Slippage of degenerate primers can cause variation in amplicon length

Amplicon Length ◽

Slippage Effect ◽

Primer Sets ◽

Metabarcoding studies often employ degenerate primers to reduce amplification bias and increase the number of detected taxa. However, degeneracy has the disadvantage of lowering binding specificity although the exact mechanisms and potential biases introduced by such off-target amplification are not fully understood. We examined sequences recovered from the ten most abundant operational taxonomic units (OTUs) in two mock communities to investigate the specificity and binding behavior of five degenerate primer sets. Our results indicate that primers frequently bound 1-2 bp upstream in taxa where a homopolymer region was present in the amplification direction. As well, although less frequent, degeneracy occasionally led to primer binding 1 bp downstream. Some widely used primer sets were severely affected by this slippage effect, while others were not. Our study shows that primer slippage can produce taxon-specific length variation in amplicons and subsequent length variation in recovered sequences. While this variation will only have small impacts on OTU designation by clustering algorithms that ignore terminal gaps, primer sets employed in metabarcoding projects should be evaluated for their sensitivity to slippage. Moreover, steps should be taken to reduce slippage by improving protocols for primer design. For example, the flanking region adjacent to the 3' end of the primer is not considered by current primer development software although GC clamps in this position could mitigate slippage. While degeneracy is important to ensure the universality of a primer, binding in homopolymer regions should be avoided.

New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil

Applied and Environmental Microbiology ◽

10.1128/aem.70.1.240-247.2004 ◽

2004 ◽

Vol 70 (1) ◽

pp. 240-247 ◽

Cited By ~ 98

Author(s):

Helmut Bürgmann ◽

Franco Widmer ◽

William Von Sigler ◽

Josef Zeyer

Keyword(s):

Marker Gene ◽

Screening Tools ◽

The Novel ◽

Degenerate Primers ◽

Free Living ◽

Universal Primer ◽

Amplification Bias ◽

Pcr Products ◽

Primer Sets ◽

Different Soils

ABSTRACT Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.

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

ISME Communications ◽

10.1038/s43705-021-00033-z ◽

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 ◽

Basic Concepts ◽

Gnotobiotic Mice ◽

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.

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

Bulletin of Entomological Research ◽

10.1017/s0007485315000681 ◽

2015 ◽

Vol 105 (6) ◽

pp. 717-727 ◽

Cited By ~ 83

Author(s):

G.-J. Brandon-Mong ◽

H.-M. Gan ◽

K.-W. Sing ◽

P.-S. Lee ◽

P.-E. Lim ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Illumina Miseq ◽

Ease Of Use ◽

Malaise Trap ◽

Detection Rates ◽

Arthropod Species ◽

Primer Sets ◽

Arthropod Biodiversity

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

Validation of COI metabarcoding primers for terrestrial arthropods

10.7287/peerj.preprints.27801v2 ◽

2019 ◽

Cited By ~ 1

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.

Validation of COI metabarcoding primers for terrestrial arthropods

10.7287/peerj.preprints.27801 ◽

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.

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 ◽

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.

Accuracy of microbial community diversity estimated by closed- and open-reference OTUs

PeerJ ◽

10.7717/peerj.3889 ◽

2017 ◽

Vol 5 ◽

pp. e3889 ◽

Cited By ~ 69

Author(s):

Robert C. Edgar

Keyword(s):

Ribosomal Rna ◽

De Novo ◽

Community Diversity ◽

Reference Database ◽

Mock Community ◽

Variable Regions ◽

Sequencing Technologies ◽

Generation Sequencing ◽

Next-generation sequencing of 16S ribosomal RNA is widely used to survey microbial communities. Sequences are typically assigned to Operational Taxonomic Units (OTUs). Closed- and open-reference OTU assignment matches reads to a reference database at 97% identity (closed), then clusters unmatched reads using a de novo method (open). Implementations of these methods in the QIIME package were tested on several mock community datasets with 20 strains using different sequencing technologies and primers. Richness (number of reported OTUs) was often greatly exaggerated, with hundreds or thousands of OTUs generated on Illumina datasets. Between-sample diversity was also found to be highly exaggerated in many cases, with weighted Jaccard distances between identical mock samples often close to one, indicating very low similarity. Non-overlapping hyper-variable regions in 70% of species were assigned to different OTUs. On mock communities with Illumina V4 reads, 56% to 88% of predicted genus names were false positives. Biological inferences obtained using these methods are therefore not reliable.

Untapped sponge microbiomes: structure specificity at host order and family levels

FEMS Microbiology Ecology ◽

10.1093/femsec/fiz136 ◽

2019 ◽

Vol 95 (9) ◽

Cited By ~ 1

Author(s):

Qi Yang ◽

Christopher M M Franco ◽

Hou-Wen Lin ◽

Wei Zhang

Keyword(s):

Species Level ◽

Rrna Gene ◽

Class Level ◽

Microbial Resources ◽

Host Phylogeny ◽

Shared Otus ◽

Primer Sets ◽

The 16S Rrna Gene ◽

Unique Otus

ABSTRACT Sponges are complex holobionts in which the structure of the microbiome has seldom been characterized above the host species level. The hypothesis tested in this study is that the structure of the sponge microbiomes is specific to the host at the order and family levels. This was done by using 33 sponge species belonging to 19 families representing five orders. A combination of three primer sets covering the V1-V8 regions of the 16S rRNA gene provided a more comprehensive coverage of the microbiomes. Both the diversity and structure of sponge microbiomes were demonstrated to be highly specific to the host phylogeny at the order and family levels. There are always dominant operational taxonomic units (OTUs) (relative abundance >1%) shared between microbial communities of sponges within the same family or order, but these shared OTUs showed high levels of dissimilarity between different sponge families and orders. The unique OTUs for a particular sponge family or order could be regarded as their ‘signature identity’. 70%–87% of these unique OTUs (class level) are unaffiliated and represent a vast resource of untapped microbiota. This study contributes to a deeper understanding on the concept of host-specificity of sponge microbiomes and highlights a hidden reservoir of sponge-associated microbial resources.

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 ◽

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.