bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/325688

New mitochondrial primers for metabarcoding of insects, designed and evaluated using in silico methods

Marquina, D.·Andersson, A. F.·Ronquist, F.
View original source ↗Export metadata ↓

Abstract

Insect metabarcoding has been mainly based on PCR amplification of short fragments within the barcoding region of the gene COI. However, because of the variability of this gene, it has been difficult to design good universal PCR primers. Most primers used today are associated with gaps in the taxonomic coverage or amplification biases that make the results less reliable and impede the detection of species that are present in the sample. We identify new primers for insect metabarcoding using computational approaches (ECOPRIMERS and DEGEPRIME) applied to the most comprehensive reference databases of mitochondrial genomes of Hexapoda assembled to date. New primers are evaluated in silico against previously published primers in terms of taxonomic coverage and resolution of the corresponding amplicons. For the latter criterion, we propose a new index, exclusive taxonomic resolution, which is a more biologically meaningful measure than the standard index used today. Our results show that the best markers are found in the ribosomal RNA genes (12S and 16S); they resolve about 90% of the genetically distinct species in the reference database. Some markers in protein-coding genes provide similar performance but only at much higher levels of primer degeneracy. Combining two of the best individual markers improves the effective taxonomic resolution with up to 10%. The resolution is strongly dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S primers detect 12% of Collembola. Our results indicate that amplicon-based metabarcoding of insect samples can be improved by choosing other primers than those commonly used today.

Source connections

Explore related subjects

bioinformaticsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Marquina, D., Andersson, A. F., Ronquist, F.. 2018-05-18. New mitochondrial primers for metabarcoding of insects, designed and evaluated using in silico methods. https://doi.org/10.1101/325688

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

The Almond Expression Atlas: A comprehensive multi-tissue transcriptomic resource for almond gene expression

Background: Improving crop adaptation and enhancing agronomic and quality traits requires a deeper understanding of gene expression regulation, for which expression atlases are valuable tools. Almond (Prunus dulcis (Mill.) D.A. Webb) is one of the most economically important tree nut crops worldwide. However, an integrated transcriptomic resource for this species is still lacking. Available data remain fragmented across independent studies, limiting their comparability and reuse. Results: Here, we present the first almond gene expression atlas: a comprehensive multi-tissue transcriptomic resource integrating publicly available RNA sequencing data. A total of 142 high-quality untreated and control samples from 17 independent projects were curated through a metadata standardization process and analyzed using a unified bioinformatics pipeline. Standardized expression profiles were generated for 25,225 annotated genes in the Prunus dulcis Texas v2.0 reference genome. The atlas integrates two complementary expression approaches: a median TPM approach capturing quantitative expression levels and a rank-based strategy implemented using the Bgee expression calling method, designed to improve comparability across heterogeneous datasets. This design enables the identification of tissue-specific candidate genes and the exploration of expression patterns across tissues and developmental stages. Its applicability is demonstrated through two use cases, highlighting its ability to identify biologically meaningful gene sets associated with bud dormancy transitions and to prioritize candidate reference genes for RT-qPCR normalization in almond. Conclusions: The Almond Expression Atlas v1.0 is publicly available through FruitDBase (fruitdbase.csic.es), providing an interactive platform for gene expression exploration and visualization. This resource supports functional genomics studies and facilitates candidate gene discovery in almond, while ensuring data traceability and promoting data reusability.

bioinformatics↗
bioRxiv2026

Leakage-Controlled Benchmarking of Variant Effect Predictors and Development of Gradient-Boosting Consensus Model

Single amino acid mutations are among the most common forms of genetic variation, with effects ranging from negligible to severe disruption of protein function. Accurate quantitative evaluation of protein variants therefore represents a key challenge in medicine and protein engineering. Computational variant effect predictors (VEPs) are widely used to prioritize missense variants, yet their fair comparison is hindered by data circularity, benchmarks restricted to human clinical annotations, and limited availability of code and training data, all of which can inflate apparent performance. Here, we reviewed 18 recent and 3 older, widely adopted variant effect predictors for reproducible benchmarking and assembled a rigorously curated, leakage-controlled Benchmark dataset by integrating clinically annotated human variants from ClinVar and Humsavar as well as cross-species variants with functional annotations. The evaluation on this dataset revealed a narrow overall performance range for the benchmarked tools (AUC 0.631-0.758) and a consistent drop from clinical to functional data. Surprisingly, several tools published two decades ago demonstrated comparable performance to mid-ranked modern predictors. Building on these results, we developed a gradient-boosting consensus model, PredictSNPprot, which achieved an AUC of 0.82 on a held-out test set, outperforming the original PredictSNP meta-predictor and the best individual state-of-the-art tool by 13.6 and 5.7 percentage points, respectively. The Benchmark dataset, including the predictions from the evaluated tools, is available at https://doi.org/10.5281/zenodo.23597130 and PredictSNPprot consensus predictor is available at https://github.com/loschmidt/PredictSNPprot.

bioinformatics↗
bioRxiv2026

MaCADEMIA: a software toolbox for single-molecule fluorescence image quantification

Single-molecule localisation microscopy (SMLM) is a powerful tool in molecular and cellular biology and part of the broad umbrella of super-resolution microscopy. A distinct feature of SMLM, is its capacity for deep quantification, beyond just spatial information, to copy number, stoichiometry, molecular interaction and dynamics in living cells. The hardware required is increasingly straightforward to implement. However, complexity remains in the image analysis where much remains fragmented across specialised software tools. Here, we present MaCADEMIA (Matlab Computational Analysis of Diffusion, Emitter Molecular Intensity and Abundance), a simple yet powerful toolbox of MATLAB functions and associated documentation to enable straightforward extraction of quantitative single-molecule information. MaCADEMIA performs ensemble particle detection achieving localisation accuracies down to 0.5 pixels Root mean squared error (RMSE) across wide signal-to-noise (SNR) ranges on standardised particle tracking benchmarks, consistently matching or exceeding the performance of 14 leading methods. Our integrated workflow enables molecular counting and stoichiometry determination through stepwise photobleaching analysis, mobility measurement via mean squared displacement, colocalisaton analysis and absolute protein copy number quantification using three-dimensional cellular models.

bioinformatics↗