scholarly journals Clinical long-read sequencing of the human mitochondrial genome for mitochondrial disease diagnostics

2019 ◽  
Author(s):  
Elizabeth Wood ◽  
Matthew D Parker ◽  
Mark J Dunning ◽  
Sirisha Hesketh ◽  
Dennis Wang ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
False Positives ◽  
Diagnostic Methods ◽  
Wilcoxon Test ◽  
Single Nucleotide Variants ◽  
Diagnostic Strategies ◽  
Sequencing Technologies ◽  
Large Deletions ◽  
Mtdna Deletions ◽  
Long Read

AbstractPurposeLong-read, third generation, sequencing technologies have the potential to improve current state of the art diagnostic strategies. In order to determine if long-read sequencing technologies are suitable for the diagnosis of mitochondrial disorders due to mitochondrial DNA (mtDNA) variants, particularly large deletions, we compared the performance of Oxford Nanopore Technologies (ONT) MinION to current diagnostic methods.MethodsWe sequenced mtDNA from nine patients with mtDNA deletion disorders and three normal controls with both ONT MinION and Illumina MiSeq. We applied a computational pipeline to estimate the positions of mtDNA deletions in patients, and subsequently validated the breakpoints using Sanger sequencing.ResultsWe were able to detect mtDNA deletions with a MinION workflow, successfully calling the disease causing event in all cases. Sequencing coverage was in most cases significantly more (p=0.03, Wilcoxon test) uniform with MinION than with MiSeq and subsequent correction of MinION reads improved breakpoint accuracy and reduced false positives. Although heteroplasmic single nucleotide variants are detectable, the high number of false positives and false negatives precludes their use in diagnostics at this time.ConclusionThe MinION is becoming an increasingly attractive diagnostic tool due to the reducing cost, increasing accuracy, and the speed at which data can be obtained.

scholarly journals Long read sequencing reveals Poxvirus evolution through rapid homogenization of gene arrays

2018 ◽  
Author(s):  
Thomas A. Sasani ◽  
Kelsey R. Cone ◽  
Aaron R. Quinlan ◽  
Nels C. Elde
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Single Nucleotide Variants ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Gene Copy Number Variation ◽  
Large Gene ◽  
Long Read ◽  
Number Variation

AbstractLarge DNA viruses rapidly evolve to defeat host defenses. Poxvirus adaptation can involve combinations of recombination-driven gene copy number variation and beneficial single nucleotide variants (SNVs) at the same locus, yet how these distinct mechanisms of genetic diversification might simultaneously facilitate adaptation to immune blocks is unknown. We performed experimental evolution with a vaccinia virus population harboring a SNV in a gene actively undergoing copy number amplification. Comparisons of virus genomes using the Oxford Nanopore Technologies sequencing platform allowed us to phase SNVs within large gene copy arrays for the first time, and uncovered a mechanism of adaptive SNV homogenization reminiscent of gene conversion, which is actively driven by selection. Our work reveals a new mechanism for the fluid gain of beneficial mutations in genetic regions undergoing active recombination in viruses, and illustrates the value of long read sequencing technologies for investigating complex genome dynamics in diverse biological systems.

scholarly journals 864. Whole Genome Sequencing is Unable to Track Candida auris Transmission

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S470-S471
Author(s):  
Scott C Roberts ◽  
Egon A Ozer ◽  
Teresa Zembower ◽  
Chao Qi
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Candida Auris ◽  
Healthcare Settings ◽  
Long Read ◽  
Outbreak Investigations ◽  
Phylogenetic Grouping

Abstract Background Candida auris (C. auris), an emerging yeast species, is often drug-resistant and has caused outbreaks in healthcare settings. Surging C. auris cases at our institution prompted whole genome sequencing (WGS) of patient and environmental specimens and comparison to local and international isolates. Methods WGS was performed on clinical and environmental isolates obtained from Northwestern Memorial Hospital (NMH) from June 2018 to December 2019. Genome sequences were compared against isolates from other institutions in the Chicagoland area obtained from a reference lab (ACL) and from the CDC. Two isolates underwent long-read sequencing on the Oxford Nanopore GridION platform to obtain closed genomes. WGS was performed on the remaining isolates with the Illumina MiSeq platform. Results Twenty isolates from NMH, five from ACL, and two from the CDC underwent WGS to yield 12.6 Mb genomes. Any two NMH isolates differed from each other by a maximum of 36 single nucleotide variants (SNV) (Figure 1). Two patients thought to be part of a transmission cluster (isolates CA06 and CA07), differed by 7 SNVs. No phylogenetic grouping between hospital systems across Chicagoland was observed. Isolates from room surfaces from a C. auris patient differed by 1-6 SNVs from each other and from 7-8 SNVs from the patient isolate. Samples taken from different body sites of another patient differed by 4-9 SNVs. Average SNV counts were lower among nosocomially acquired cases when compared to C. auris isolates present on admission (Figure 2). All NMH isolates were fluconazole sensitive, but a fluconazole resistant ACL isolate differed from a sensitive NMH isolate by only 4 SNVs. Figure 1: Phylogenetic tree of all NMH and ACL isolates with fluconazole sensitivities Figure 2: Observed pairwise SNP differences between nosocomial and POA strains Conclusion WGS of C. auris did not reveal identical isolates in any instance, even from the same patient or the patients and their environment. Generally, lower numbers of SNVs were observed for intra- versus inter-institutional isolates. More work is needed to determine the use, if any, of WGS in outbreak investigations. Disclosures All Authors: No reported disclosures

scholarly journals Long read sequencing reveals poxvirus evolution through rapid homogenization of gene arrays

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Thomas A Sasani ◽  
Kelsey R Cone ◽  
Aaron R Quinlan ◽  
Nels C Elde
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Single Nucleotide Variants ◽  
Sequencing Technologies ◽  
Gene Copy Number Variation ◽  
Large Gene ◽  
Oxford Nanopore ◽  
Long Read ◽  
Number Variation

Poxvirus adaptation can involve combinations of recombination-driven gene copy number variation and beneficial single nucleotide variants (SNVs) at the same loci. How these distinct mechanisms of genetic diversification might simultaneously facilitate adaptation to host immune defenses is unknown. We performed experimental evolution with vaccinia virus populations harboring a SNV in a gene actively undergoing copy number amplification. Using long sequencing reads from the Oxford Nanopore Technologies platform, we phased SNVs within large gene copy arrays for the first time. Our analysis uncovered a mechanism of adaptive SNV homogenization reminiscent of gene conversion, which is actively driven by selection. This study reveals a new mechanism for the fluid gain of beneficial mutations in genetic regions undergoing active recombination in viruses and illustrates the value of long read sequencing technologies for investigating complex genome dynamics in diverse biological systems.

scholarly journals Longshot enables accurate variant calling in diploid genomes from single-molecule long read sequencing

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Peter Edge ◽  
Vikas Bansal
Keyword(s):  
Single Molecule ◽  
Variant Calling ◽  
Small Scale ◽  
Whole Genome ◽  
Limited Information ◽  
Single Nucleotide Variants ◽  
Pacific Biosciences ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read

Abstract Whole-genome sequencing using sequencing technologies such as Illumina enables the accurate detection of small-scale variants but provides limited information about haplotypes and variants in repetitive regions of the human genome. Single-molecule sequencing (SMS) technologies such as Pacific Biosciences and Oxford Nanopore generate long reads that can potentially address the limitations of short-read sequencing. However, the high error rate of SMS reads makes it challenging to detect small-scale variants in diploid genomes. We introduce a variant calling method, Longshot, which leverages the haplotype information present in SMS reads to accurately detect and phase single-nucleotide variants (SNVs) in diploid genomes. We demonstrate that Longshot achieves very high accuracy for SNV detection using whole-genome Pacific Biosciences data, outperforms existing variant calling methods, and enables variant detection in duplicated regions of the genome that cannot be mapped using short reads.

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

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.

scholarly journals Targeted Transcriptome Analysis using Synthetic Long Read Sequencing Uncovers Isoform Reprograming in the Progression of Colon Cancer

2020 ◽  
Author(s):  
Silvia Liu ◽  
Indira Wu ◽  
Yan-Ping Yu ◽  
Michael Balamotis ◽  
Baoguo Ren ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Progression ◽  
Large Scale ◽  
Metastatic Cancer ◽  
Error Rates ◽  
Single Nucleotide Variants ◽  
Mrna Isoforms ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read

AbstractDiversity in human gene expression stems, to a large extent, from splicing exons into multiple mRNA isoforms. Characterization of isoforms requires accurate long-read sequencing. However, read lengths, high error rates, low throughput and large input requirements are some of the challenges that remain to be addressed in sequencing technologies.In this study, we used a barcoding-based synthetic long read (SLR) isoform sequencing approach, LoopSeq, to generate sequencing reads sufficiently long and accurate to identify isoforms using standard short read Illumina sequencers. The method identifies isoforms from control RNA samples with 99.4% accuracy and a 0.01% per-base error rate, exceeding the accuracy reported for other long-read sequencing technologies.Applied to targeted transcriptome sequencing of over 10,000 genes from colon cancers and their metastatic counterparts, LoopSeq revealed large scale isoform redistributions from benign colon mucosa to primary colon cancer and metastatic cancer and identified several novel gene fusion isoforms in the colon cancer samples. Strikingly, our data showed that most single nucleotide variants (SNV’s) occurred dominantly in specific isoforms and that some SNVs underwent isoform switching in cancer progression.The ability to use short read sequencers to generate accurate long-read isoform information as the raw unit of transcriptional information holds promise as a new and widely accessible approach in RNA isoform analyses.

scholarly journals Model research of the pig’s microbiome based on “One Health” concept in the light of the shared human and animal health

2021 ◽  
Vol 75 ◽  
pp. 297-303
Author(s):  
Marta Satora ◽  
Anna Rząsa ◽  
Krzysztof Rypuła ◽  
Katarzyna Płoneczka-Janeczko
Keyword(s):  
Health Status ◽  
Animal Health ◽  
Human Microbiome ◽  
Illumina Miseq ◽  
Diagnostic Methods ◽  
Diagnostic Tools ◽  
Personal Genome ◽  
Intestinal Microbes ◽  
Sequencing Technologies ◽  
The Impact

The human microbiome in terms of the number of bacteria exceeds the number of cells in the human body. It is defined as an additional “forgotten organ” and plays a key role in maintaining a high health status, which is conditioned by the maintenance of certain proportions and natural relations between bacteria and cells of the host organism. New diagnostic methods can enable profiling not only the human microbiome, but also livestock. An innovative analytical method, which is next generation sequencing (NGS), is increasingly used in the study of the microbiome. Many bacteria are referred to as “uncultivated” or “non-culturable” and metagenomics has played an important role in detecting these bacteria and has contributed to the development of new media for their cultivation. The main application of NGS in microbiology is to replace the conventional characterization of pathogens based on the assessment of morphology, staining properties and metabolic traits with their genome related characteristics. There are several platforms, i.e. “diagnostic tools”, that use a variety of DNA sequencing technologies, among others Ion Torrent Personal Genome Machine (PGM), Pacific Biosciences (PacBio) and Illumina MiSeq. In the case of swine microbiome, studies of the microbiome with the use of modern sequencing technologies seem to be particularly interesting in the aspect of the upcoming, inevitable changes in preventive and therapeutic procedures in animals. Analyses of this type integrate with the concept of the shared human and animal health and enable an in-depth assessment of the impact of specific factors on the population of intestinal microbes and learning how to “form” the composition of the microbiome in order to improve the quality of husbandry and to maintain the pig’s proper health status.

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

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.

scholarly journals Deep repeat resolution—the assembly of the Drosophila Histone Complex

2018 ◽  
Vol 47 (3) ◽  
pp. e18-e18 ◽  
Author(s):  
Philipp Bongartz ◽  
Siegfried Schloissnig
Keyword(s):  
De Novo ◽  
Machine Learning Algorithms ◽  
Single Nucleotide Variants ◽  
Major Step ◽  
Base Pairs ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Wide Range ◽  
Long Read ◽  
Genome Assemblies

Abstract Though the advent of long-read sequencing technologies has led to a leap in contiguity of de novo genome assemblies, current reference genomes of higher organisms still do not provide unbroken sequences of complete chromosomes. Despite reads in excess of 30 000 base pairs, there are still repetitive structures that cannot be resolved by current state-of-the-art assemblers. The most challenging of these structures are tandemly arrayed repeats, which occur in the genomes of all eukaryotes. Untangling tandem repeat clusters is exceptionally difficult, since the rare differences between repeat copies are obscured by the high error rate of long reads. Solving this problem would constitute a major step towards computing fully assembled genomes. Here, we demonstrate by example of the Drosophila Histone Complex that via machine learning algorithms, it is possible to exploit the underlying distinguishing patterns of single nucleotide variants of repeats from very noisy data to resolve a large and highly conserved repeat cluster. The ideas explored in this paper are a first step towards the automated assembly of complex repeat structures and promise to be applicable to a wide range of eukaryotic genomes.

scholarly journals Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

2021 ◽  
Author(s):  
Alexander Zee ◽  
Dori Zhi Qian Deng ◽  
Matthew Adams ◽  
Kayla D. Schimke ◽  
Russell Corbett-Detig ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Illumina Miseq ◽  
High Accuracy ◽  
Error Rates ◽  
Short Read ◽  
Rolling Circle ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Real Time Analysis ◽  
Long Read

High-throughput short-read sequencing has taken on a central role in research and diagnostics. Literally hundreds of different assays exist today to take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities, and the inertia associated with the research enterprise as a whole have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the long-read ONT MinION by circularizing and rolling circle amplifying the short library molecules using the R2C2 method. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into millions of high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, as well as regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow.

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