In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

AbstractThis last decade, environmental DNA metabarcoding approaches have been developed and improved to minimize biological and technical biases; some challenges, however, remain, as the design of primers. Here we have performed a comprehensive assessment of ten COI and two 16S primer sets. We have combined in silico, in vivo-mock community of 33 arthropod taxa from 16 orders and guano analyses to identify primer sets that should maximize arthropod detection and taxonomic identification, whilst identifying bat species and minimizing labour time and cost. We have focused on two insectivorous bat species living in mixed-colonies, the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy’s bat (Myotis emarginatus). We have found that the level of primer degeneracy is the main factor influencing arthropod detection for in silico and mock community analyses, while the amplicon length is critical for the detection of arthropods from degraded DNA samples. Our results confirm the importance of performing predator detection and taxonomic identification, simultaneously with arthropod sequencing, as faeces samples can be contaminated by different insectivorous species. Moreover, amplifying bat DNA does not affect the primers’ capacity to detect arthropods. We therefore recommend the systematic simultaneous identification of predator and prey. Finally, we evidenced that one third of the prey occurrences are unreliable and probably not of primary interest in diet studies, which might decrease the relevance of combining several primer sets instead of using one efficient primer set. In conclusion, this study provides general criteria enabling the selection of primers whilst considering different scientific and methodological constraints.

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Short COI markers for freshwater macroinvertebrate metabarcoding

10.7287/peerj.preprints.3037v2 ◽

2017 ◽

Author(s):

Ecaterina Edith Vamos ◽

Vasco Elbrecht ◽

Florian Leese

Keyword(s):

In Silico ◽

Detection Efficiency ◽

In Silico Analysis ◽

Environmental Dna ◽

Degraded Dna ◽

Macroinvertebrate Communities ◽

Universal Primer ◽

Primer Sets ◽

Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

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In Silico and Experimental Evaluation of Primer Sets for Species-Level Resolution of the Vaginal Microbiota Using 16S Ribosomal RNA Gene Sequencing

The Journal of Infectious Diseases ◽

10.1093/infdis/jiy508 ◽

2018 ◽

Vol 219 (2) ◽

pp. 305-314 ◽

Cited By ~ 10

Author(s):

William J Van Der Pol ◽

Ranjit Kumar ◽

Casey D Morrow ◽

Eugene E Blanchard ◽

Christopher M Taylor ◽

...

Keyword(s):

Ribosomal Rna ◽

Experimental Evaluation ◽

In Silico ◽

Gene Sequencing ◽

Species Level ◽

Ribosomal Rna Gene ◽

Operational Taxonomic Units ◽

16S Ribosomal Rna Gene ◽

Primer Sets ◽

Level Identification

V4 sequence reads clustered at 99% identity and assigned to operational taxonomic units using the 99% clustered, extended Greengenes database provided optimal species-level identification of vaginal bacteria. This method provided results similar to those obtained with DADA2 and/or using the SILVA database.

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Short COI markers for freshwater macroinvertebrate metabarcoding

10.7287/peerj.preprints.3037v1 ◽

2017 ◽

Author(s):

Ecaterina Edith Vamos ◽

Vasco Elbrecht ◽

Florian Leese

Keyword(s):

In Silico ◽

In Silico Analysis ◽

Degraded Dna ◽

Macroinvertebrate Communities ◽

Universal Primer ◽

Primer Sets ◽

Clear Differentiation ◽

Silico Analysis ◽

Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using Cytochrome c oxidase I (COI) metabarcoding. However, in cases where only degraded DNA is available, e.g. from poorly conserved museum specimens, eDNA filtered from water or gut content analyses, universal primer sets that amplify only a short COI fragment are advantageous. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vivo by sequencing previously used freshwater macroinvertebrate mock communities of known composition and three monitoring samples from Romanian streams. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vivo , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2/BR2) revealed that efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

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BioLaboro: A bioinformatics system for detecting molecular assay signature erosion and designing new assays in response to emerging and reemerging pathogens

10.1101/2020.04.08.031963 ◽

2020 ◽

Author(s):

Mitchell Holland ◽

Daniel Negrón ◽

Shane Mitchell ◽

Nate Dellinger ◽

Mychal Ivancich ◽

...

Keyword(s):

In Silico ◽

Sequence Data ◽

Use Cases ◽

Detection Accuracy ◽

The Novel ◽

Nucleoprotein Gene ◽

Pcr Assays ◽

Primer Sets ◽

Novel Coronavirus ◽

Pathogen Genome

AbstractBackgroundEmerging and reemerging infectious diseases such as the novel Coronavirus disease, COVID-19 and Ebola pose a significant threat to global society and test the public health community’s preparedness to rapidly respond to an outbreak with effective diagnostics and therapeutics. Recent advances in next generation sequencing technologies enable rapid generation of pathogen genome sequence data, within 24 hours of obtaining a sample in some instances. With these data, one can quickly evaluate the effectiveness of existing diagnostics and therapeutics using in silico approaches. The propensity of some viruses to rapidly accumulate mutations can lead to the failure of molecular detection assays creating the need for redesigned or newly designed assays.ResultsHere we describe a bioinformatics system named BioLaboro to identify signature regions in a given pathogen genome, design PCR assays targeting those regions, and then test the PCR assays in silico to determine their sensitivity and specificity. We demonstrate BioLaboro with two use cases: Bombali Ebolavirus (BOMV) and the novel Coronavirus 2019 (SARS-CoV-2). For the BOMV, we analyzed 30 currently available real-time reverse transcription-PCR assays against the three available complete genome sequences of BOMV. Only two met our in silico criteria for successful detection and neither had perfect matches to the primer/probe sequences. We designed five new primer sets against BOMV signatures and all had true positive hits to the three BOMV genomes and no false positive hits to any other sequence. Four assays are closely clustered in the nucleoprotein gene and one is located in the glycoprotein gene. Similarly, for the SARS-CoV-2, we designed five highly specific primer sets that hit all 145 whole genomes (available as of February 28, 2020) and none of the near neighbors.ConclusionsHere we applied BioLaboro in two real-world use cases to demonstrate its capability; 1) to identify signature regions, 2) to assess the efficacy of existing PCR assays to detect pathogens as they evolve over time, and 3) to design new assays with perfect in silico detection accuracy, all within hours, for further development and deployment. BioLaboro is designed with a user-friendly graphical user interface for biologists with limited bioinformatics experience.

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Lessons and Considerations for the Creation of Universal Primers Targeting Non-Conserved, Horizontally Mobile Genes

Applied and Environmental Microbiology ◽

10.1128/aem.02181-20 ◽

2020 ◽

Author(s):

Damon C. Brown ◽

Raymond J. Turner

Keyword(s):

In Silico ◽

Primer Design ◽

Detection Methods ◽

Universal Primers ◽

Sequence Alignments ◽

Universal Primer ◽

Conserved Genes ◽

Primer Sets ◽

Conserved Core ◽

Core Genes

Effective and accurate primer design is an increasingly important skill as the use of PCR-based diagnostics in clinical and environmental settings is on the rise. While universal primer sets have been successfully designed for highly conserved core genes such as 16S rRNA and characteristic genes such as dsrAB and dnaJ, primer sets for mobile, accessory genes such as multidrug resistance efflux pumps (MDREP) have not been explored. Here, we describe an approach to create universal primer sets for select MDREP genes chosen from five superfamilies (SMR, MFS, MATE, ABC and RND) identified in a model community of six members (Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica). Using sequence alignments and in silico PCR analyses, a new approach for creating universal primers sets targeting mobile, non-conserved genes has been developed and compared to more traditional approaches used for highly conserved genes. A discussion of the potential shortfalls of the primer sets designed this way are described. The approach described here can be adapted to any unique gene set and aid in creating a wider, more robust library of primer sets to detect less conserved genes and improve the field of PCR-based screening research. IMPORTANCE Increasing use of molecular detection methods, specifically PCR and qPCR, requires utmost confidence in the results while minimizing false positives and negatives due to poor primer designs. Frequently, these detection methods are focused on conserved, core genes which limits their applications. These screening methods are being used in various industries for specific genetic targets or key organisms such as viral or infectious strains, or characteristic genes indicating the presence of key metabolic processes. The significance of this work is to improve primer design approaches to broaden the scope of detectable genes. The use of the techniques explored here will improve detection of non-conserved genes through unique primer design approaches. Additionally, the approaches here highlight additional, important information which can be gleaned during the in silico phase of primer design which will improve our gene annotations based on percent identities.

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Short COI markers for freshwater macroinvertebrate metabarcoding

10.7287/peerj.preprints.3037 ◽

2017 ◽

Cited By ~ 1

Author(s):

Ecaterina Edith Vamos ◽

Vasco Elbrecht ◽

Florian Leese

Keyword(s):

In Silico ◽

Detection Efficiency ◽

In Silico Analysis ◽

Environmental Dna ◽

Degraded Dna ◽

Macroinvertebrate Communities ◽

Universal Primer ◽

Primer Sets ◽

Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

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

10.7287/peerj.preprints.2044v1 ◽

2016 ◽

Cited By ~ 1

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.

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Lessons and considerations for the creation of universal primers targeting non-conserved, horizontally mobile genes

10.1101/2020.03.30.017442 ◽

2020 ◽

Author(s):

Damon C. Brown ◽

Raymond J. Turner

Keyword(s):

In Silico ◽

Primer Design ◽

Detection Methods ◽

Universal Primers ◽

Sequence Alignments ◽

Universal Primer ◽

Conserved Genes ◽

Primer Sets ◽

Conserved Core ◽

Core Genes

AbstractEffective and accurate primer design is an increasingly important skill as the use of PCR-based diagnostics in clinical and environmental settings is on the rise. While universal primer sets have been successfully designed for highly conserved core genes such as 16S rRNA and characteristic genes such as dsrAB and dnaJ, primer sets for mobile, accessory genes such as multidrug resistance efflux pumps (MDREP) have not been explored. Here, we describe an approach to create universal primer sets for select MDREP genes chosen from five superfamilies (SMR, MFS, MATE, ABC and RND) identified in a model community of six members (Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica). Using sequence alignments and in silico PCR analyses, a new approach for creating universal primers sets targeting mobile, non-conserved genes has been developed and compared to more traditional approaches used for highly conserved genes. A discussion of the potential shortfalls of the primer sets designed this way are described. The approach described here can be adapted to any unique gene set and aid in creating a wider, more robust library of primer sets to detect less conserved genes and improve the field of PCR-based screening research.ImportanceIncreasing use of molecular detection methods, specifically PCR and qPCR, requires utmost confidence in the results while minimizing false positives and negatives due to poor primer designs. Frequently, these detection methods are focused on conserved, core genes which limits their applications. These screening methods are being used in various industries for specific genetic targets or key organisms such as viral or infectious strains, or characteristic genes indicating the presence of key metabolic processes. The significance of this work is to improve primer design approaches to broaden the scope of detectable genes. The use of the techniques explored here will improve detection of non-conserved genes through unique primer design approaches. Additionally, the approaches here highlight additional, important information which can be gleaned during the in silico phase of primer design which will improve our gene annotations based on sequence homologies.

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In Silico Toxicology

10.1039/9781849732093 ◽

2010 ◽

Cited By ~ 30

Keyword(s):

In Silico

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