scholarly journals Metabarcoding data allow for reliable biomass estimates in the most abundant animals on earth

2019 ◽  
Vol 3 ◽  
Author(s):  
Janina Schenk ◽  
Stefan Geisen ◽  
Nils Kleinboelting ◽  
Walter Traunspurger
Keyword(s):  
Nematode Species ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Nematode Communities ◽  
Biogeochemical Models ◽  
Flow Through ◽  
Primer Sets ◽  
User Friendly ◽  
Ribosomal Rrna ◽  
Mock Communities

Microscopic organisms are the dominant and most diverse organisms on Earth. Nematodes, as part of this microscopic diversity, are by far the most abundant animals and their diversity is equally high. Molecular metabarcoding is often applied to study the diversity of microorganisms, but has yet to become the standard to determine nematode communities. As such, the information metabarcoding provides, such as in terms of species coverage, taxonomic resolution and especially if sequence reads can be linked to the abundance or biomass of nematodes in a sample, has yet to be determined. Here, we applied metabarcoding using three primer sets located within ribosomal rRNA gene regions to target assembled mock-communities consisting of 18 different nematode species that we established in 9 different compositions. We determined abundances and biomass of all species added to examine if relative sequence abundance or biomass can be linked to relative sequence reads. We found that nematode communities are not equally represented by the three different primer sets and we found that relative read abundances almost perfectly correlated positively with relative species biomass for two of the primer sets. This strong biomass-read number correlation suggests that metabarcoding reads can reveal biomass information even amongst more complex nematode communities as present in the environment and possibly can be transferred to better study other groups of organisms. This biomass-read link is of particular importance for more reliably assessing nutrient flow through food-webs, as well as adjusting biogeochemical models through user-friendly and easily obtainable metabarcoding data.

scholarly journals A novel metabarcoding strategy for studying nematode communities

2020 ◽  
Author(s):  
Md. Maniruzzaman Sikder ◽  
Mette Vestergård ◽  
Rumakanta Sapkota ◽  
Tina Kyndt ◽  
Mogens Nicolaisen
Keyword(s):  
18S Rrna ◽  
High Throughput Sequencing ◽  
Soil Samples ◽  
Coi Gene ◽  
Rrna Gene ◽  
Nematode Communities ◽  
Sequencing Platform ◽  
Spiked Soil ◽  
Primer Sets ◽  
Mock Communities

AbstractNematodes are widely abundant soil metazoa and often referred to as indicators of soil health. While recent advances in next-generation sequencing technologies have accelerated research in microbial ecology, the ecology of nematodes remains poorly elucidated, partly due to the lack of reliable and validated sequencing strategies. Objectives of the present study were (i) to compare commonly used primer sets and to identify the most suitable primer set for metabarcoding of nematodes; (ii) to establish and validate a high-throughput sequencing strategy for nematodes using Illumina paired-end sequencing. In this study, we tested four primer sets for amplicon sequencing: JB3/JB5 (mitochondrial, I3-M11 partition); SSU_04F/SSU_22R (18S rRNA, V1-V2 region); Nemf/18Sr2b (18S rRNA, V6-V8 region) from earlier studies; and MMSF/MMSR (18S rRNA, V4-V5 region), a newly developed primer set from this study. In order to test the primer sets, we used 22 samples of individual nematode species, 20 mock communities, 20 soil samples, 20 spiked soil samples (mock communities in soil), and 4 root/rhizosphere soil samples. We successfully amplified the target regions (I3-M11 partition of the COI gene; V1-V2, V4-V8 region of 18S rRNA gene) from these 86 DNA samples with the four different primer combinations and sequenced the amplicons on an Illumina MiSeq sequencing platform. We found that the MMSF/MMSR and Nemf/18Sr2b were efficient in detecting nematode compared to JB and SSU primer sets based on annotation of sequence reads at genus and in some cases at species level. Therefore, these primer sets are suggested for studies of nematode communities in agricultural environments.

Development of an efficient PCR-based diagnosis protocol for the identification of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Nematology ◽  
2004 ◽  
Vol 6 (2) ◽  
pp. 279-285 ◽  
Author(s):  
Jae Soon Kang ◽  
Kwang Sik Choi ◽  
Sang Chul Shin ◽  
Il Sung Moon ◽  
Sang Gil Lee ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
5S Rrna ◽  
Rrna Gene ◽  
Pinewood Nematode ◽  
Pine Wood ◽  
5S Rrna Gene ◽  
Primer Sets ◽  
Species Specific

Abstract Pine wood wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus , has been a serious problem in the southern regions of Korea. Efficient diagnosis of B. xylophilus from infected pine wood specimens is critical for the management of this pest. Traditional microscopic examination often results in an erroneous identification because a closely related non-pathogenic species, B. mucronatus, has a great degree of morphological similarity to B. xylophilus. In an attempt to search for reliable molecular markers for the discrimination of these species, we have cloned the 5S rRNA genomic DNA fragments containing both coding and intergenic spacer (IGS) regions from B. xylophilus and B. mucronatus through a homology-probing PCR strategy. Sequence analyses revealed that coding sequences of the 5S rRNA gene from the two species are almost identical (98.3% homology) but that the IGS sequences differ substantially between the species. Based on the IGS sequence differences (69.7% homology), we designed species-specific primer sets and developed a PCR-based diagnosis protocol for the identification and discrimination of the two nematode species on a molecular basis.

scholarly journals Long-read DNA metabarcoding of ribosomal rRNA in the analysis of fungi from aquatic environments

2018 ◽  
Author(s):  
Felix Heeger ◽  
Elizabeth C. Bourne ◽  
Christiane Baschien ◽  
Andrey Yurkov ◽  
Boyke Bunk ◽  
...  
Keyword(s):  
De Novo ◽  
Error Rates ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
Sequencing Error ◽  
Rrna Gene ◽  
Dna Metabarcoding ◽  
Long Read ◽  
Reference Sequences ◽  
Ribosomal Rrna

ABSTRACTDNA metabarcoding is now widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints have limited most studies to marker lengths of ca. 300-600 bp. Longer sequencing reads of several 5 thousand bp are now possible with third-generation sequencing. The increased marker lengths provide greater taxonomic resolution and enable the use of phylogenetic methods of classifcation, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most well-established bioinformatics tools for DNA metabarcoding were originally 10 designed for short reads and are therefore not suitable. Here we used Pacifc Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote ribosomal SSU and LSU rRNA genes and the ITS spacer region. We developed a long-read analysis pipeline that reduced error rates to levels 15 comparable to short-read platforms. Validation using fungal isolates and a mock community indicated that our pipeline detected 98% of chimeras de novo i.e., even in the absence of reference sequences. We recovered 947 OTUs from water and sediment samples in a natural lake, 848 of which could be classifed to phylum, 486 to family, 397 to genus and 330 to species. By 20 allowing for the simultaneous use of three global databases (Unite, SILVA, RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 25 non-fungal OTUs indicate that long-read DNA metabarcoding holds promise for the study of eukaryotic diversity more broadly.

scholarly journals New Insights Into Nematode DNA-metabarcoding as Revealed by the Characterization of Artificial and Spiked Nematode Communities

Diversity ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 52 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nancy de Sutter ◽  
Nicole Viaene ◽  
Annelies Haegeman
Keyword(s):  
Species Richness ◽  
18S Rrna ◽  
Nematode Species ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Soil Nematode ◽  
Mock Community ◽  
Nematode Communities ◽  
Dna Metabarcoding ◽  
Soil Nematode Communities

Nematodes are ideal biological indicators to monitor soil biodiversity and ecosystem functioning. For this reason, they have been receiving increasing attention from a broad range of scientists. The main method to characterize soil nematode communities until at least genus level is still based on microscopic observations of nematode morphology. Such an approach is time-consuming, labor-intensive, and requires specialized personnel. The first studies on the potential use of DNA-metabarcoding to characterize nematode communities showed some shortcomings: under- or overestimation of species richness caused by failure to detect a number of nematode species or caused by intraspecific sequence variants increasing the number of OTUs (operational taxonomic units) or ‘molecular’ species, and flaws in quantification. We set up experiments to optimize this metabarcoding approach. Our results provided new insights such as the drastic effect of different DNA-extraction methods on nematode species richness due to variation in lysis efficacy. Our newly designed primer set (18S rRNA gene, V4-V5 region) showed in silico an improved taxonomic coverage compared with a published primer set (18S rRNA gene, V6-V8 region). However, results of DNA-metabarcoding with the new primer set showed less taxonomic coverage, and more non-nematode reads. Thus, the new primer set might be more suitable for whole soil faunal analysis. Species-specific correction factors calculated from a mock community with equal amounts of different nematode species were applied on another mock community with different amounts of the same nematode species and on a biological sample spiked with four selected nematode species. Results showed an improved molecular quantification. In conclusion, DNA-metabarcoding of soil nematode communities is useful for monitoring shifts in nematode composition but the technique still needs further optimization to enhance its precision.

scholarly journals rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude OGIER ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Matthieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

Abstract Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

scholarly journals rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude OGIER ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Matthieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

Abstract Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

scholarly journals rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude Ogier ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Mathieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

AbstractBackgroundMicrobiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene.ResultsWe first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii.ConclusionsOur results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

scholarly journals Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

2021 ◽  
Author(s):  
Masayuki K. Sakata ◽  
Mone U. Kawata ◽  
Atsushi Kurabayashi ◽  
Takaki Kurita ◽  
Masatoshi Nakamura ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Biodiversity Monitoring ◽  
Natural Ecosystems ◽  
Universal Primer ◽  
Primer Sets

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding-analysis of extra-organismal DNA released into the environment-allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set Amph16S had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

scholarly journals Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas C. A. Hitch ◽  
Nicole Treichel ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Careful Attention ◽  
Operational Taxonomic Units ◽  
Basic Concepts ◽  
Gnotobiotic Mice ◽  
Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

scholarly journals Detection of Food Spoilage and Pathogenic Bacteria Based on Ligation Detection Reaction Coupled to Flow-Through Hybridization on Membranes

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
K. Böhme ◽  
P. Cremonesi ◽  
M. Severgnini ◽  
Tomás G. Villa ◽  
I. C. Fernández-No ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Cost Effective ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Dot Blot ◽  
Culture Collections ◽  
Ligation Detection Reaction ◽  
Flow Through ◽  
Food Safety And Quality

Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific forAeromonasspp.,Pseudomonasspp.,Shewanellaspp., andMorganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.

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