ARDEP, a Rapid Degenerate Primer Design Pipeline Based on k-mers for Amplicon Microbiome Studies

The survey of microbial diversity in various environments has relied upon the widespread use of well-evaluated amplification primers for taxonomic marker genes (e.g., prokaryotic 16S and fungal ITS). However, it is urgent to develop a fast and accurate bioinformatic program to design primers for microbial functional genes to explore more mechanisms in the microbial community. Here, we provide a rapid degenerate primer design pipeline (ARDEP) based on the k-mer algorithm, which can bypass the time-consuming step of sequence alignment to greatly reduce run times while ensuring accuracy. In addition, we developed an open-access platform for the implementation of primer design projects that could also calculate the amplification product length, GC content, Annealing Temperature (Tm), and ΔG of primer self-folding, and identify covered species and functional groups. Using this new platform, we designed primers for several functional genes in the nitrogen cycle, including napA and amoA. Our newly designed primers achieved higher coverage than the commonly used primers for all tested genes. The program and the associated platform that applied the k-mer algorithm could greatly enhance the design and evaluation of primers for environmental microbiome studies.

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Degenerate primer design system for gene biodiversity study using dynamic pattern matching

2012 7th International Symposium on Health Informatics and Bioinformatics ◽

10.1109/hibit.2012.6209050 ◽

2012 ◽

Author(s):

Weeris Treeratanajaru ◽

Supawin Watcharamul ◽

Rajalida Lipikorn

Keyword(s):

Pattern Matching ◽

Primer Design ◽

Degenerate Primer ◽

Design System ◽

Dynamic Pattern ◽

Biodiversity Study

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GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring

Frontiers in Microbiology ◽

10.3389/fmicb.2021.787651 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ilona A. Ruhl ◽

Andriy Sheremet ◽

Chantel C. Furgason ◽

Susanne Krause ◽

Robert M. Bowers ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Phylogenetic Trees ◽

Hot Spring ◽

Gc Content ◽

Gene Copy ◽

Marker Genes ◽

Rrna Gene ◽

Separate Species ◽

Distinct Subgroup

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.

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MDC-Analyzer: A novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites

BioTechniques ◽

10.2144/000114177 ◽

2014 ◽

Vol 56 (6) ◽

Cited By ~ 11

Author(s):

Lixia Tang ◽

Xiong Wang ◽

Beibei Ru ◽

Hengfei Sun ◽

Jian Huang ◽

...

Keyword(s):

Primer Design ◽

Design Tool ◽

Degenerate Primer

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Accumulating Variation at Conserved Sites in Potyvirus Genomes Is Driven by Species Discovery and Affects Degenerate Primer Design

PLoS ONE ◽

10.1371/journal.pone.0001586 ◽

2008 ◽

Vol 3 (2) ◽

pp. e1586 ◽

Cited By ~ 25

Author(s):

Linda Zheng ◽

Paul J. Wayper ◽

Adrian J. Gibbs ◽

Mathieu Fourment ◽

Brendan C. Rodoni ◽

...

Keyword(s):

Primer Design ◽

Degenerate Primer ◽

Species Discovery

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The Coupling of Carbon, Nitrogen and Sulphur Transformational Processes in River Sediments Based on Correlationship among the Functional Genes

10.5194/bg-2018-369 ◽

2018 ◽

Author(s):

Mingzhu Zhang ◽

Yang Li ◽

Qingye Sun ◽

Piaoxue Chen ◽

Xuhao Wei

Keyword(s):

River Sediments ◽

Coupled System ◽

Coupled Systems ◽

Functional Gene ◽

Coupled Processes ◽

Functional Genes ◽

Marker Genes ◽

Ecological Processes ◽

Sediment Samples ◽

Reference Index

Abstract. Microorganisms in sediments play an important role in C-, N- and S-cycles by regulating forms and contents of these elements. The coupled system or synergistic reaction among three elemental cycles can effectively alleviate the pollution of C, N, and S in sediments. However, ecological processes coupling C-, N- and S-cycles in sediments are still poorly understood. In order to understand the ecological processes mediated by microorganisms living in river sediments, a total of 135 sediment samples were collected from Huaihe River and its branches located in the Northern of Anhui Province, the abundance of functional marker genes (mcrA, pmoA, cmo, amoA, hzo, nirK, nirS, nosZ, dsrB, aprA), involving in C-, N- and S-transformation, were determined by qPCR. The correlation among functional genes from 135 river sediment samples was calculated. We supposed that the correlationship among functional genes could be used as a reference index speculating the coupled systems of C-N-S in this reasearch, then the distinct coupling relation of C-N-S was revealed, and probable genetic mechanisms were also expounded based on the hypothesis. The study found that amoA-AOA and dsrB possibly played a secondary role, while S-functional gene (aprA), C-functional gene (mcrA) and N-functional gene (hzo) were the key functional genes that participate in the coupled processes in the elemental biogeochemical cycle. The results also demonstrated that C, N might have combined effects on the coupling of carbon, nitrogen and sulphur transformation.

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Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer degeneracy

PeerJ ◽

10.7717/peerj.7121 ◽

2019 ◽

Vol 7 ◽

pp. e7121

Author(s):

Ankur Naqib ◽

Silvana Poggi ◽

Stefan J. Green

Keyword(s):

Polymerase Chain Reaction ◽

Community Structure ◽

Microbial Community ◽

Annealing Temperature ◽

Degenerate Primer ◽

Chain Reaction ◽

Degenerate Primers ◽

Polymerase Chain ◽

Dna Template ◽

Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias. In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

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Trait-trait relationships and functional tradeoffs vary with genome size in prokaryotes

10.1101/2021.07.23.453341 ◽

2021 ◽

Author(s):

Sara Beier ◽

Johannes Werner ◽

Thierry Bouvier ◽

Nicolas Mouquet ◽

Cyrille Violle

Keyword(s):

Resource Use ◽

Marker Genes ◽

Ecological Strategies ◽

Base Pairs ◽

History Of ◽

Productivity Gradients ◽

Resilience Framework ◽

Taxonomic Marker ◽

Trait Correlations ◽

Usage Efficiency

We report genomic traits that have been associated with the life history of prokaryotes and highlight conflicting findings concerning earlier observed trait correlations and tradeoffs. In order to address possible explanations for these contradictions we examined trait-trait variations of 11 genomic traits from ~ 17,000 sequenced genomes. The studied trait-trait variations suggested: (i) the predominance of two resistance and resilience-related orthogonal axes , (ii) an overlap between a resilience axis and an axis of resource usage efficiency. These findings imply that resistance associated traits of prokaryotes are globally decoupled from resilience and resource use efficiencies associated traits. However, further inspection of pairwise scatterplots showed that resistance and resilience traits tended to be positively related for genomes up to roughly five million base pairs and negatively for larger genomes. This in turn precludes a globally consistent assignment of prokaryote genomic traits to the competitor - stress-tolerator -ruderal (CSR) schema that sorts species depending on their location along disturbance and productivity gradients into three ecological strategies and may serve as an explanation for conflicting findings from earlier studies. All reviewed genomic traits featured significant phylogenetic signals and we propose that our trait table can be applied to extrapolate genomic traits from taxonomic marker genes. This will enable to empirically evaluate the assembly of these genomic traits in prokaryotic communities from different habitats and under different productivity and disturbance scenarios as predicted via the resistance-resilience framework formulated here.

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