scholarly journals Evaluation of a custom QIAseq targeted DNA panel with 164 ancestry informative markers sequenced with the Illumina MiSeq

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
D. Truelsen ◽  
A. Freire-Aradas ◽  
M. Nazari ◽  
A. Aliferi ◽  
D. Ballard ◽  
...  
Keyword(s):  
Middle Eastern ◽  
Illumina Miseq ◽  
Poor Quality ◽  
Ancestry Informative Markers ◽  
Sequencing Platform ◽  
Overall Performance ◽  
Dna Profiles ◽  
Drop Outs ◽  
European Populations ◽  
Forensic Genetic

AbstractIntroduction of new methods requires meticulous evaluation before they can be applied to forensic genetic case work. Here, a custom QIAseq Targeted DNA panel with 164 ancestry informative markers was assessed using the MiSeq sequencing platform. Concordance, sensitivity, and the capability for analysis of mixtures were tested. The assay gave reproducible and nearly concordant results with an input of 10 and 2 ng DNA. Lower DNA input led to an increase in both locus and allele drop-outs, and a higher variation in heterozygote balance. Locus or allele drop-outs in the samples with less than 2 ng DNA input were not necessarily associated with the overall performance of a locus. Thus, the QIAseq assay will be difficult to implement in a forensic genetic setting where the sample material is often scarce and of poor quality. With equal or near equal mixture ratios, the mixture DNA profiles were easily identified by an increased number of imbalanced heterozygotes. For more skewed mixture ratios, the mixture DNA profiles were identified by an increased noise level. Lastly, individuals from Great Britain and the Middle East were investigated. The Middle Eastern individuals showed a greater affinity with South European populations compared to North European populations.

Genome sequence of Aspergillus flavus A7, a marine-derived fungus with antibacterial activity

Genome ◽  
2020 ◽  
Author(s):  
Yaru Gao ◽  
Xinyang Du ◽  
Huanhuan Li ◽  
Ying Wang
Keyword(s):  
Aspergillus Flavus ◽  
Structural Diversity ◽  
Gene Clusters ◽  
Illumina Miseq ◽  
Physicochemical Characteristics ◽  
Antibacterial Activities ◽  
Chemical Diversity ◽  
Genome Comparison ◽  
Marine Microorganisms ◽  
Sequencing Platform

Due to the specific properties of the marine environment, marine microorganisms have exclusive physicochemical characteristics that are different from those of terrestrial microorganisms, which can produce various secondary metabolites (SMs) with considerable structural diversity and biological activity. In this study, three strains of coepiphytic Aspergillus with potential antibacterial activities, A7 (Aspergillus flavus), B27 (Aspergillus flavipes) and R12 (Aspergillus sydowii), were isolated from the South China Sea. Via the Illumina MiSeq sequencing platform, the genomes of the three strains were sequenced, and genome comparison showed the highest diversity of the biosynthetic gene clusters (BGCs) in A7. Meanwhile, a comparison of physiological and genomic characteristics between A7 and other Aspergillus flavus strains demonstrated the superior environmental adaptability of A7, which is apparently consistent with the genetic richness of BGCs. By assigning reads to known BGCs, putative BGCs were allocated in A7 that corresponded to various SMs, including naphthopyrone, pyranonigrin E, cyclopiazonic acids, etc. Based on gene homology analysis, we surmise that a region is involved in the biosynthesis of ustiloxin-like RiPPs, a less thoroughly studied SM in fungi. Our results provide genetic information for the investigation of marine Aspergillus sp., which may help to elucidate their chemical diversity and adaptive strategies.

scholarly journals Genomic microsatellites identify shared Jewish ancestry intermediate between Middle Eastern and European populations

BMC Genetics ◽  
2009 ◽  
Vol 10 (1) ◽  
Author(s):  
Naama M Kopelman ◽  
Lewi Stone ◽  
Chaolong Wang ◽  
Dov Gefel ◽  
Marcus W Feldman ◽  
...  
Keyword(s):  
Middle Eastern ◽  
Jewish Ancestry ◽  
Genomic Microsatellites ◽  
European Populations

scholarly journals CD-HIT-OTU-MiSeq, an Improved Approach for Clustering and Analyzing Paired End MiSeq 16S rRNA Sequences

2017 ◽  
Author(s):  
Weizhong Li ◽  
Yuanyuan Chang
Keyword(s):  
16S Rrna ◽  
High Speed ◽  
De Novo ◽  
Sequence Data ◽  
Illumina Miseq ◽  
Poor Quality ◽  
Reference Database ◽  
Rrna Gene ◽  
Variable Regions ◽  
Novel Approach

AbstractIn recent years, Illumina MiSeq sequencers replaced pyrosequencing platforms and became dominant in 16S rRNA sequencing. One unique feature of MiSeq technology, compared with Pyrosequencing, is the Paired End (PE) reads, with each read can be sequenced to 250-300 bases to cover multiple variable regions on the 16S rRNA gene. However, the PE reads need to be assembled into a single contig at the beginning of the analysis. Although there are many methods capable of assembling PE reads into contigs, a big portion of PE reads can not be accurately assembled because the poor quality at the 3’ ends of both PE reads in the overlapping region. This causes that many sequences are discarded in the analysis. In this study, we developed a novel approach for clustering and annotation MiSeq-based 16S sequence data, CD-HIT-OTU-MiSeq. This new approach has four distinct novel features. (1) The package can clustering PE reads without joining them into contigs. (2) Users can choose a high quality portion of the PE reads for analysis (e.g. first 200 / 150 bases from forward / reverse reads), according to base quality profile. (3) We implemented a tool that can splice out the target region (e.g. V3-V4) from a full-length 16S reference database into the PE sequences. CD-HIT-OTU-MiSeq can cluster the spliced PE reference database together with samples, so we can derive Operational Taxonomic Units (OTUs) and annotate these OTUs concurrently. (4) Chimeric sequences are effectively identified through de novo approach. The package offers high speed and high accuracy. The software package is freely available as open source package and is distributed along with CD-HIT from http://cd-hit.org. Within the CD-HIT package, CD-HIT-OTU-MiSeq is within the usecase folder.

scholarly journals Changes In The Gut Microbiota After Hepatitis C Virus Eradication

2021 ◽  
Author(s):  
Takashi Honda ◽  
Masatoshi Ishigami ◽  
Kenta Yamamoto ◽  
Tomoaki Takeyama ◽  
Takanori Ito ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Gut Microbiota ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Virus Eradication ◽  
Sequencing Platform ◽  
Direct Acting Antiviral ◽  
Faecal Samples ◽  
Before And After

Abstract The gut microbiota interacts with infectious diseases and affects host immunity. It has also been reported that liver disease is associated with changes in the gut microbiota. To investigate changes in the gut microbiota before and after eradication of hepatitis C virus (HCV) by direct-acting antiviral (DAA) treatment in patients with chronic hepatitis C (CHC), we investigated 42 samples from 14 CHC patients who received DAA therapy for HCV. Faecal samples were obtained before treatment (Pre), when treatment ended (EOT), and 24 weeks after treatment ended (Post24). The target V3–4 region of the 16S rRNA gene from faecal samples was amplified using the Illumina Miseq sequencing platform. The diversity of the gut microbiota did not differ significantly between Pre, EOT, and Post24. PCoA analysis showed that for each patient, the values at Pre, EOT, and Post 24 were concentrated within a small area. LEfSe analysis showed that the relative abundances of Faecalibacterium and Bacillus were increased at EOT and further increased at Post24, and these genera were significantly increased at Post24 compared to Pre. These results suggest that changes in the gut microbiota need to be considered as among the various effects on living organisms observed after HCV elimination.

scholarly journals A comprehensive germline variant and expression analyses of ACE2, TMPRSS2 and SARS-CoV-2 activator FURIN genes from the Middle East: Combating SARS-CoV-2 with precision medicine

2020 ◽  
Author(s):  
Fahd Al-Mulla ◽  
Anwar Mohammad ◽  
Ashraf Al Madhoun ◽  
Dania Haddad ◽  
Hamad Ali ◽  
...  
Keyword(s):  
Viral Entry ◽  
Middle Eastern ◽  
East Asian ◽  
Cell Receptor ◽  
Mortality Rates ◽  
Whole Exome ◽  
African Populations ◽  
A Cell ◽  
Natural Variations ◽  
European Populations

AbstractThe severity of the new COVID-19 pandemic caused by the SARS-CoV-2 virus is strikingly variable in different global populations. SARS-CoV-2 uses ACE2 as a cell receptor, TMPRSS2 protease, and FURIN peptidase to invade human cells. Here, we investigated 1,378 whole-exome sequences of individuals from the Middle Eastern populations (Kuwait, Qatar, and Iran) to explore natural variations in the ACE2, TMPRSS2, and FURIN genes. We identified two activating variants (K26R and N720D) in the ACE2 gene that are more common in Europeans than in the Middle Eastern, East Asian, and African populations. We postulate that K26R can activate ACE2 and facilitate binding to S-protein RBD while N720D enhances TMPRSS2 cutting and, ultimately, viral entry. We also detected deleterious variants in FURIN that are frequent in the Middle Eastern but not in the European populations. This study highlights specific genetic variations in the ACE2 and FURIN genes that may explain SARS-CoV-2 clinical disparity. We showed structural evidence of the functionality of these activating variants that increase the SARS-CoV-2 aggressiveness. Finally, our data illustrate a significant correlation between ACE2 variants identified in people from Middle Eastern origins that can be further explored to explain the variation in COVID-19 infection and mortality rates globally.

scholarly journals Gut Microbiome Analysis In Adult Tropical Gars (Atractosteus tropicus)

2019 ◽  
Author(s):  
Roberto Méndez-Pérez ◽  
Rodrigo García-López ◽  
J. Santiago Bautista-López ◽  
Jorge F. Vázquez-Castellanos ◽  
Emyr S. Peña-Marín ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Gut Microbiome ◽  
Aeromonas Hydrophila ◽  
Illumina Miseq ◽  
Gut Contents ◽  
Estuarine Fish ◽  
Southern Mexico ◽  
Bacterial Gene ◽  
Sequencing Platform ◽  
Atractosteus Tropicus

AbstractTropical gar (Atractosteus tropicus), is freshwater and estuarine fish that has inhabited the Earth since the Mesozoic era, undergoing limited physiological variation ever since. This omnivorous fish is endemic to southern Mexico and part of Central America. Besides its recognized cultural and scientific relevance, the species has seen remarkable growth in its economic impact due to pisciculture. Previous studies have highlighted the role of microbial communities in fish, particularly those in the gut microbiome, in maintaining their host homeostasis or disease. In this study, we present the first report of the whole taxonomic composition of microbial communities in gut contents of adults’A. tropicus, by sex (female/male) and origin (wild/cultivated). Using culture-independent techniques, we extracted metagenomic DNA that was used for high throughput 16S rDNA profiling by amplifying the V4 – V5 hypervariable regions of the bacterial gene. A total of 364,735 total paired-end reads were obtained on an Illumina MiSeq sequencing platform, belonging to 508 identified genera, with the most and least abundant areCetobacterium, Edwardsiella, Serratia, Clostridium sensu stricto, PaludibacterandCampylobacter, Snodgrassella, Albirhodobacter, Lentilitoribacter, respectively. We detected that, by sex and origin, Proteobacteria, Fusobacteria, Firmicutes and Bacteroidetes phyla are the core gut microbiome of the adults’A. tropicus. We discover the Deinococcus-Thermus phylum sequence, wildtype males only, with extremophile capacity in another freshwater fish. We also identified the speciesLactococcus lactisstrains CAU929 and CAU6600, Cp6 and CAU9951,Cetobacteriumstrain H69,Aeromonas hydrophilastrains P5 and WR-5-3-2,Aeromonas sobriastrain CP DC28 andAeromonas hydrophilawith probiotic potential in aquaculture within the three dominant phyla, especially in wild-type organisms.

scholarly journals Understanding and mitigating some limitations of Illumina© MiSeq for environmental sequencing of Fungi

2017 ◽  
Author(s):  
Dan Thomas ◽  
Roo Vandegrift ◽  
Graham Bailes ◽  
Bitty Roy
Keyword(s):  
Pcr Amplification ◽  
Its Region ◽  
Illumina Miseq ◽  
Current Standard ◽  
Sequencing Platform ◽  
Variance Stabilization ◽  
Standard Tool ◽  
Illumina Sequence ◽  
Community Controls ◽  
Two Stages

AbstractITS-amplicon metabarcode studies using the illumina MiSeq sequencing platform are the current standard tool for fungal ecology studies. Here we report on some of the particular challenges experienced while creating and using a ribosomal RNA gene (rDNA) amplicon library for an ecological study. Two significant complications were encountered. First, artificial differences in read abundances among OTUs were observed, apparently resulting from bias at two stages: PCR amplification of genomic DNA with ITS-region Illumina-sequence-adapted-primers, and during Illumina sequencing. These differential read abundances were only partially corrected by a common variance-stabilization method. Second, tag-switching (or the shifting of amplicons to incorrect sample indices) occurred at high levels in positive mock-community controls. An example of a bioinformatic method to estimate the rate of tag switching is shown, some recommendations on the use of positive controls and primer choice are given, and one approach to reducing potential false positives resulting from these technological biases is presented.

scholarly journals The leaf bacterial microbiota of female and male kiwifruit plants in distinct seasons: assessing the impact of Pseudomonas syringae pv. actinidiae

2021 ◽  
Author(s):  
Aitana Ares ◽  
Joana Pereira ◽  
Eva Garcia ◽  
Joana Costa ◽  
Igor Tiago
Keyword(s):  
Bacterial Community ◽  
Pseudomonas Syringae ◽  
Relative Abundance ◽  
Bacterial Communities ◽  
Defense Mechanisms ◽  
Abiotic Factors ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Sequencing Platform ◽  
The Impact

The pandemic Pseudomonas syringae pv. actinidiae (Psa) has been compromising the production of the kiwifruit industry in major producing countries. Abiotic factors and plant gender are known to influence the disease outcome. To better understand their impact, we have determined the diversity of the leafs bacterial communities using the V5-V6 region of the 16S rRNA gene amplicon on the Illumina MiSeq sequencing platform. Healthy and diseased female and male kiwifruit plants were analyzed in two consecutive seasons: spring and autumn. This work describes whether the season, plant gender and the presence of Psa can affect the leaves bacterial community. Fifty bacterial operational taxonomic units (OTUs) were identified and assigned to five phyla distributed by 14 different families and 23 genera. The leaves of healthy female and male kiwi plants share most of the identified bacterial populations, that undergoes major seasonal changes. In both cases a substantial increase of the relative abundance of genus Methylobacterium is observed in autumn. The presence of Psa induced profound changes on leaves bacterial communities structure translated into a reduction in the relative abundance of previously dominant genera that had been found in healthy plants, namely Hymenobacter, Sphingomonas and Massilia. The impact of Psa was less pronounced in the bacterial community structure of male plants in both seasons. Some of the naturally occurring genera have the potential to act as an antagonist or as enhancers of the defense mechanisms paving the way for environmentally friendly and sustainable disease control.

scholarly journals Culture-independent detection and characterisation of Mycobacterium tuberculosis and M. africanum in sputum samples using shotgun metagenomics on a benchtop sequencer

2014 ◽  
Author(s):  
Emma L Doughty ◽  
Martin J Sergeant ◽  
Ifedayo M.O Adetifa ◽  
Martin Antonio ◽  
Mark J Pallen
Keyword(s):  
Mycobacterium Tuberculosis ◽  
West Africa ◽  
Dna Extraction ◽  
Illumina Miseq ◽  
Diagnostic Methods ◽  
Insertion Element ◽  
Sensitivity Testing ◽  
Shotgun Metagenomics ◽  
Sequencing Platform ◽  
Specific Amplification

Tuberculosis remains a major global health problem. Laboratory diagnostic methods that allow effective, early detection of cases are central to management of tuberculosis in the individual patient and in the community. Since the 1880s, laboratory diagnosis of tuberculosis has relied primarily on microscopy and culture. However, microscopy fails to provide species- or lineage-level identification and culture-based workflows for diagnosis of tuberculosis remain complex, expensive, slow, technically demanding and poorly able to handle mixed infections. We therefore explored the potential of shotgun metagenomics, sequencing of DNA from samples without culture or target-specific amplification or capture, to detect and characterise strains from the Mycobacterium tuberculosis complex in smear-positive sputum samples obtained from The Gambia in West Africa. Eight smear- and culture-positive sputum samples were investigated using a differential-lysis protocol followed by a kit-based DNA extraction method, with sequencing performed on a benchtop sequencing instrument, the Illumina MiSeq. The number of sequence reads in each sputum-derived metagenome ranged from 989,442 to 2,818,238. The proportion of reads in each metagenome mapping against the human genome ranged from 20% to 99%. We were able to detect sequences from the M. tuberculosis complex in all eight samples, with coverage of the H37Rv reference genome ranging from 0.002X to 0.7X. By analysing the distribution of large sequence polymorphisms (deletions and the locations of the insertion element IS6110) and single nucleotide polymorphisms (SNPs), we were able to assign seven of eight metagenome-derived genomes to a species and lineage within the M. tuberculosis complex. Two metagenome-derived mycobacterial genomes were assigned to M. africanum, a species largely confined to West Africa; the others that could be assigned belonged to lineages T, H or LAM within the clade of “modern” M. tuberculosis strains. We have provided proof of principle that shotgun metagenomics can be used to detect and characterise M. tuberculosis sequences from sputum samples without culture or target-specific amplification or capture, using an accessible benchtop-sequencing platform, the Illumina MiSeq, and relatively simple DNA extraction, sequencing and bioinformatics protocols. In our hands, sputum metagenomics does not yet deliver sufficient depth of coverage to allow sequence-based sensitivity testing; it remains to be determined whether improvements in DNA extraction protocols alone can deliver this or whether culture, capture or amplification steps will be required. Nonetheless, we can foresee a tipping point when a unified automated metagenomics-based workflow might start to compete with the plethora of methods currently in use in the diagnostic microbiology laboratory.

scholarly journals Evaluation of Oxford Nanopore MinIONTM Sequencing for 16S rRNA Microbiome Characterization

2017 ◽  
Author(s):  
Xiao Ma ◽  
Elyse Stachler ◽  
Kyle Bibby
Keyword(s):  
16S Rrna ◽  
Pure Culture ◽  
Community Sample ◽  
Operational Taxonomic Unit ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Ribosomal Database Project ◽  
Nanopore Sequencing ◽  
Sequencing Platform ◽  
Oxford Nanopore

AbstractIn this manuscript we evaluate the potential for microbiome characterization by sequencing of near-full length 16S rRNA gene region fragments using the Oxford Nanopore MinION (hereafter ‘Nanopore’) sequencing platform. We analyzed pure-culture E. coli and P. fluorescens, as well as a low-diversity mixed community sample from hydraulic fracturing produced water. Both closed and open reference operational taxonomic unit (OTU) picking failed, necessitating the direct use of sequences without OTU picking. The Ribosomal Database Project classifier against the Green Genes database was found to be the optimal annotation approach, with average pure-culture annotation accuracies of 93.8% and 82.0% at the phyla and genus levels, respectively. Comparative analysis of an environmental sample using Nanopore and Illumina MiSeq sequencing identified high taxonomic similarity when using a weighted metric (Bray-Curtis), and significantly reduced similarity when using an unweighted metric (Jaccard). These results highlight the great potential of Nanopore sequencing to analyze broad microbial community trends, and the challenge of applying Nanopore sequencing to discern rare taxa in mixed microbial communities. Finally, we observed that between-run carryover following washes on the same flowcell accounted for >10% of sequence reads, necessitating future development to either prevent carryover or filter sequences of interest (e.g. barcoding).

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