Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from ...nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and nearly complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the United States or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside California, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.
Evolving resistance to arte misi ni n-based compounds threatens to derail attempts to control malaria. Resistance has been confirmed in western Cambodia and has recently emerged in western Thailand, ...but is absent from neighboring Laos. Artemisinin resistance results in reduced parasite clearance rates (CRs) after treatment. We used a two-phase strategy to identify genome region(s) underlying this ongoing selective event. Geographical differentiation and haplotype structure at 6969 polymorphic single-nucleotide polymorphisms (SNPs) in 91 parasites from Cambodia, Thailand, and Laos identified 33 genome regions under strong selection. We screened SNPs and microsatellites within these regions in 715 parasites from Thailand, identifying a selective sweep on chromosome 13 that shows strong association (P = 10⁻⁶ to 10⁻¹²) with slow CRs, illustrating the efficacy of targeted association for identifying the genetic basis of adaptive traits.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The microbiota that colonize the human gut and other tissues are dynamic, varying both in composition and functional state between individuals and over time. Gene expression measurements can provide ...insights into microbiome composition and function. However, efficient and unbiased removal of microbial ribosomal RNA (rRNA) presents a barrier to acquiring metatranscriptomic data. Here we describe a probe set that achieves efficient enzymatic rRNA removal of complex human-associated microbial communities. We demonstrate that the custom probe set can be further refined through an iterative design process to efficiently deplete rRNA from a range of human microbiome samples. Using synthetic nucleic acid spike-ins, we show that the rRNA depletion process does not introduce substantial quantitative error in gene expression profiles. Successful rRNA depletion allows for efficient characterization of taxonomic and functional profiles, including during the development of the human gut microbiome. The pan-human microbiome enzymatic rRNA depletion probes described here provide a powerful tool for studying the transcriptional dynamics and function of the human microbiome.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Drug resistant strains of the malaria parasite, Plasmodium falciparum, have rendered chloroquine ineffective throughout much of the world. In parts of Africa and Asia, the coordinated shift from ...chloroquine to other drugs has resulted in the near disappearance of chloroquine-resistant (CQR) parasites from the population. Currently, there is no molecular explanation for this phenomenon. Herein, we employ metabolic quantitative trait locus mapping (mQTL) to analyze progeny from a genetic cross between chloroquine-susceptible (CQS) and CQR parasites. We identify a family of hemoglobin-derived peptides that are elevated in CQR parasites and show that peptide accumulation, drug resistance, and reduced parasite fitness are all linked in vitro to CQR alleles of the P. falciparum chloroquine resistance transporter (pfcrt). These findings suggest that CQR parasites are less fit because mutations in pfcrt interfere with hemoglobin digestion by the parasite. Moreover, our findings may provide a molecular explanation for the reemergence of CQS parasites in wild populations.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations ...with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.
Resistance to the cytostatic activity of the antimalarial drug chloroquine (CQ) is becoming well understood, however, resistance to cytocidal effects of CQ is largely unexplored. We find that PfCRT ...mutations that almost fully recapitulate P. falciparum cytostatic CQ resistance (CQR(CS)) as quantified by CQ IC50 shift, account for only 10-20% of cytocidal CQR (CQR(CC)) as quantified by CQ LD50 shift. Quantitative trait loci (QTL) analysis of the progeny of a chloroquine sensitive (CQS; strain HB3)×chloroquine resistant (CQR; strain Dd2) genetic cross identifies distinct genetic architectures for CQR(CS) vs CQR(CC) phenotypes, including identification of novel interacting chromosomal loci that influence CQ LD50. Candidate genes in these loci are consistent with a role for autophagy in CQR(CC), leading us to directly examine the autophagy pathway in intraerythrocytic CQR parasites. Indirect immunofluorescence of RBC infected with synchronized CQS vs CQR trophozoite stage parasites reveals differences in the distribution of the autophagy marker protein PfATG8 coinciding with CQR(CC). Taken together, the data show that an unusual autophagy-like process is either activated or inhibited for intraerythrocytic trophozoite parasites at LD50 doses (but not IC50 doses) of CQ, that the pathway is altered in CQR P. falciparum, and that it may contribute along with mutations in PfCRT to confer the CQR(CC) phenotype.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gene expression DNA microarrays have been vital for characterizing whole-genome transcriptional profiles. Nevertheless, their effectiveness relies heavily on the accuracy of genome sequences, the ...annotation of gene structures, and the sequence-dependent performance of individual probes. Currently available gene expression arrays for the malaria parasite Plasmodium falciparum rely on an average of 2 probes per gene, usually positioned near the 3' end of genes; consequently, existing designs are prone to measurement bias and cannot capture complexities such as the occurrence of transcript isoforms arising from alternative splicing or alternative start/ stop sites. Here, we describe two novel gene expression arrays with exon-focused probes designed with an average of 12 and 20 probes spanning each gene. This high probe density minimizes signal noise inherent in probe-to-probe sequence-dependent hybridization intensity. We demonstrate that these exon arrays accurately profile genome-wide expression, comparing favorably to currently available arrays and RNA-seq profiling, and can detect alternatively spliced transcript isoforms as well as non-coding RNAs (ncRNAs). Of the 964 candidate alternate splicing events from published RNA-seq studies, 162 are confirmed using the exon array. Furthermore, the exon array predicted 330 previously unidentified alternate splicing events. Gene expression microarrays continue to offer a cost-effective alternative to RNA-seq for the simultaneous monitoring of gene expression and alternative splicing events. Microarrays may even be preferred in some cases due to their affordability and the rapid turn-around of results when hundreds of samples are required for fine-scale systems biology investigations, including the monitoring of the networks of gene co-expression in the emergence of drug resistance.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The determinants of transcriptional regulation in malaria parasites remain elusive. The presence of a well-characterized gene expression cascade shared by different Plasmodium falciparum strains ...could imply that transcriptional regulation and its natural variation do not contribute significantly to the evolution of parasite drug resistance. To clarify the role of transcriptional variation as a source of stain-specific diversity in the most deadly malaria species and to find genetic loci that dictate variations in gene expression, we examined genome-wide expression level polymorphisms (ELPs) in a genetic cross between phenotypically distinct parasite clones. Significant variation in gene expression is observed through direct co-hybridizations of RNA from different P. falciparum clones. Nearly 18% of genes were regulated by a significant expression quantitative trait locus. The genetic determinants of most of these ELPs resided in hotspots that are physically distant from their targets. The most prominent regulatory locus, influencing 269 transcripts, coincided with a Chromosome 5 amplification event carrying the drug resistance gene, pfmdr1, and 13 other genes. Drug selection pressure in the Dd2 parental clone lineage led not only to a copy number change in the pfmdr1 gene but also to an increased copy number of putative neighboring regulatory factors that, in turn, broadly influence the transcriptional network. Previously unrecognized transcriptional variation, controlled by polymorphic regulatory genes and possibly master regulators within large copy number variants, contributes to sweeping phenotypic evolution in drug-resistant malaria parasites.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum) genome is crucial for understanding its evolution; however the 81% (A+T) genome ...poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX) high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites.
We present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS) sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. De novo assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ≤ 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs) and many non crossover (NCO) gene conversions throughout the genome.
By sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Copy number is a major source of genome variation with important evolutionary implications. Consequently, it is essential to determine copy number variant (CNV) behavior, distributions and ...frequencies across genomes to understand their origins in both evolutionary and generational time frames. We use comparative genomic hybridization (CGH) microarray and the resolution provided by a segregating population of cloned progeny lines of the malaria parasite, Plasmodium falciparum, to identify and analyze the inheritance of 170 genome-wide CNVs.
We describe CNVs in progeny clones derived from both Mendelian (i.e. inherited) and non-Mendelian mechanisms. Forty-five CNVs were present in the parent lines and segregated in the progeny population. Furthermore, extensive variation that did not conform to strict Mendelian inheritance patterns was observed. 124 CNVs were called in one or more progeny but in neither parent: we observed CNVs in more than one progeny clone that were not identified in either parent, located more frequently in the telomeric-subtelomeric regions of chromosomes and singleton de novo CNVs distributed evenly throughout the genome. Linkage analysis of CNVs revealed dynamic copy number fluctuations and suggested mechanisms that could have generated them. Five of 12 previously identified expression quantitative trait loci (eQTL) hotspots coincide with CNVs, demonstrating the potential for broad influence of CNV on the transcriptional program and phenotypic variation.
CNVs are a significant source of segregating and de novo genome variation involving hundreds of genes. Examination of progeny genome segments provides a framework to assess the extent and possible origins of CNVs. This segregating genetic system reveals the breadth, distribution and dynamics of CNVs in a surprisingly plastic parasite genome, providing a new perspective on the sources of diversity in parasite populations.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
