrated copy number and mutational analysis is underway to establish their inter-relationships and relative contributions to clonal evolution during tumourigenesis. In summary, single-cell analysis has enabled the resolution of common mutational and copy number drivers, alongside sub-clonal events and distinct patterns of clonal and spatial evolution, in medulloblastoma development. We anticipate these findings will provide a critical foundation for future improved biomarker selection, and the development of targeted therapies.

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