scholarly journals Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community

2018 ◽  
Author(s):  
Thomas W. A. Braukmann ◽  
Natalia V. Ivanova ◽  
Sean W. J. Prosser ◽  
Vasco Elbrecht ◽  
Dirk Steinke ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Ion Torrent ◽  
Mock Community ◽  
Species Recovery ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Terrestrial Arthropods ◽  
Dna Metabarcoding ◽  
Powerful Approach ◽  
Read Abundance

AbstractDNA metabarcoding is an attractive approach for monitoring biodiversity. However, it is subject to biases that often impede detection of all species in a sample. In particular, the proportion of sequences recovered from each species depends on its biomass, mitome copy number, and primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 BINs, a species proxy. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body partitions (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicon generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, other variables did. As expected, the best recovery was obtained from the Single Leg treatment, but the Bulk Abdomen produced a more uniform read abundance than the Bulk Leg or Composite Leg samples. Primer choice also influenced species recovery. Our results reveal how variation in protocols can have substantive impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote. Although metabarcoding is a powerful approach, further optimization of analytical protocols is crucial to obtain reproducible results and increase its cost-effectiveness.

scholarly journals Impact of DNA Sequencing and Analysis Methods on 16S rRNA Gene Bacterial Community Analysis of Dairy Products

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Zhengyao Xue ◽  
Mary E. Kable ◽  
Maria L. Marco
Keyword(s):  
16S Rrna ◽  
Dna Sequencing ◽  
Dairy Products ◽  
Bacterial Species ◽  
Ion Torrent ◽  
Mock Community ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Sequencing Method ◽  
Mock Communities

ABSTRACT DNA sequencing and analysis methods were compared for 16S rRNA V4 PCR amplicon and genomic DNA (gDNA) mock communities encompassing nine bacterial species commonly found in milk and dairy products. The two communities comprised strain-specific DNA that was pooled before (gDNA) or after (PCR amplicon) the PCR step. The communities were sequenced on the Illumina MiSeq and Ion Torrent PGM platforms and then analyzed using the QIIME 1 (UCLUST) and Divisive Amplicon Denoising Algorithm 2 (DADA2) analysis pipelines with taxonomic comparisons to the Greengenes and Ribosomal Database Project (RDP) databases. Examination of the PCR amplicon mock community with these methods resulted in operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) that ranged from 13 to 118 and were dependent on the DNA sequencing method and read assembly steps. The additional 4 to 109 OTUs/ASVs (from 9 OTUs/ASVs) included assignments to spurious taxa and sequence variants of the 9 species included in the mock community. Comparisons between the gDNA and PCR amplicon mock communities showed that combining gDNAs from the different strains prior to PCR resulted in up to 8.9-fold greater numbers of spurious OTUs/ASVs. However, the DNA sequencing method and paired-end read assembly steps conferred the largest effects on predictions of bacterial diversity, with effect sizes of 0.88 (Bray-Curtis) and 0.32 (weighted Unifrac), independent of the mock community type. Overall, DNA sequencing performed with the Ion Torrent PGM and analyzed with DADA2 and the Greengenes database resulted in the most accurate predictions of the mock community phylogeny, taxonomy, and diversity. IMPORTANCE Validated methods are urgently needed to improve DNA sequence-based assessments of complex bacterial communities. In this study, we used 16S rRNA PCR amplicon and gDNA mock community standards, consisting of nine, dairy-associated bacterial species, to evaluate the most commonly applied 16S rRNA marker gene DNA sequencing and analysis platforms used in evaluating dairy and other bacterial habitats. Our results show that bacterial metataxonomic assessments are largely dependent on the DNA sequencing platform and read curation method used. DADA2 improved sequence annotation compared with QIIME 1, and when combined with the Ion Torrent PGM DNA sequencing platform and the Greengenes database for taxonomic assignment, the most accurate representation of the dairy mock community standards was reached. This approach will be useful for validating sample collection and DNA extraction methods and ultimately investigating bacterial population dynamics in milk- and dairy-associated environments.

scholarly journals Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

2019 ◽  
Author(s):  
Rachel L. Marine ◽  
Laura C. Magaña ◽  
Christina J. Castro ◽  
Kun Zhao ◽  
Anna M. Montmayeur ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Ion Torrent ◽  
Library Preparation ◽  
Genome Sequences ◽  
Sequencing Platform ◽  
Viral Genomes ◽  
Ion Torrent Pgm ◽  
Miseq Platform ◽  
Sequencing Platforms ◽  
The Cost

ABSTRACTNext-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

scholarly journals Impact of DNA sequencing and analysis methods on 16S rRNA gene bacterial community analysis in dairy products

2018 ◽  
Author(s):  
Zhengyao Xue ◽  
Mary E Kable ◽  
Maria L Marco
Keyword(s):  
16S Rrna ◽  
Dna Sequencing ◽  
Dairy Products ◽  
Bacterial Species ◽  
Ion Torrent ◽  
Mock Community ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Sequencing Method ◽  
Mock Communities

AbstractDNA sequencing and analysis methods were compared for 16S rRNA V4 PCR amplicon and gDNA mock communities encompassing nine bacterial species commonly found in milk and dairy products. The communities were examined using Illumina MiSeq and Ion Torrent PGM DNA sequencing methods followed by the QIIME 1 (UCLUST) and Divisive Amplicon Denoising Algorithm 2 (DADA2) data analysis pipelines including taxonomic comparisons to the Greengenes and Ribosomal Database Project (RDP) databases. Examination of the PCR amplicon mock community with these methods resulted in Operation Taxonomy Units (OTUs) and Amplicon Sequence Variants (ASVs) that ranged from a low of 13 to high of 118 and were dependent on the DNA sequencing method and read assembly step. The elevated numbers of OTUs and ASVs included assignments to spurious taxa as well as sequence variants of the nine species included in the mock community. Comparisons between the gDNA and PCR amplicon mock communities showed that combining gDNA from the different strains prior to PCR resulted in up to 8.9-fold greater numbers of spurious OTUs and ASVs. However, the DNA sequencing method and initial data assembly steps conferred the largest effects on predictions of bacterial diversity, independent of the mock community type (PCR amplicon or gDNA; Bray-Curtis R2 = 0.88 and weighted Unifrac, R2 = 0.32). Overall, DNA sequencing performed with the Ion Torrent PGM and analyzed with DADA2 and the Greengenes database resulted in the most accurate predictions of the mock community phylogeny, taxonomy, and diversity.ImportanceValidated methods are urgently needed to improve DNA-sequence based assessments of complex bacterial communities. In this study, we used 16S rRNA PCR amplicon and gDNA mock community standards, consisting of nine, dairy-associated bacterial species, to evaluate the most commonly applied 16S rRNA marker gene DNA sequencing and analysis platforms used in evaluating dairy and other bacterial habitats. Our results show that bacterial metataxonomic assessments are largely dependent on the DNA sequencing platform and read curation method used. DADA2 improved sequence annotation compared with QIIME 1, and when combined with the Ion Torrent PGM DNA sequencing platform and the Greengenes database for taxonomic assignment, the most accurate representation of the dairy mock community standards was reached. This approach will be useful for validating sample collection and DNA extraction methods and ultimately investigating bacterial population dynamics in milk and dairy-associated environments.

scholarly journals Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

2020 ◽  
Vol 280 ◽  
pp. 113865 ◽  
Author(s):  
Rachel L. Marine ◽  
Laura C. Magaña ◽  
Christina J. Castro ◽  
Kun Zhao ◽  
Anna M. Montmayeur ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Ion Torrent ◽  
Ion Torrent Pgm

scholarly journals Validation of COI metabarcoding primers for terrestrial arthropods

2019 ◽  
Author(s):  
Vasco Elbrecht ◽  
Thomas WA Braukmann ◽  
Natalia V Ivanova ◽  
Sean WJ Prosser ◽  
Mehrdad Hajibabaei ◽  
...  
Keyword(s):  
Taxonomic Resolution ◽  
Minor Effect ◽  
Malaise Trap ◽  
Mock Community ◽  
Sequencing Platform ◽  
Amplification Bias ◽  
Terrestrial Arthropods ◽  
Primer Sets ◽  
Taxonomic Groups ◽  
Trap Sample

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids ecosystem assessment. However , it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. Thus this study tests the performance of 36 primer sets on a mock community containing 374 species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets where also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from the 21 amplicon pools of the mock community and Malaise trap sample were used to select four primer sets for metabarcoding evaluation at different annealing temperatures (40-60 Co) using the mock community. Temperature did only have a minor effect on taxa recovery that varied with the specific primer pair. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrated that certain primer sets can recover most taxa in a diverse species assemblage. Thus there is no need to employ several primer sets targeting the same amplicon. While we identified several suited primer sets for arthropod metabarcoding, the primer selection depends on the targeted taxonomic groups, as well as DNA quality, desired taxonomic resolution, and sequencing platform employed for analysis.

scholarly journals Validation of COI metabarcoding primers for terrestrial arthropods

2019 ◽  
Author(s):  
Vasco Elbrecht ◽  
Thomas WA Braukmann ◽  
Natalia V Ivanova ◽  
Sean WJ Prosser ◽  
Mehrdad Hajibabaei ◽  
...  
Keyword(s):  
Taxonomic Resolution ◽  
Minor Effect ◽  
Malaise Trap ◽  
Mock Community ◽  
Sequencing Platform ◽  
Amplification Bias ◽  
Terrestrial Arthropods ◽  
Primer Sets ◽  
Taxonomic Groups ◽  
Trap Sample

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids ecosystem assessment. However , it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. Thus this study tests the performance of 36 primer sets on a mock community containing 374 species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets where also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from the 21 amplicon pools of the mock community and Malaise trap sample were used to select four primer sets for metabarcoding evaluation at different annealing temperatures (40-60 Co) using the mock community. Temperature did only have a minor effect on taxa recovery that varied with the specific primer pair. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrated that certain primer sets can recover most taxa in a diverse species assemblage. Thus there is no need to employ several primer sets targeting the same amplicon. While we identified several suited primer sets for arthropod metabarcoding, the primer selection depends on the targeted taxonomic groups, as well as DNA quality, desired taxonomic resolution, and sequencing platform employed for analysis.

scholarly journals A new oomycete metabarcoding method using the rps10 gene

2021 ◽  
Author(s):  
Zachary S. L. Foster ◽  
Felipe E Albornoz ◽  
Valerie J Fieland ◽  
Meredith M Larsen ◽  
Frank Andrew Jones ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Illumina Miseq ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Mock Community ◽  
Operational Taxonomic Units ◽  
Wide Range ◽  
Dna Metabarcoding ◽  
Improved Methods

Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as a locus useful for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on available reference sequences from a wide range of taxa. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Both the sequence and predicted taxonomy of ASVs and OTUs were compared to the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1 bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest the proposed rps10 locus results in substantially less amplification of non-target organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing.

scholarly journals Validation of COI metabarcoding primers for terrestrial arthropods

2019 ◽  
Author(s):  
Vasco Elbrecht ◽  
Thomas WA Braukmann ◽  
Natalia V Ivanova ◽  
Sean WJ Prosser ◽  
Mehrdad Hajibabaei ◽  
...  
Keyword(s):  
Taxonomic Resolution ◽  
Minor Effect ◽  
Malaise Trap ◽  
Mock Community ◽  
Sequencing Platform ◽  
Amplification Bias ◽  
Terrestrial Arthropods ◽  
Primer Sets ◽  
Taxonomic Groups ◽  
Trap Sample

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids ecosystem assessment. However , it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. Thus this study tests the performance of 36 primer sets on a mock community containing 374 species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets where also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from the 21 amplicon pools of the mock community and Malaise trap sample were used to select four primer sets for metabarcoding evaluation at different annealing temperatures (40-60 Co) using the mock community. Temperature did only have a minor effect on taxa recovery that varied with the specific primer pair. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrated that certain primer sets can recover most taxa in a diverse species assemblage. Thus there is no need to employ several primer sets targeting the same amplicon. While we identified several suited primer sets for arthropod metabarcoding, the primer selection depends on the targeted taxonomic groups, as well as DNA quality, desired taxonomic resolution, and sequencing platform employed for analysis.

Efficiency of Corynebacterium pseudotuberculosis Cp31 Genome Assembly with the Hi-Q Enzyme on an Ion Torrent PGM Sequencing Platform

2014 ◽  
Vol 7 (12) ◽  
Author(s):  
Adonney AO Veras Pablo HCG de Sa ◽  
Kenny C Pinheiro Diego Assis ◽  
das Gracas Rafael ◽  
Azevedo Barauna Maria Paula ◽  
Cruz Schneider Vasco Azevedo ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Corynebacterium Pseudotuberculosis ◽  
Ion Torrent ◽  
Sequencing Platform ◽  
Ion Torrent Pgm
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