scholarly journals Analyzing Low-Level mtDNA Heteroplasmy—Pitfalls and Challenges from Bench to Benchmarking

2021 ◽  
Vol 22 (2) ◽  
pp. 935
Author(s):  
Federica Fazzini ◽  
Liane Fendt ◽  
Sebastian Schönherr ◽  
Lukas Forer ◽  
Bernd Schöpf ◽  
...  
Keyword(s):  
Variant Calling ◽  
Illumina Miseq ◽  
Bioinformatic Analysis ◽  
Low Level ◽  
Sequencing Technologies ◽  
Laboratory Setup ◽  
Dna Mixture ◽  
The Individual ◽  
Highly Sensitive Detection ◽  
Sensitivity Specificity

Massive parallel sequencing technologies are promising a highly sensitive detection of low-level mutations, especially in mitochondrial DNA (mtDNA) studies. However, processes from DNA extraction and library construction to bioinformatic analysis include several varying tasks. Further, there is no validated recommendation for the comprehensive procedure. In this study, we examined potential pitfalls on the sequencing results based on two-person mtDNA mixtures. Therefore, we compared three DNA polymerases, six different variant callers in five mixtures between 50% and 0.5% variant allele frequencies generated with two different amplification protocols. In total, 48 samples were sequenced on Illumina MiSeq. Low-level variant calling at the 1% variant level and below was performed by comparing trimming and PCR duplicate removal as well as six different variant callers. The results indicate that sensitivity, specificity, and precision highly depend on the investigated polymerase but also vary based on the analysis tools. Our data highlight the advantage of prior standardization and validation of the individual laboratory setup with a DNA mixture model. Finally, we provide an artificial heteroplasmy benchmark dataset that can help improve somatic variant callers or pipelines, which may be of great interest for research related to cancer and aging.

scholarly journals The application of Nanopore sequencing for variant calling on the human mitochondrial DNA

2021 ◽  
Vol 66 (2) ◽  
Author(s):  
Anton Shikov ◽  
Viktoriya Tsay ◽  
Mikhail Fedyakov ◽  
Yuri Eismont ◽  
Alena Rudnik ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
False Negative ◽  
Variant Calling ◽  
Illumina Miseq ◽  
Routine Practice ◽  
Jaccard Coefficient ◽  
High Coverage ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read

The emergence of long-read sequencing technologies has made a revolutionary step in genome biology and medicine. However, long reads are characterized by a relatively high error rate, impairing their usage for variant calling as a part of routine practice. Thus, we here examine different popular variant callers on long-read sequences of the human mitochondrial genome, convenient in terms of small size and easily obtained high coverage. The sequencing of mitochondrial DNA from 8 patients was conducted via Illumina (MiSeq) and the Oxford Nanopore platform (MinION), with the former utilized as a gold standard when evaluating variant calling’s accuracy. We used a conventional GATK3-BWA-based pipeline for paired-end reads and Guppy basecaller coupled with minimap2 for MinION data, respectively. We then compared the outputs of Clairvoyante, Nanopolish, GATK3, Longshot, DeepVariant, and Varscan tools applied on long-read alignments by analyzing false-positive and false-negative rates. While for most callers, raw signals represented false positives due to homopolymeric errors, Nanopolish demonstrated both high similarity (Jaccard coefficient of 0.82) and a comparable number of calls with the Illumina data (140 vs. 154) with the best performance according to AUC (area under ROC curve, 0.953) as well. In sum, our results, despite being obtained from a small dataset, provide evidence that sufficient coverage coupled with an optimal pipeline could make long reads of mitochondrial DNA applicable for variant calling.

Chlorinated Organics in Nearshore Waters and Tributaries of the St. Clair River

1986 ◽  
Vol 21 (3) ◽  
pp. 344-350 ◽  
Author(s):  
Barry G. Oliver ◽  
Klaus L.E. Kaiser
Keyword(s):  
United States ◽  
Suspended Sediment ◽  
Waste Disposal ◽  
Large Volume ◽  
Water Samples ◽  
Chemical Properties ◽  
Low Level ◽  
Chemical Company ◽  
Physical Chemical ◽  
The Individual

Abstract The concent rat ions of hexachloroethane (HCE), hexachlorobutadiene (HCBD), pentachlorobenzene (QCB), hexachlorobenzene (HCB) and octachlorostyrene (OCS) in large volume water samples show that the major sources of these chemicals to the St. Clair River are Dow Chemical Company effluents and, to a lesser degree, Sarnia’s Township ditch which drains one of Dow’s waste disposal sites. Tributaries entering the river on both sides of the Canada/United States border contain measurable concentrations of these chemicals indicating low level contamination throughout the area. The degree of water/suspended sediment partitioning of the chemicals (Kp) was studied. Kp values for the individual chemicals changed in a manner consistent with changes in their physical-chemical properties.

scholarly journals PrimerPooler: automated primer pooling to prepare library for targeted sequencing

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Silas S. Brown ◽  
Yun-Wen Chen ◽  
Ming Wang ◽  
Alexandra Clipson ◽  
Eguzkine Ochoa ◽  
...  
Keyword(s):  
Variant Calling ◽  
Pcr Amplification ◽  
Illumina Miseq ◽  
Degenerate Primers ◽  
Cross Hybridization ◽  
Targeted Next Generation Sequencing ◽  
Target Sequencing ◽  
A Genome ◽  
Experimental Protocols ◽  
Minimal Depth

Abstract Targeted next-generation sequencing based on PCR amplification involves pooling of hundreds to thousands of primers, for preamplification and subsequent parallel single/multiplex PCR. It is often necessary to allocate the set of primers into subpools, a common issue being potential cross-hybridization. For smaller numbers of primers, pool division can be done manually using trial and error to minimize potential hybridization, but this becomes inefficient and time consuming with increasing numbers of primer pairs. We developed PrimerPooler that automates swapping of primer pairs between any user-defined number of subpools to obtain combinations with low-potential interactions. PrimerPooler performs inter-/intra-primer hybridization analysis to identify the adverse interactions, as well as simultaneous mapping of all primers onto a genome sequence in a single run without requiring a prior index of the genome. This allows detection of overlapping primer pairs and allocation of these primer pairs into separate subpools where tiling approaches are used. Using PrimerPooler, 1153 primer pairs were assigned to three preamplification pools (388, 389 and 376 primer pairs each), then 144 subpools of six- to nine-plex PCR for Fluidigm Access Array PCR, followed by Illumina MiSeq sequencing. With optimized experimental protocols, an average of 3269 reads was achieved for the targeted regions, with 95% of targets covered by at least 50 reads, the minimal depth of reads for confident variant calling. PrimerPooler provides a fast and highly efficient stratification of primer pairs for targeted enrichment, thus ensuring representative amplification of the targeted sequences. PrimerPooler is also able to analyse degenerate primers, and is thus also useful for microbiological identification and related target sequencing.

scholarly journals RECORD: Reference-Assisted Genome Assembly for Closely Related Genomes

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Krisztian Buza ◽  
Bartek Wilczynski ◽  
Norbert Dojer
Keyword(s):  
Reference Genome ◽  
De Novo ◽  
Real Data ◽  
Reference Sequence ◽  
Individual Genome ◽  
Single Experiment ◽  
Sequencing Technologies ◽  
Sequencing Cost ◽  
The Individual ◽  
Assembly Software

Background. Next-generation sequencing technologies are now producing multiple times the genome size in total reads from a single experiment. This is enough information to reconstruct at least some of the differences between the individual genome studied in the experiment and the reference genome of the species. However, in most typical protocols, this information is disregarded and the reference genome is used.Results. We provide a new approach that allows researchers to reconstruct genomes very closely related to the reference genome (e.g., mutants of the same species) directly from the reads used in the experiment. Our approach applies de novo assembly software to experimental reads and so-called pseudoreads and uses the resulting contigs to generate a modified reference sequence. In this way, it can very quickly, and at no additional sequencing cost, generate new, modified reference sequence that is closer to the actual sequenced genome and has a full coverage. In this paper, we describe our approach and test its implementation called RECORD. We evaluate RECORD on both simulated and real data. We made our software publicly available on sourceforge.Conclusion. Our tests show that on closely related sequences RECORD outperforms more general assisted-assembly software.

scholarly journals Analysis of Carbohydrate Metabolism Genes of Spongospora subterranea Using 454 Pyrosequencing

2014 ◽  
Vol 67 (2) ◽  
pp. 7247-7260 ◽  
Author(s):  
Pablo Andrés Gutiérrez Sánchez ◽  
Juan Fernando Alzate ◽  
Mauricio Marín Montoya
Keyword(s):  
Dna Sequences ◽  
454 Pyrosequencing ◽  
Plant Pathogens ◽  
Plasmodiophora Brassicae ◽  
Bioinformatic Analysis ◽  
Spongospora Subterranea ◽  
Sequencing Technologies ◽  
Intron Structure ◽  
Basic Biology

Spongospora subterranea, the causal agent of Potato powdery scab, is an important soil-borne obligate protozoan commonly found in Andean soils. This is a serious problem that causes cosmetic damage on the skin of tubers and induces root gall formation, diminishing the yield and commercial value of the potato. Genetic studies on S. subterranea are difficult due to its obligate parasitism, which explains the lack of available knowledge on its basic biology. S. subterranea is a member of the Plasmodiophorida order, a protist taxa that includes other important plant pathogens such as Plasmodiophora brassicae and Spongospora nasturtii. Little is known about the genomes of Plasmodiophorida; however, with the use of Next-Generation Sequencing technologies combined with appropriate bioinformatic techniques, it is possible to obtain genomic sequences from obligate pathogens such as S. subterranea. To gain a better understanding of the biology of this pathogen and Plasmodiophorida in general, DNA sequences from a cystosori-enriched sample of S. subterranea were obtained using 454 pyrosequencing technology. As a first step in understanding the nutritional requirements of S. subterranea as well as its infective and resistance structures, we present a bioinformatic analysis of 24 contigs related to genes involved in the glycolysis, starch, celullose and chitin metabolism. Intron structure and codon usage is also discussed. The genes analyzed in this study are a good source of information for studies aimed at characterizing these enzymes in vitro, as well as the generation of new methods for the molecular detection of S. subterranea in either soils or infected plants.

scholarly journals The Impact of Dietary Grape Seed Meal on Healthy and Aflatoxin B1 Afflicted Microbiota of Pigs after Weaning

Toxins ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 25 ◽  
Author(s):  
Iulian A. Grosu ◽  
Gina C. Pistol ◽  
Ionelia Taranu ◽  
Daniela E. Marin
Keyword(s):  
Large Intestine ◽  
Relative Abundance ◽  
Aflatoxin B1 ◽  
Illumina Miseq ◽  
Grape Seed ◽  
Antagonistic Effect ◽  
Seed Meal ◽  
Control Group ◽  
The Individual ◽  
The Impact

The study investigated the effect of grape seed (GS) meal, aflatoxin (AFB1), or their combination on the large intestine microbiota of weanling piglets. Twenty-four piglets were allocated into four groups based on diet composition: (1) Control group; (2) AFB1 (320 g/kg feed) group; (3) GS group (8% inclusion in the diet); (4) AFB1 + GS group. After 30 days of experiment, the colon content was used for microbiota analyses; after isolation of total bacterial genomic DNA, V3/V4 regions of the 16S rRNA amplicons were sequenced using the Illumina MiSeq platform. The raw sequences were analyzed using the v.1.9.1 QIIME pipeline software. 157 numbers of OTUs were identified among all four dietary groups with 26 of them being prevalent above 0.05% in the total relative abundance. GS and AFB1 increase the relative abundance of phylum Bacteroidetes and Proteobacteria, while decreasing the Firmicutes abundance in a synergic manner as compared with the individual treatments. An additive or synergistic action of the two treatments was identified for Lactobacillus, Prevotella and Campylobacter, while rather an antagonistic effect was observed on Lachnospira. The action mechanisms of aflatoxin B1 and grape seed meal that drive the large intestine microbiota to these changes are not known and need further investigations.

scholarly journals Evaluation of MT Family Isoforms as Potential Biomarker for Predicting Progression and Prognosis in Gastric Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-15
Author(s):  
Mingfu Tong ◽  
Wenquan Lu ◽  
Hao Liu ◽  
Jian Wu ◽  
Mingzuo Jiang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Mrna Expression ◽  
Promoter Region ◽  
Gene Promoter ◽  
Distinct Type ◽  
Bioinformatic Analysis ◽  
Kaplan Meier ◽  
Potential Biomarker ◽  
Wide Range ◽  
The Individual

Background. Metallothioneins (MTs) family comprises many isoforms, most of which are frequently dysregulated in a wide range of cancers. However, the expression pattern and exact role of each distinct MT family isoform which contributes to tumorigenesis, progression, and drug resistance of gastric cancer (GC) are still unclear. Methods. Publicly available databases including Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan-Meier plotter, SurvExpress, MethHC, cBioportal, and GeneMANIA were accessed to perform an integrated bioinformatic analysis and try to detect fundamental relationships between each MT family member and GC. Results. Bioinformatic data indicated that the mRNA expression of all MT family members was almost lowly expressed in GC compared with normal gastric tissue (P<0.05), and patients with reduced mRNA expression of each individual MT member had inconsistent prognostic value (OS, FP, PPS), which depended on the individual isoform of MT. A negative correlation between the methylation in promoter region of majority of MT members and their mRNA expression was detected from MethHC database (p<0.001). Data downloaded from TCGA revealed that MTs were rarely mutated in GC patients and MT2A was frequently regulated by other three genes (FOS, JUN, SP1) in GC patients. Conclusion. MTs were nearly downregulated, and distinct type of MT harbored different prognostic role in GC patients. Methylation in gene promoter region of MTs partially contributed to their reduced expression in GC. Our comprehensive analyses from multiple independent databases may further lead researches to explore MT-targeting reagents or potential diagnostic and prognostic markers for GC patients.

Sorption Kinetics of Uranium-238, Neptunium-237, Caesium-134, and Strontium-85 on a Glacial Deposit

1997 ◽  
Vol 506 ◽  
Author(s):  
L.N. Moyes ◽  
D.J. Bunker ◽  
J.T. Smith ◽  
F.R. Livens ◽  
C.R. Hughes ◽  
...  
Keyword(s):  
Waste Disposal ◽  
Kinetic Models ◽  
Sorption Kinetics ◽  
Kinetic Control ◽  
Glacial Deposit ◽  
Sorption Behaviour ◽  
Low Level ◽  
The Individual ◽  
Kinetics Of ◽  
Uptake Mechanisms

ABSTRACTBatch sorption experiments have been used to assess the sorption behaviour of four radionuclides, important in the context of low-level waste disposal, on a glacial substrate. Data for sorption of 238U, 237Np, 134Cs and 85Sr are compared and agree well with independent studies. A series of well-established kinetic models have been used to describe the individual uptake mechanisms and rate parameters reported. Sorption occurs via both equilibrium and kinetically controlled pathways, with neptunium sorption being under kinetic control to the greatest extent.

scholarly journals X-in-the-Loop Testing of a Thermal Management System Intended for an Electric Vehicle with In-Wheel Motors

Energies ◽  
2020 ◽  
Vol 13 (23) ◽  
pp. 6452
Author(s):  
Ilya Kulikov ◽  
Kirill Karpukhin ◽  
Rinat Kurmaev
Keyword(s):  
Electric Vehicle ◽  
Thermal Management ◽  
Electric Drive ◽  
Management System ◽  
Driving Cycle ◽  
Vehicle Speed ◽  
Laboratory Setup ◽  
Thermal Management System ◽  
Physical Part ◽  
The Individual

The article describes an elaboration of the X-in-the-loop (XiL) testing environment for a thermal management system (TMS) intended for the traction electric drive of an electric vehicle, which has each of its wheels driven by an in-wheel motor. The TMS features the individual thermal regulation of each electric drive using a hydraulic layout with parallel pipelines and electrohydraulic pumps embedded into them. The XiL system is intended as a tool for studying and developing the TMS design and controls. It consists of the virtual part and the physical part. The former simulates the vehicle operating in a driving cycle with the heat power dissipated by the electric drive components, which entails the change in their temperature regimes. The physical part includes the TMS itself consisting of a radiator, pipelines, and pumps. The physical part also features devices intended for simulation of the electric drive components in terms of their thermal and hydraulic behaviors, as well as devices that simulate airflow induced by the vehicle motion. Bilateral, real-time interactions are established between the two said parts combining them into a cohesive system, which models the studied electric vehicle and its components. The article gives a description of a laboratory setup, which implements the XiL environment including the mathematical models, hardware devices, as well as the control loops that establish the interaction of those components. An example of using this system in a driving cycle test shows the interaction between its parts and operation of the TMS in conditions simulated in both virtual and physical domains. The results constitute calculated and measured quantities including vehicle speed, operating parameters of the electric drives, coolant and air flow rates, and temperatures of the system components.

scholarly journals Correlations between microsatellite instability and the biological behaviour of tumours

2019 ◽  
Vol 145 (12) ◽  
pp. 2891-2899 ◽  
Author(s):  
Guang Yang ◽  
Ru-yi Zheng ◽  
Zai-shun Jin
Keyword(s):  
Microsatellite Instability ◽  
Relevant Literature ◽  
Treatment Strategies ◽  
Repair System ◽  
Biological Behaviour ◽  
Low Level ◽  
Coding Regions ◽  
The Individual ◽  
High Level ◽  
Frameshift Peptides

Abstract Purpose Microsatellites are widely distributed repetitive DNA motifs, accounting for approximately 3% of the genome. Due to mismatch repair system deficiency, insertion or deletion of repetitive units often occurs, leading to microsatellite instability. In this review, we aimed to explore the relationship between MSI and biological behaviour of colorectal carcinoma, gastric carcinoma, lymphoma/leukaemia and endometrial carcinoma, as well as the application of frameshift peptide vaccines in cancer therapy. Methods The relevant literature from PubMed and Baidu Xueshu were reviewed in this article. The ClinicalTrials.gov database was searched for clinical trials related to the specific topic. Results Microsatellite instability is divided into three subtypes: high-level, low-level microsatellite instability, and stable microsatellites. The majority of tumour patients with high-level microsatellite instability often show a better efficacy and prognosis than those with low-level microsatellite instability or stable microsatellites. In coding regions, especially for genes involved in tumourigenesis, microsatellite instability often results in inactivation of proteins and contributes to tumourigenesis. Moreover, the occurrence of microsatellite instability in coding regions can also cause the generation of frameshift peptides that are thought to be unknown and novel to the individual immune system. Thus, these frameshift peptides have the potential to be biomarkers to raise tumour-specific immune responses. Conclusion MSI has the potential to become a key predictor for evaluating the degree of malignancy, efficacy and prognosis of tumours. Clinically, MSI patterns will provide more valuable information for clinicians to create optimal individualized treatment strategies based on frameshift peptides vaccines.

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