scholarly journals Validation and development of COI metabarcoding primers for freshwater macroinvertebrate bioassessment

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Human Impacts ◽  
Biodiversity Loss ◽  
Freshwater Ecosystems ◽  
Amplification Efficiency ◽  
Mock Community ◽  
Sequence Alignments ◽  
Freshwater Invertebrate ◽  
Dna Metabarcoding

A central challenge in the present era of biodiversity loss is to assess and manage human impacts on freshwater ecosystems. Macroinvertebrates are an important group for bioassessment as many taxa show specific responses to environmental conditions. However, generating accurate macroinvertebrate inventories based on larval morphology is difficult and error-prone. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify invertebrates in bulk samples to the species level, has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for bioassessment critically relies on carefully evaluating primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 most globally relevant freshwater invertebrate groups for stream assessment. Using these sequence alignments, we developed four primer combinations optimized for freshwater macrozoobenthos. All primers were evaluated by sequencing ten mock community samples, each consisting of 52 freshwater invertebrate taxa. Additionally, popular metabarcoding primers from the literature and the developed primers were tested in silico against the 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the number of detected taxa, yet all detected more taxa than standard ‘Folmer’ barcoding primers. Two new primer combinations showed more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that some primers currently used in metabarcoding studies may not be suitable for amplification of freshwater macroinvertebrates. Therefore, careful primer evaluation and more region / ecosystem specific primers are needed before DNA metabarcoding can be used for routine bioassessment of freshwater ecosystems.

scholarly journals Validation and development of freshwater invertebrate metabarcoding COI primers for Environmental Impact Assessment

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Human Impacts ◽  
Biodiversity Loss ◽  
Freshwater Ecosystems ◽  
Amplification Efficiency ◽  
Mock Community ◽  
Sequence Alignments ◽  
Freshwater Invertebrate ◽  
Dna Metabarcoding

A central challenge in the present era of biodiversity loss is to assess and manage human impacts on freshwater ecosystems. Macroinvertebrates are an ideal group for Environmental Impacts Assessment (EIA). However, generating accurate macroinvertebrate inventories based on larval morphology is difficult and error-prone. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify invertebrates in bulk samples at the species level, has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for EIA critically relies on carefully evaluating primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 most globally relevant freshwater invertebrate groups in EIAs. Using these sequence alignments, we developed four primer combinations optimized for freshwater macrozoobenthos. All primers were evaluated by sequencing ten mock community samples each consisting of 52 freshwater invertebrate taxa. Additionally, popular metabarcoding primers from the literature and the developed primers were tested in silico against the 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the number of detected taxa, yet all detected more taxa than standard ‘Folmer’ barcoding primers. Two new primer combinations showed more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that some primers currently used in metabarcoding studies may not be suitable for amplification of insect and freshwater taxa. Therefore, careful primer evaluation and more region / ecosystem specific primers are needed before DNA metabarcoding can be used for routine EIA of freshwater ecosystems.

scholarly journals Validation and development of freshwater invertebrate metabarcoding COI primers for Environmental Impact Assessment

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Human Impacts ◽  
Biodiversity Loss ◽  
Freshwater Ecosystems ◽  
Amplification Efficiency ◽  
Mock Community ◽  
Sequence Alignments ◽  
Freshwater Invertebrate ◽  
Dna Metabarcoding

To assess and manage human impacts on freshwater ecosystems is a central challenge in the present era of biodiversity loss. Macroinvertebrates are an ideal group for Environmental Impacts Assessment (EIA). However, generating accurate macroinvertebrate inventories is difficult and error-prone based on larval morphology. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify bulk invertebrates on species level has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for EIA critically relies on carefully evaluated primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 globally most relevant freshwater invertebrate groups in EIAs. Using these sequence alignments, we developed four primers combinations optimized for freshwater macrozoobenthos. All primers were evaluated by sequencing ten mock community samples each consisting of 52 freshwater invertebrate taxa. Additionally popular metabarcoding primers from the literature and the here developed primers were tested in silico against the 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the amount of detected taxa, yet all retrieved more taxa than standard ‘Folmer’ barcoding primers. Two new primer combinations showed more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that not all primers currently used in metabarcoding studies are likely not suitable for amplification of insect and freshwater taxa. Thus careful primer evaluation and more region / ecosystem specific primers might be needed, before DNA metabarcoding can be used for routine EIA of freshwater ecosystems.

scholarly journals Validation and development of COI metabarcoding primers for freshwater macroinvertebrate bioassessment

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Human Impacts ◽  
Biodiversity Loss ◽  
Freshwater Ecosystems ◽  
Amplification Efficiency ◽  
Mock Community ◽  
Sequence Alignments ◽  
Freshwater Invertebrate ◽  
Dna Metabarcoding

A central challenge in the present era of biodiversity loss is to assess and manage human impacts on freshwater ecosystems. Macroinvertebrates are an important group for bioassessment as many taxa show specific responses to environmental conditions. However, generating accurate macroinvertebrate inventories based on larval morphology is difficult and error-prone. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify invertebrates in bulk samples to the species level, has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for bioassessment critically relies on carefully evaluating primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 most globally relevant freshwater invertebrate groups for stream assessment. Using these sequence alignments, we developed four primer combinations optimized for freshwater macrozoobenthos. All primers were evaluated by sequencing ten mock community samples, each consisting of 52 freshwater invertebrate taxa. Additionally, popular metabarcoding primers from the literature and the developed primers were tested in silico against the 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the number of detected taxa, yet all detected more taxa than standard ‘Folmer’ barcoding primers. Two new primer combinations showed more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that some primers currently used in metabarcoding studies may not be suitable for amplification of freshwater macroinvertebrates. Therefore, careful primer evaluation and more region / ecosystem specific primers are needed before DNA metabarcoding can be used for routine bioassessment of freshwater ecosystems.

scholarly journals Development and validation of DNA metabarcoding COI primers for aquatic invertebrates using the R package "PrimerMiner"

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Dna Barcode ◽  
R Package ◽  
Gene Marker ◽  
Amplification Efficiency ◽  
Mock Community ◽  
Freshwater Invertebrate ◽  
Dna Metabarcoding ◽  
High Base

1) DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. However, typically used barcoding genes, such as the cytochrome c oxidase subunit I (COI) region for animals, are highly variable. Thus, different taxa in communities under study are often not amplified equally well and some might even remain undetected due to primer bias. To reduce these problems, optimized metabarcoding primers for the typical communities found in certain geographic regions- and/or ecosystems are necessary. 2) We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxonomic groups and then applies sequence clustering to reduce biases introduced by the different number of available sequences per species. We downloaded COI data for the 15 most relevant freshwater invertebrate groups for stream ecosystem assessment and developed four primer sets with high base degeneracy based on that. Primer performance was tested by sequencing ten mock community samples each consisting of 52 freshwater invertebrate taxa. Additionally, we used PrimerMiner to evaluate the developed primers against other metabarcoding primers in silico. 3) The developed primers varied in amplification efficiency and the amount of detected taxa, yet all retrieved more taxa than standard Folmer barcoding primers. Additionally, the BF/BR primers amplified taxa very consistently, with the BF2+BR2 and BF2+BR1 primer combinations showing better amplification than a previously tested ribosomal marker (16S). Except for the BF1+BR1 primers all BF/BR primers combinations detected all 42 insect taxa present in the mock community samples. In silico evaluation of the developed primers demonstrates their suitability for metabarcoding of non-aquatic insect samples. 4) With PrimerMiner we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. Our sequence datasets generated with the newly developed metabarcoding primers demonstrate that the design of optimized primers with high base degeneracy is superior to classical markers and enables us to detect almost 100% of animal taxa present in a sample using the standard COI barcoding gene. Therefore, the PrimerMiner package and the developed primers are useful beyond biodiversity assessment in aquatic ecosystems.

scholarly journals Development and validation of DNA metabarcoding COI primers for aquatic invertebrates using the R package "PrimerMiner"

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Dna Barcode ◽  
R Package ◽  
Gene Marker ◽  
Amplification Efficiency ◽  
Mock Community ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
High Base

1) DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. However, typically used barcoding genes, such as the cytochrome c oxidase subunit I (COI) region for animals, are highly variable. Thus, different taxa in communities under study are often not amplified equally well and some might even remain undetected due to primer bias. To reduce these problems, optimized region- and/or ecosystem- specific metabarcoding primers are necessary. 2) We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxa and then applies sequence clustering to reduce biases introduced by differed number of available sequences per species. To design primers targeted for freshwater invertebrates, we downloaded COI data for the 15 most important invertebrate groups relevant for stream ecosystem assessment. Four primer sets with high base degeneracy were developed and their performance tested by sequencing ten mock community samples consisting each of 52 freshwater invertebrate taxa. Additionally, we evaluated the developed primers against other metabarcoding primers in silico using PrimerMiner. 3) Amplification and sequencing was successful for all ten mock community samples with the four different primer combinations. The developed primers varied in amplification efficiency and amount of taxa detected, but all primer sets detected more taxa than standard Folmer barcoding primers. Additionally, the BF / BR primers amplified taxa very consistently, the BF2+BR2 and BF2+BR1 primer combination even better than a previously tested ribosomal marker (16S). Except for the BF1+BR1 primer combination, all BF / BR primers detected all 42 insect taxa present in the mock samples. In silico evaluation of the developed primers showed that they are also likely to work very well on other non aquatic invertebrate samples. 4) With PrimerMiner, we here provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. Our sequence datasets generated with the newly developed metabarcoding primers demonstrate that the design of optimized primers with high base degeneracy is superior to classical markers and enable us to detect almost 100% of animal taxa present in a sample using the standard COI barcoding gene. Therefore, the PrimerMiner package and primers developed using this tool are useful beyond assessment of biodiversity in aquatic ecosystems.

scholarly journals PrimerMiner: an R package for development and in silico validation of DNA metabarcoding primers

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Dna Barcode ◽  
R Package ◽  
Primer Design ◽  
Reference Sequence ◽  
Gene Marker ◽  
Sequence Alignments ◽  
Variable Binding ◽  
Dna Metabarcoding

1) DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that serve as a backbone for primer design is currently available. 2) We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico, which are in our opinion more realistic then the strategy employed in another available software for that purpose, ecoPCR. 3) We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both databases, we were able to increase the amount of reference data 249-fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner. 4) With PrimerMiner we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU based reference alignments generated with PrimerMiner can be used for manual primer design, or processed with bioinformatic tools for primer development.

scholarly journals PrimerMiner: an R package for development and in silico validation of DNA metabarcoding primers

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Dna Barcode ◽  
R Package ◽  
Primer Design ◽  
Reference Sequence ◽  
Gene Marker ◽  
Sequence Alignments ◽  
Variable Binding ◽  
Dna Metabarcoding

1) DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that serve as a backbone for primer design is currently available. 2) We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico, which are in our opinion more realistic then the strategy employed in another available software for that purpose, ecoPCR. 3) We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both databases, we were able to increase the amount of reference data 249-fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner. 4) With PrimerMiner we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU based reference alignments generated with PrimerMiner can be used for manual primer design, or processed with bioinformatic tools for primer development.

scholarly journals Whole genome characterization of strains belonging to the Ralstonia solanacearum species complex and in silico analysis of TaqMan assays for detection in this heterogenous species complex

2021 ◽  
Author(s):  
Viola Kurm ◽  
Ilse Houwers ◽  
Claudia E. Coipan ◽  
Peter Bonants ◽  
Cees Waalwijk ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
In Silico ◽  
Species Complex ◽  
Sequence Data ◽  
In Silico Analysis ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genome Sequences ◽  
Pcr Assays

AbstractIdentification and classification of members of the Ralstonia solanacearum species complex (RSSC) is challenging due to the heterogeneity of this complex. Whole genome sequence data of 225 strains were used to classify strains based on average nucleotide identity (ANI) and multilocus sequence analysis (MLSA). Based on the ANI score (>95%), 191 out of 192(99.5%) RSSC strains could be grouped into the three species R. solanacearum, R. pseudosolanacearum, and R. syzygii, and into the four phylotypes within the RSSC (I,II, III, and IV). R. solanacearum phylotype II could be split in two groups (IIA and IIB), from which IIB clustered in three subgroups (IIBa, IIBb and IIBc). This division by ANI was in accordance with MLSA. The IIB subgroups found by ANI and MLSA also differed in the number of SNPs in the primer and probe sites of various assays. An in-silico analysis of eight TaqMan and 11 conventional PCR assays was performed using the whole genome sequences. Based on this analysis several cases of potential false positives or false negatives can be expected upon the use of these assays for their intended target organisms. Two TaqMan assays and two PCR assays targeting the 16S rDNA sequence should be able to detect all phylotypes of the RSSC. We conclude that the increasing availability of whole genome sequences is not only useful for classification of strains, but also shows potential for selection and evaluation of clade specific nucleic acid-based amplification methods within the RSSC.

scholarly journals A First Glimpse on Cold-Adapted PCB-Oxidizing Bacteria in Edmonson Point Lakes (Northern Victoria Land, Antarctica)

Water ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 109
Author(s):  
Maria Papale ◽  
Angelina Lo Giudice ◽  
Alessandro Ciro Rappazzo ◽  
Maurizio Azzaro ◽  
Carmen Rizzo
Keyword(s):  
Human Impacts ◽  
Current Knowledge ◽  
Freshwater Ecosystems ◽  
Trophic Levels ◽  
Cold Adapted ◽  
Remote Areas ◽  
Victoria Land ◽  
Long Lifetime ◽  
Aroclor 1242 ◽  
Edmonson Point

Antarctic freshwater ecosystems are especially vulnerable to human impacts. Polychlorobiphenyls (PCBs) are persistent organic pollutants that have a long lifetime in the environment. Despite their use having either been phased out or restricted, they are still found in nature, also in remote areas. Once in the environment, the fate of PCBs is strictly linked to bacteria which represent the first step in the transfer of toxic compounds to higher trophic levels. Data on PCB-oxidizing bacteria from polar areas are still scarce and fragmented. In this study, the occurrence of PCB-oxidizing cold-adapted bacteria was evaluated in water and sediment of four coastal lakes at Edmonson Point (Northern Victoria Land, Antarctica). After enrichment with biphenyl, 192 isolates were obtained with 57 of them that were able to grow in the presence of the PCB mixture Aroclor 1242, as the sole carbon source. The catabolic gene bphA, as a proxy for PCB degradation potential, was harbored by 37 isolates (out of 57), mainly affiliated to the genera Salinibacterium, Arthrobacter (among Actinobacteria) and Pusillimonas (among Betaproteobacteria). Obtained results enlarge our current knowledge on cold-adapted PCB-oxidizing bacteria and pose the basis for their potential application as a valuable eco-friendly tool for the recovery of PCB-contaminated cold sites.

scholarly journals Diversity of echinostomes (Digenea: Echinostomatidae) in their snail hosts at high latitudes

Parasite ◽  
2021 ◽  
Vol 28 ◽  
pp. 59
Author(s):  
Camila Pantoja ◽  
Anna Faltýnková ◽  
Katie O’Dwyer ◽  
Damien Jouet ◽  
Karl Skírnisson ◽  
...  
Keyword(s):  
North America ◽  
Dna Sequences ◽  
Sequence Data ◽  
Molecular Data ◽  
Migratory Bird ◽  
Parasite Fauna ◽  
Life Cycles ◽  
Freshwater Ecosystems ◽  
Trematode Species ◽  
Host Life

The biodiversity of freshwater ecosystems globally still leaves much to be discovered, not least in the trematode parasite fauna they support. Echinostome trematode parasites have complex, multiple-host life-cycles, often involving migratory bird definitive hosts, thus leading to widespread distributions. Here, we examined the echinostome diversity in freshwater ecosystems at high latitude locations in Iceland, Finland, Ireland and Alaska (USA). We report 14 echinostome species identified morphologically and molecularly from analyses of nad1 and 28S rDNA sequence data. We found echinostomes parasitising snails of 11 species from the families Lymnaeidae, Planorbidae, Physidae and Valvatidae. The number of echinostome species in different hosts did not vary greatly and ranged from one to three species. Of these 14 trematode species, we discovered four species (Echinoparyphium sp. 1, Echinoparyphium sp. 2, Neopetasiger sp. 5, and Echinostomatidae gen. sp.) as novel in Europe; we provide descriptions for the newly recorded species and those not previously associated with DNA sequences. Two species from Iceland (Neopetasiger islandicus and Echinoparyphium sp. 2) were recorded in both Iceland and North America. All species found in Ireland are new records for this country. Via an integrative taxonomic approach taken, both morphological and molecular data are provided for comparison with future studies to elucidate many of the unknown parasite life cycles and transmission routes. Our reports of species distributions spanning Europe and North America highlight the need for parasite biodiversity assessments across large geographical areas.

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