scholarly journals DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

2015 ◽  
Vol 105 (6) ◽  
pp. 717-727 ◽  
Author(s):  
G.-J. Brandon-Mong ◽  
H.-M. Gan ◽  
K.-W. Sing ◽  
P.-S. Lee ◽  
P.-E. Lim ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Ease Of Use ◽  
Malaise Trap ◽  
Detection Rates ◽  
Operational Taxonomic Units ◽  
Arthropod Species ◽  
Primer Sets ◽  
Arthropod Biodiversity

AbstractMetabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

scholarly journals Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 241-252
Author(s):  
Dyah Asri Handayani Taroepratjeka ◽  
Tsuyoshi Imai ◽  
Prapaipid Chairattanamanokorn ◽  
Alissara Reungsang
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
High Salinity ◽  
Amplicon Sequencing ◽  
Spatial Proximity ◽  
Lignocellulosic Waste ◽  
Evaporation Pond ◽  
Operational Taxonomic Units ◽  
Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

scholarly journals Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding

MycoKeys ◽  
2018 ◽  
Vol 39 ◽  
pp. 29-40 ◽  
Author(s):  
Sten Anslan ◽  
R. Henrik Nilsson ◽  
Christian Wurzbacher ◽  
Petr Baldrian ◽  
Leho Tedersoo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Computation Time ◽  
Potential Effect ◽  
Data Sets ◽  
Sequencing Data ◽  
Operational Taxonomic Units ◽  
High Throughput Sequencing Data ◽  
Recent Developments

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

Analysis of the effects of nanosilver on bacterial community in the intestinal fluid of silkworms using high-throughput sequencing

2019 ◽  
Vol 110 (3) ◽  
pp. 309-320
Author(s):  
Chen Lin ◽  
Zhou Wei ◽  
Zhou Yi ◽  
Tan Tingting ◽  
Du Huamao ◽  
...  
Keyword(s):  
Bacterial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Intestinal Bacteria ◽  
Illumina Miseq ◽  
The Body ◽  
Dependent Manner ◽  
Negative Effects ◽  
Silkworm Larvae ◽  
Intestinal Bacterial Community

AbstractNanosilver is an environment-friendly, harmless alternative of traditional disinfectants which can be potentially applied in the sericulture industry. However, the effects of nanosilver on the intestinal bacterial community of the silkworms (Bombyx mori L.) are unclear. In this study, Illumina MiSeq high-throughput sequencing technology was used to assess the intestinal bacterial community in both male and female silkworms while treated with different concentrations of nanosilver. We found that nanosilver significantly influenced the composition of silkworm intestinal bacterial community on the different taxonomic levels. Most conspicuously, the abundance of Firmicutes was increased by the treatment of 20 mg L−1 nanosilver but decreased by that of 100 mg L−1 nanosilver at the phylum level. The same trend was observed in Bacilli at the class level and in Enterococcus at the genus level. In some extreme cases, application of nanosilver eliminated the bacterium, e.g., Brevibacillus, but increased the population of several other bacteria in the host intestine, such as Blautia, Terrisporobacter, Faecalibacterium, and some bacteria could only be found in nanosilver treatment groups, e.g., Dialister. In addition, although nanosilver generally showed negative effects on the cocooning rate in a dose-dependent manner, we found that 20 mg L−1 nanosilver treatment significantly increased the body weight of silkworms and did not show negative effects on the survival rate. These results indicated that the intestinal bacteria community of silkworm larvae was significantly changed after nanosilver treatment which might consequently influence host growth and development.

Effect of Plantaricin 163 in Combination with Thymol and Surfactin on Crucian Carp (Carassius auratus)

2019 ◽  
Vol 82 (8) ◽  
pp. 1283-1291 ◽  
Author(s):  
HECHAO DU ◽  
XIUXIU LI ◽  
ZHAOXIN LU ◽  
XIAOMEI BIE ◽  
HAIZHEN ZHAO ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Crucian Carp ◽  
Thiobarbituric Acid ◽  
Commercial Application ◽  
Operational Taxonomic Units ◽  
Spoilage Bacteria ◽  
Plate Counts ◽  
Natural Preservatives ◽  
Control Samples

ABSTRACT The use of natural preservatives has attracted considerable attention owing to their generally safe and environmentally friendly properties. In this study, we investigated the effects of the preservative A1, composed of plantaricin 163, thymol, and surfactin, on bacterial communities and storage quality of refrigerated crucian carp. A total of 522 operational taxonomic units belonging to 20 phyla and 272 genera were identified by high-throughput sequencing, showing a comprehensive coverage of bacterial composition of crucian carp. In untreated samples after spoilage, Brochothrix was the predominant genus, followed by Aeromonas and Pseudomonas. After treatment with A1, the growth of these spoilage bacteria was significantly inhibited according to high-throughput sequencing and plate counts, and Lactococcus became the most abundant organism at the end of storage. Meanwhile, compared with control samples, the shelf life of A1-treated samples extended from 3 to 12 days on the basis of the sensory evaluation and the total viable counts. Furthermore, the total volatile basic nitrogen, thiobarbituric acid, and pH values for A1-treated samples were significantly lower than that of control samples. The results indicate that preservative A1 has potential commercial application in the preservation of refrigerated crucian carp.

scholarly journals The promise of a DNA taxonomy

2004 ◽  
Vol 359 (1444) ◽  
pp. 669-679 ◽  
Author(s):  
Mark L. Blaxter
Keyword(s):  
High Throughput ◽  
Small Proportion ◽  
High Throughput Sequencing ◽  
Meta Analysis ◽  
Molecular Data ◽  
Similar Extent ◽  
Molecular Barcoding ◽  
Dna Taxonomy ◽  
Biologically Relevant ◽  
Operational Taxonomic Units

Not only is the number of described species a very small proportion of the estimated extant number of taxa, but it also appears that all concepts of the extent and boundaries of ‘species’ fail in many cases. Using conserved molecular sequences it is possible to define and diagnose molecular operational taxonomic units (MOTU) that have a similar extent to traditional ‘species’. Use of a MOTU system not only allows the rapid and effective identification of most taxa, including those not encountered before, but also allows investigation of the evolution of patterns of diversity. A MOTU approach is not without problems, particularly in the area of deciding what level of molecular difference defines a biologically relevant taxon, but has many benefits. Molecular data are extremely well suited to re–analysis and meta–analysis, and data from multiple independent studies can be readily collated and investigated by using new parameters and assumptions. Previous molecular taxonomic efforts have focused narrowly. Advances in high–throughput sequencing methodologies, however, place the idea of a universal, multi–locus molecular barcoding system in the realm of the possible.

scholarly journals Sorting things out - assessing effects of unequal specimen biomass on DNA metabarcoding

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Bianca Peinert ◽  
Florian Leese
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequencing Depth ◽  
West Germany ◽  
Coi Gene ◽  
Mountain Stream ◽  
Detection Rates ◽  
Size Classes ◽  
Dna Metabarcoding ◽  
Size Sorting

1) Environmental bulk samples often contain many taxa with biomass differences of several orders of magnitude. This can be problematic in DNA metabarcoding and metagenomic high throughput sequencing approaches, as large specimens contribute over proportionally much DNA template. Thus a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples and balancing the amounts of tissue used per size fraction should improve detection rates, but has not been systematically tested. 2) Here we tested the effects of size sorting on taxa detection using freshwater macroinvertebrates. Kick sampling was performed at two locations of a low-mountain stream in West Germany, specimens were morphologically identified and sorted into small, medium and large size classes (< 2.5x5, 5x10 and up to 10x20 mm). Tissue from the 3 size categories was extracted individually, and pooled to simulate bulk samples that were not sorted and samples which were sorted and then pooled proportionately by specimen size. DNA from all 5 extractions of both samples was amplified using 4 different freshwater primer sets for the COI gene and sequenced on a HiSeq Illumina sequencer. 3) Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples, at the same sequencing depth. Our results imply that sequencing depth can be decreased ~ 5 fold when sorting the samples into three size classes. 4) Our results demonstrate that even a coarse size sorting can substantially improve detection rates. While high throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass is a simple yet efficient method to reduce current sequencing costs.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals Using high-throughput sequencing of ITS2 to describe Symbiodinium metacommunities in St. John, US Virgin Islands

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3472 ◽  
Author(s):  
Ross Cunning ◽  
Ruth D. Gates ◽  
Peter J. Edmunds
Keyword(s):  
Coral Reef ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Virgin Islands ◽  
Us Virgin Islands ◽  
Operational Taxonomic Units ◽  
The North ◽  
Application Specific ◽  
North And South ◽  
Symbiotic Microalgae

Symbiotic microalgae (Symbiodinium spp.) strongly influence the performance and stress-tolerance of their coral hosts, making the analysis of Symbiodinium communities in corals (and metacommunities on reefs) advantageous for many aspects of coral reef research. High-throughput sequencing of ITS2 nrDNA offers unprecedented scale in describing these communities, yet high intragenomic variability at this locus complicates the resolution of biologically meaningful diversity. Here, we demonstrate that generating operational taxonomic units by clustering ITS2 sequences at 97% similarity within, but not across, samples collapses sequence diversity that is more likely to be intragenomic, while preserving diversity that is more likely interspecific. We utilize this ‘within-sample clustering’ to analyze Symbiodinium from ten host taxa on shallow reefs on the north and south shores of St. John, US Virgin Islands. While Symbiodinium communities did not differ between shores, metacommunity network analysis of host-symbiont associations revealed Symbiodinium lineages occupying ‘dominant’ and ‘background’ niches, and coral hosts that are more ‘flexible’ or ‘specific’ in their associations with Symbiodinium. These methods shed new light on important questions in coral symbiosis ecology, and demonstrate how application-specific bioinformatic pipelines can improve the analysis of metabarcoding data in microbial metacommunity studies.

Sign in / Sign up

Export Citation Format

Share Document