Genomic Analysis of Human Noroviruses Using Hybrid Illumina-Nanopore Data

Whole genome sequence (WGS) analysis of noroviruses is routinely performed by employing a metagenomic approach. While this methodology has several advantages, such as allowing for examination of co-infection, it has some limitations such as the requirement of high viral load to achieve full-length or near full-length genomic sequences. In this study, we used an amplification approach to obtain full-length genomic amplicons from 39 Canadian GII isolates followed by deep sequencing on Illumina and Oxford Nanopore platforms. This approach significantly reduced the required viral titre to obtain full-genome coverage. Herein, we compared the coverage and sequences obtained by both platforms and provided an in-depth genomic analysis of the obtained sequences, including the presence of single nucleotide variants (SNVs) and recombination events.

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Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

10.1101/2021.12.14.472689 ◽

2021 ◽

Author(s):

Zachary L Skidmore ◽

Jason Kunisaki ◽

Yiing Lin ◽

Kelsy C Cotto ◽

Erica K Barnell ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Copy Number Variants ◽

Genomic Analysis ◽

Structural Variants ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Tert Promoter Mutations ◽

Somatic Alterations ◽

Differential Gene ◽

Promoter Mutations

Background: Liver cancer is the second leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) risk factors include chronic hepatitis, cirrhosis, and alcohol abuse, whereby tumorigenesis is induced through inflammation and subsequent fibrotic response. However, a subset of HCC arises in non-cirrhotic livers. We characterized the genomic and transcriptomic landscape of non-cirrhotic HCC to identify features underlying the disease's development and progression. Methods: Whole genome and transcriptome sequencing was performed on 30 surgically resectable tumors comprised of primarily of non-cirrhotic HCC and adjacent normal tissue. Using somatic variants, capture reagents were created and employed on an additional 87 cases of mixed cirrhotic/non-cirrhotic HCC. Cases were analyzed to identify viral integrations, single nucleotide variants (SNVs), insertions and deletions (INDELS), copy number variants, loss of heterozygosity, gene fusions, structural variants, and differential gene expression. Results: We detected 3,750 SNVs/INDELS and extensive CNVs and expression changes. Recurrent TERT promoter mutations occurred in >52% of non-cirrhotic discovery samples. Frequently mutated genes included TP53, CTNNB1, and APOB. Cytochrome P450 mediated metabolism was significantly downregulated. Structural variants were observed at MACROD2, WDPCP, and NCKAP5 in >20% of samples. Furthermore, NR1H4 fusions involving gene partners EWSR1, GNPTAB, and FNIP1 were detected and validated in 2 non-cirrhotic samples. Conclusion: Genomic analysis can elucidate mechanisms that may contribute to non-cirrhotic HCC tumorigenesis. The comparable mutational landscape between cirrhotic and non-cirrhotic HCC supports previous work suggesting a convergence at the genomic level during disease progression. It is therefore possible genomic-based treatments can be applied to both HCC subtypes with progressed disease.

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Whole genome sequencing of orofacial cleft trios from the Gabriella Miller Kids First Pediatric Research Consortium identifies a new locus on chromosome 21

10.1101/743526 ◽

2019 ◽

Author(s):

Nandita Mukhopadhyay ◽

Madison Bishop ◽

Michael Mortillo ◽

Pankaj Chopra ◽

Jacqueline B. Hetmanski ◽

...

Keyword(s):

Birth Defects ◽

Large Scale ◽

Pediatric Research ◽

Chromosome 21 ◽

Whole Genome Sequence ◽

Whole Genome ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Family Based ◽

Kids First

AbstractOrofacial clefts (OFCs) are one of the most common birth defects worldwide and create a significant health burden. The majority of OFCs are non-syndromic, and the genetic component has been only partially determined. Here, we analyze whole genome sequence (WGS) data for association with risk of OFCs in European and Colombian families selected from a multicenter family-based OFC study. Part of the Gabriella Miller Kids First Pediatric Research Program, this is the first large-scale WGS study of OFC in parent-offspring trios. WGS provides deeper and more specific genetic data than currently available using imputation on single nucleotide polymorphic (SNP) marker panels. Here, association analysis of genome-wide single nucleotide variants (SNV) and short insertions and deletions (indels) identified a new locus on chromosome 21 in Colombian families, within a region known to be expressed during craniofacial development. This study reinforces the ancestry differences seen in the genetic etiology of OFCs, and the need for larger samples when for studying OFCs and other birth defects in admixed populations.

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Comparative genomic analysis of the principal Cryptosporidium species that infect humans

PeerJ ◽

10.7717/peerj.10478 ◽

2020 ◽

Vol 8 ◽

pp. e10478

Author(s):

Laura M. Arias-Agudelo ◽

Gisela Garcia-Montoya ◽

Felipe Cabarcas ◽

Ana L. Galvan-Diaz ◽

Juan F. Alzate

Keyword(s):

Genomic Analysis ◽

Comparative Genomic Analysis ◽

Comparative Genomic ◽

Single Nucleotide Variants ◽

Nucleotide Substitutions ◽

Genomic Comparison ◽

Single Nucleotide ◽

Associated Proteins ◽

Species Specific ◽

Ngs Data

Cryptosporidium parasites are ubiquitous and can infect a broad range of vertebrates and are considered the most frequent protozoa associated with waterborne parasitic outbreaks. The intestine is the target of three of the species most frequently found in humans: C. hominis, C. parvum, and. C. meleagridis. Despite the recent advance in genome sequencing projects for this apicomplexan, a broad genomic comparison including the three species most prevalent in humans have not been published so far. In this work, we downloaded raw NGS data, assembled it under normalized conditions, and compared 23 publicly available genomes of C. hominis, C. parvum, and C. meleagridis. Although few genomes showed highly fragmented assemblies, most of them had less than 500 scaffolds and mean coverage that ranged between 35X and 511X. Synonymous single nucleotide variants were the most common in C. hominis and C. meleagridis, while in C. parvum, they accounted for around 50% of the SNV observed. Furthermore, deleterious nucleotide substitutions common to all three species were more common in genes associated with DNA repair, recombination, and chromosome-associated proteins. Indel events were observed in the 23 studied isolates that spanned up to 500 bases. The highest number of deletions was observed in C. meleagridis, followed by C. hominis, with more than 60 species-specific deletions found in some isolates of these two species. Although several genes with indel events have been partially annotated, most of them remain to encode uncharacterized proteins.

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A fully phased accurate assembly of an individual human genome

10.1101/855049 ◽

2019 ◽

Cited By ~ 19

Author(s):

David Porubsky ◽

Peter Ebert ◽

Peter A. Audano ◽

Mitchell R. Vollger ◽

William T. Harvey ◽

...

Keyword(s):

Genome Assembly ◽

De Novo ◽

Consensus Sequence ◽

Error Rates ◽

Single Individual ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Consensus Sequences ◽

Oxford Nanopore ◽

Genome Assemblies

The prevailing genome assembly paradigm is to produce consensus sequences that “collapse” parental haplotypes into a consensus sequence. Here, we leverage the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing (Strand-seq)1,2 and combine them with high-fidelity (HiFi) long sequencing reads3, in a novel reference-free workflow for diploid de novo genome assembly. Employing this strategy, we produce completely phased de novo genome assemblies separately for each haplotype of a single individual of Puerto Rican origin (HG00733) in the absence of parental data. The assemblies are accurate (QV > 40), highly contiguous (contig N50 > 25 Mbp) with low switch error rates (0.4%) providing fully phased single-nucleotide variants (SNVs), indels, and structural variants (SVs). A comparison of Oxford Nanopore and PacBio phased assemblies identifies 150 regions that are preferential sites of contig breaks irrespective of sequencing technology or phasing algorithms.

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Analysis of cell-free circulating tumor DNA in 419 patients with glioblastoma and other primary brain tumors

CNS Oncology ◽

10.2217/cns-2018-0015 ◽

2019 ◽

Vol 8 (2) ◽

pp. CNS34 ◽

Cited By ~ 26

Author(s):

David E Piccioni ◽

Achal Singh Achrol ◽

Lesli A Kiedrowski ◽

Kimberly C Banks ◽

Najee Boucher ◽

...

Keyword(s):

Brain Tumors ◽

Genomic Analysis ◽

Circulating Tumor Dna ◽

Single Nucleotide Variants ◽

Viable Option ◽

Single Nucleotide ◽

Primary Brain Tumors ◽

Somatic Alteration ◽

Tumor Sequencing ◽

Tumor Dna

Aim: Genomically matched trials in primary brain tumors (PBTs) require recent tumor sequencing. We evaluated whether circulating tumor DNA (ctDNA) could facilitate genomic interrogation in these patients. Methods: Data from 419 PBT patients tested clinically with a ctDNA NGS panel at a CLIA-certified laboratory were analyzed. Results: A total of 211 patients (50%) had ≥1 somatic alteration detected. Detection was highest in meningioma (59%) and gliobastoma (55%). Single nucleotide variants were detected in 61 genes, with amplifications detected in ERBB2, MET, EGFR and others. Conclusion: Contrary to previous studies with very low yields, we found half of PBT patients had detectable ctDNA with genomically targetable off-label or clinical trial options for almost 50%. For those PBT patients with detectable ctDNA, plasma cfDNA genomic analysis is a clinically viable option for identifying genomically driven therapy options.

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Comparison of single-nucleotide variants identified by Illumina and Oxford Nanopore technologies in the context of a potential outbreak of Shiga toxin–producing Escherichia coli

GigaScience ◽

10.1093/gigascience/giz104 ◽

2019 ◽

Vol 8 (8) ◽

Cited By ~ 8

Author(s):

David R Greig ◽

Claire Jenkins ◽

Saheer Gharbia ◽

Timothy J Dallman

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Variant Calling ◽

Reference Database ◽

Sequencing Data ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Oxford Nanopore ◽

Variant Filtering ◽

Oxford Nanopore Technologies

Abstract Background We aimed to compare Illumina and Oxford Nanopore Technology sequencing data from the 2 isolates of Shiga toxin–producing Escherichia coli (STEC) O157:H7 to determine whether concordant single-nucleotide variants were identified and whether inference of relatedness was consistent with the 2 technologies. Results For the Illumina workflow, the time from DNA extraction to availability of results was ∼40 hours, whereas with the ONT workflow serotyping and Shiga toxin subtyping variant identification were available within 7 hours. After optimization of the ONT variant filtering, on average 95% of the discrepant positions between the technologies were accounted for by methylated positions found in the described 5-methylcytosine motif sequences, CC(A/T)GG. Of the few discrepant variants (6 and 7 difference for the 2 isolates) identified by the 2 technologies, it is likely that both methodologies contain false calls. Conclusions Despite these discrepancies, Illumina and Oxford Nanopore Technology sequences from the same case were placed on the same phylogenetic location against a dense reference database of STEC O157:H7 genomes sequenced using the Illumina workflow. Robust single-nucleotide polymorphism typing using MinION-based variant calling is possible, and we provide evidence that the 2 technologies can be used interchangeably to type STEC O157:H7 in a public health setting.

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Detecting and phasing minor single-nucleotide variants from long-read sequencing data

Nature Communications ◽

10.1038/s41467-021-23289-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhixing Feng ◽

Jose C. Clemente ◽

Brandon Wong ◽

Eric E. Schadt

Keyword(s):

Genetic Heterogeneity ◽

Error Rates ◽

Metagenomic Data ◽

Sequencing Data ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

Technological Limitations

AbstractCellular genetic heterogeneity is common in many biological conditions including cancer, microbiome, and co-infection of multiple pathogens. Detecting and phasing minor variants play an instrumental role in deciphering cellular genetic heterogeneity, but they are still difficult tasks because of technological limitations. Recently, long-read sequencing technologies, including those by Pacific Biosciences and Oxford Nanopore, provide an opportunity to tackle these challenges. However, high error rates make it difficult to take full advantage of these technologies. To fill this gap, we introduce iGDA, an open-source tool that can accurately detect and phase minor single-nucleotide variants (SNVs), whose frequencies are as low as 0.2%, from raw long-read sequencing data. We also demonstrate that iGDA can accurately reconstruct haplotypes in closely related strains of the same species (divergence ≥0.011%) from long-read metagenomic data.

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Detecting and phasing minor single-nucleotide variants from long-read sequencing data

10.1101/2020.09.25.314252 ◽

2020 ◽

Author(s):

Zhixing Feng ◽

Jose Clemente ◽

Brandon Wong ◽

Eric E. Schadt

Keyword(s):

Genetic Heterogeneity ◽

Error Rates ◽

Metagenomic Data ◽

Significant Advance ◽

Sequencing Data ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read

AbstractCellular genetic heterogeneity is common in many biological conditions including cancer, microbiome, co-infection of multiple pathogens. Detecting and phasing minor variants, which is to determine whether multiple variants are from the same haplotype, play an instrumental role in deciphering cellular genetic heterogeneity, but are still difficult because of technological limitations. Recently, long-read sequencing technologies, including those by Pacific Biosciences and Oxford Nanopore, have provided an unprecedented opportunity to tackle these challenges. However, high error rates make it difficult to take full advantage of these technologies. To fill this gap, we introduce iGDA, an open-source tool that can accurately detect and phase minor single-nucleotide variants (SNVs), whose frequencies are as low as 0.2%, from raw long-read sequencing data. We also demonstrated that iGDA can accurately reconstruct haplotypes in closely-related strains of the same species (divergence ≥ 0.011%) from long-read metagenomic data. Our approach, therefore, presents a significant advance towards the complete deciphering of cellular genetic heterogeneity.

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MRSA Transmission in Intensive Care Units: Genomic Analysis of Patients, Their Environments, and Healthcare Workers

Clinical Infectious Diseases ◽

10.1093/cid/ciaa731 ◽

2020 ◽

Author(s):

Kyle J Popovich ◽

Stefan J Green ◽

Koh Okamoto ◽

Yoona Rhee ◽

Mary K Hayden ◽

...

Keyword(s):

Intensive Care ◽

Healthcare Workers ◽

Genomic Analysis ◽

Contact Tracing ◽

Patient Encounter ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Contact Precautions ◽

Cross Transmission ◽

Genomic Clusters

Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA)—and now USA300 MRSA—is a significant intensive care unit (ICU) pathogen; healthcare worker (HCW) contamination may lead to patient cross-transmission. Methods From September 2015 to February 2016, to study the spread of MRSA, we enrolled HCWs in 4 adult ICUs caring for patients on MRSA contact precautions. Samples were collected from patient body sites and high-touch surfaces in patient rooms. HCW hands, gloves, and personal protective equipment were sampled pre/post-patient encounter. Whole genome sequencing (WGS) was used to compare isolates from patients, HCWs, and environment. Results There were 413 MRSA isolates sequenced (38% USA300, 52% USA100) from 66 patient encounters. Six of 66 HCWs were contaminated with MRSA prior to room entry. Isolates from a single patient encounter were typically either USA100 or USA300; in 8 (12%) encounters both USA300 and USA100 were isolated. WGS demonstrated that isolates from patients, HCWs, and environment often were genetically similar, although there was substantial between-encounter diversity. Strikingly, there were 5 USA100 and 1 USA300 clusters that contained similar strains (<22 single-nucleotide variants [SNVs], with most <10 SNVs) within the cluster despite coming from different encounters, suggesting intra- and inter-ICU spread of strains, that is, 4 of these genomic clusters were from encounters in the same ICU; 5 of 6 clusters occurred within 1 week. Conclusions We demonstrated frequent spread of MRSA USA300 and USA100 strains among patients, environment, and HCWs. WGS identified possible spread within and even between ICUs. Future analysis with detailed contact tracing in conjunction with genomic data may further elucidate pathways of MRSA spread and points for intervention.

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Pedigree-based estimation of human mobile element retrotransposition rates

10.1101/506691 ◽

2018 ◽

Cited By ~ 1

Author(s):

Julie Feusier ◽

W. Scott Watkins ◽

Jainy Thomas ◽

Andrew Farrell ◽

David J. Witherspoon ◽

...

Keyword(s):

De Novo ◽

Phylogenetic Analyses ◽

Mobile Element ◽

Whole Genome Sequence ◽

Structural Variants ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Alu Elements ◽

Depth Analysis ◽

Parent Of Origin

AbstractGermline mutation rates in humans have been estimated for a variety of mutation types, including single nucleotide and large structural variants. Here we directly measure the germline retrotransposition rate for the three active retrotransposon elements: L1, Alu, and SVA. We utilized three tools for calling Mobile Element Insertions (MEIs) (MELT, RUFUS, and TranSurVeyor) on blood-derived whole genome sequence (WGS) data from 603 CEPH individuals, comprising 33 three-generation pedigrees. We identified 27 de novo MEIs in 440 births. The retrotransposition rate estimates for Alu elements, one in 40, is roughly half the rate estimated using phylogenetic analyses, a difference in magnitude similar to that observed for single nucleotide variants. The L1 retrotransposition rate is one in 62 births and is within range of previous estimates (1:20-1:200 births). The SVA retrotransposition rate, one in 55 births, is much higher than the previous estimate of one in 900 births. Our large, three-generation pedigrees allowed us to assess parent-of-origin effects and the timing of insertion events in either gametogenesis or early embryonic development. We find a statistically significant paternal bias in Alu retrotransposition. Our study represents the first in-depth analysis of the rate and dynamics of human retrotransposition from WGS data in three-generation human pedigrees.

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