scholarly journals Epiphytic Diatom-Based Biomonitoring in Mediterranean Ponds: Traditional Microscopy versus Metabarcoding Approaches

Water ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1351
Author(s):  
María Borrego-Ramos ◽  
Eloy Bécares ◽  
Pedro García ◽  
Alejandro Nistal ◽  
Saúl Blanco
Keyword(s):  
Water Quality ◽  
Light Microscopy ◽  
High Throughput ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Reference Database ◽  
Morphological Identification ◽  
Quality Class ◽  
Shallow Ponds ◽  
Relative Abundances

Benthic diatoms have traditionally been used as bioindicators of aquatic ecosystems. Because diatom-based monitoring of water quality is required by European legislation, molecular-based methods had emerged as useful alternatives to classical methods based on morphological identification using light microscopy. The aim of this study was to test the reliability of DNA metabarcoding combined with High-Throughput Sequencing (HTS) techniques in the bioassessment of the trophic status of 22 Mediterranean shallow ponds in NW Spain. For each pond, the Trophic Diatom Index (TDI) was calculated from inventories obtained by identification using light microscopy (LM) followed by high-throughput sequencing (HTS) at the molecular level. Ponds were subsequently classified into five water quality classes. The results showed a good correspondence between both methods, especially after applying a correction factor that depended on the biovolume of the cells. This correspondence led to the assignment to the same quality class in 59% of the ponds. The determination and quantification of valves or DNA sequences was one of the main pitfalls, which mainly included those related to the variability in the relative abundances of some species. Accordingly, ponds with similar relative abundances for the dominant species were assigned to the same quality class. Moreover, other difficulties leading the discrepancies were the misidentification of some species due to the presence of semi-cryptic taxa, the incompleteness of the reference database and the bioinformatic protocol. Thus, the validation of DNA-based methods for the identification of freshwater diatoms represents an important goal, as an alternative to using traditional methods in Mediterranean shallow ponds.

scholarly journals An account of epiphytic diatoms in mediterranean wetlands: comparing morphological and molecular assessments

2021 ◽  
Vol 4 ◽  
Author(s):  
María Borrego-Ramos ◽  
Eloy Bécares ◽  
Pedro García-García ◽  
Alejandro Nistal ◽  
Saúl Blanco
Keyword(s):  
Water Quality ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Reference Database ◽  
Good Correspondence ◽  
Quality Class ◽  
Dna Metabarcoding ◽  
Shallow Ponds ◽  
Relative Abundances ◽  
Cryptic Taxa

Benthic diatoms are well known bioindicators of water quality, used in many aquatic ecosystems. Since diatom-based monitoring of water quality is required by European legislation, the search for methods that facilitate this task has become more relevant. The aim of this study was to test the reliability of DNA metabarcoding combined with high-throughput sequencing (HTS) techniques in the bioassessment of 22 Mediterranean shallow ponds in Spain. For each pond, Trophic Diatom Index (TDI) was calculated from inventories obtained by using light microscopy, and then molecular (HTS) methods. Ponds were subsequently classified into five water quality classes. Our results showed a good correspondence between both methods, especially after applying a correction factor depending on the biovolume of the cells. This correspondence led to the assignment to the same quality class in 59% of the ponds. The determination and quantification of valves or DNA sequences was one of the main pitfalls, mainly those related to the variability in the relative abundances of some species. Accordingly, ponds with similar relative abundances for the dominant species were assigned to the same quality class. Moreover, other difficulties leading the discrepancies were the misidentification of some species due to the presence of semi-cryptic taxa, the incompleteness of the reference database and the bioinformatic protocol. Therefore, the validation of DNA-based methods for the identification of freshwater diatoms represents an important goal, as an alternative to traditional ones in Mediterranean shallow ponds.

scholarly journals Application of Broccoli Residues to Soil can Suppress Verticillium Wilt of Cotton by Regulating the Bacterial Community Structure of the Rhizosphere

2021 ◽  
Author(s):  
Weisong Zhao ◽  
Qinggang Guo ◽  
Shezeng Li ◽  
Xiuyun Lu ◽  
Peipei Wang ◽  
...  
Keyword(s):  
Community Structure ◽  
Bacterial Community ◽  
High Throughput ◽  
Verticillium Wilt ◽  
Bacterial Community Structure ◽  
High Throughput Sequencing ◽  
Field Experiments ◽  
Cotton Seeds ◽  
Relative Abundances ◽  
And Control

Abstract [Aims] Verticillium wilt (VW) of cotton was effectively controlled by application of broccoli residues (BR) to soil. Information regarding the variation in bacterial communities in rhizosphere of cotton cultivars with different VW resistance levels under BR treatment is still lacking and finally to provide guidance for screening effective biocontrol bacteria. [Methods] Real-time fluorescence quantitative PCR was used to determine the population of Verticillium dahliae, the effects of BR on the bacterial community structure in rhizosphere were determined by high-throughput sequencing technology. [Results] Results showed that control effects for susceptible cultivar (cv. EJ-1) and resistant cultivar (cv. J863) on VW after BR treatment were 51.76% and 86.15%, the population of V. dahliae decreased by 18.88% and 30.27%, respectively. High-throughput sequencing showed that ACE and Chao1 indices were increased by application of BR. Actinobacteria, Proteobacteria, Bacteroidetes, Gemmatimonadetes, Acidobacteria, and Firmicutes were the most dominant phyla, and relative abundances of these bacterial taxa significantly differed between cultivars. Additionally, Bacillus stably increased in rhizosphere following BR treatment. Redundancy analysis (RDA) showed that relative abundances of Bacillus, Lysobacter, Streptomyces, Rubrobacter, Gemmatimonas, Bryobacter and Nocardioides were correlated with occurrence of VW. Field experiments demonstrated that dressing cotton seeds with Bacillus subtilis NCD-2 could successfully reduce occurrence of VW, and control effects for EJ-1 and J863 were 35.26% and 31.02%, respectively. [Conclusions] The application of BR changed the bacterial community structure in cotton rhizosphere, decreased the population of V. dahliae in soil, and increased the abundance of beneficial microorganisms, thus significantly reducing the occurrence of VW.

scholarly journals Use of High Throughput Sequencing and Light Microscopy Show Contrasting Results in a Study of Phytoplankton Occurrence in a Freshwater Environment

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e106510 ◽  
Author(s):  
Xi Xiao ◽  
Hanne Sogge ◽  
Karin Lagesen ◽  
Ave Tooming-Klunderud ◽  
Kjetill S. Jakobsen ◽  
...  
Keyword(s):  
Light Microscopy ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Freshwater Environment

Next Generation High Throughput Sequencing to Assess Microbial Communities: An Application Based on Water Quality

2021 ◽  
Vol 106 (5) ◽  
pp. 727-733
Author(s):  
Gowher A. Wani ◽  
Mohd Asgar Khan ◽  
Mudasir A. Dar ◽  
Manzoor A. Shah ◽  
Zafar A. Reshi
Keyword(s):  
Water Quality ◽  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Next Generation

scholarly journals Field-based species identification in eukaryotes using real-time nanopore sequencing

2017 ◽  
Author(s):  
Joe Parker ◽  
Andrew J. Helmstetter ◽  
Dion Devey ◽  
Alexander S.T. Papadopulos
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Reference Database ◽  
Nanopore Sequencing ◽  
Technological Advances ◽  
A Genome ◽  
Hybrid Genome

Advances in DNA sequencing and informatics have revolutionised biology over the past four decades, but technological limitations have left many applications unexplored1,2. Recently, portable, real-time, nanopore sequencing (RTnS) has become available. This offers opportunities to rapidly collect and analyse genomic data anywhere3–5. However, the generation of datasets from large, complex genomes has been constrained to laboratories6,7. The portability and long DNA sequences of RTnS offer great potential for field-based species identification, but the feasibility and accuracy of these technologies for this purpose have not been assessed. Here, we show that a field-based RTnS analysis of closely-related plant species (Arabidopsis spp.)8 has many advantages over laboratory-based high-throughput sequencing (HTS) methods for species level identification-by-sequencing and de novo phylogenomics. Samples were collected and sequenced in a single day by RTnS using a portable, “al fresco” laboratory. Our analyses demonstrate that correctly identifying unknown reads from matches to a reference database with RTnS reads enables rapid and confident species identification. Individually annotated RTnS reads can be used to infer the evolutionary relationships of A. thaliana. Furthermore, hybrid genome assembly with RTnS and HTS reads substantially improved upon a genome assembled from HTS reads alone. Field-based RTnS makes real-time, rapid specimen identification and genome wide analyses possible. These technological advances are set to revolutionise research in the biological sciences9 and have broad implications for conservation, taxonomy, border agencies and citizen science.

scholarly journals In silico assessment of primers for eDNA studies using PrimerTree and application to characterize the biodiversity surrounding the Cuyahoga River

2015 ◽  
Author(s):  
M.V. Cannon ◽  
J. Hester ◽  
A. Shalkhauser ◽  
E.R. Chan ◽  
K. Logue ◽  
...  
Keyword(s):  
High Throughput ◽  
Dna Sequences ◽  
Water Samples ◽  
High Throughput Sequencing ◽  
Biological Diversity ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Asian Carp ◽  
Broad Perspective ◽  
Wide Range

Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semiaquatic animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity.

scholarly journals ChiTaH: a fast and accurate tool for identifying known human chimeric sequences from high-throughput sequencing data

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Rajesh Detroja ◽  
Alessandro Gorohovski ◽  
Olawumi Giwa ◽  
Gideon Baum ◽  
Milana Frenkel-Morgenstern
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Complex Disease ◽  
Single Cells ◽  
Reference Database ◽  
Sequencing Data ◽  
Sequencing Technologies ◽  
High Throughput Sequencing Data ◽  
Chimeric Rnas ◽  
Sensitivity Specificity

Abstract Fusion genes or chimeras typically comprise sequences from two different genes. The chimeric RNAs of such joined sequences often serve as cancer drivers. Identifying such driver fusions in a given cancer or complex disease is important for diagnosis and treatment. The advent of next-generation sequencing technologies, such as DNA-Seq or RNA-Seq, together with the development of suitable computational tools, has made the global identification of chimeras in tumors possible. However, the testing of over 20 computational methods showed these to be limited in terms of chimera prediction sensitivity, specificity, and accurate quantification of junction reads. These shortcomings motivated us to develop the first ‘reference-based’ approach termed ChiTaH (Chimeric Transcripts from High–throughput sequencing data). ChiTaH uses 43,466 non–redundant known human chimeras as a reference database to map sequencing reads and to accurately identify chimeric reads. We benchmarked ChiTaH and four other methods to identify human chimeras, leveraging both simulated and real sequencing datasets. ChiTaH was found to be the most accurate and fastest method for identifying known human chimeras from simulated and sequencing datasets. Moreover, especially ChiTaH uncovered heterogeneity of the BCR-ABL1 chimera in both bulk and single-cells of the K-562 cell line, which was confirmed experimentally.

scholarly journals Indel-correcting DNA barcodes for high-throughput sequencing

2018 ◽  
Vol 115 (27) ◽  
pp. E6217-E6226 ◽  
Author(s):  
John A. Hawkins ◽  
Stephen K. Jones ◽  
Ilya J. Finkelstein ◽  
William H. Press
Keyword(s):  
High Throughput ◽  
Dna Sequences ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Gc Content ◽  
Error Correcting Codes ◽  
Dna Barcodes ◽  
Library Size ◽  
Sequencing Errors ◽  
Insertion And Deletion

Many large-scale, high-throughput experiments use DNA barcodes, short DNA sequences prepended to DNA libraries, for identification of individuals in pooled biomolecule populations. However, DNA synthesis and sequencing errors confound the correct interpretation of observed barcodes and can lead to significant data loss or spurious results. Widely used error-correcting codes borrowed from computer science (e.g., Hamming, Levenshtein codes) do not properly account for insertions and deletions (indels) in DNA barcodes, even though deletions are the most common type of synthesis error. Here, we present and experimentally validate filled/truncated right end edit (FREE) barcodes, which correct substitution, insertion, and deletion errors, even when these errors alter the barcode length. FREE barcodes are designed with experimental considerations in mind, including balanced guanine-cytosine (GC) content, minimal homopolymer runs, and reduced internal hairpin propensity. We generate and include lists of barcodes with different lengths and error correction levels that may be useful in diverse high-throughput applications, including >106 single-error–correcting 16-mers that strike a balance between decoding accuracy, barcode length, and library size. Moreover, concatenating two or more FREE codes into a single barcode increases the available barcode space combinatorially, generating lists with >1015 error-correcting barcodes. The included software for creating barcode libraries and decoding sequenced barcodes is efficient and designed to be user-friendly for the general biology community.

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