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14 results for “universal primer”
Data from: Major histocompatibility complex class I evolution in songbirds: universal primers, rapid evolution and base compositional shifts in exon 3
Genes of the Major Histocompatibility Complex (MHC) have become an important marker for the investigation of adaptive genetic variation in vertebrates because of their critical role in pathogen resistance. However, despite significant advances in the last few years the characterization of MHC variation in non-model species still remains a challenging task due to the redundancy and high variation of this gene complex. Here we report the utility of a single pair of primers for the cross-amplification of the third exon of MHC class I genes, which encodes the more polymorphic half of the peptide-binding region (PBR), in oscine passerines (songbirds; Aves: Passeriformes), a group especially challenging for MHC characterization due to the presence of large and complex MHC multigene families. In our survey, although the primers failed to amplify exon 3 from two suboscine passerine birds, they amplified exon 3 of multiple MHC class I genes in all 16 species of oscine songbirds tested, yielding a total of 120 sequences. The 16 songbird species belong to 14 different families, primarily within the Passerida, but also in the Corvida. Using a conservative approach based on the analysis of cloned amplicons (n = 16) from each species, we found between 3 and 10 MHC sequences per individual. Each allele repertoire was highly divergent, with the overall number of polymorphic sites per species ranging from 33 to 108 (out of 264 sites) and the average number of nucleotide differences between alleles ranging from 14.67 to 43.67. Our survey in songbirds allowed us to compare macroevolutionary dynamics of exon 3 between songbirds and non-passerine birds. We found compelling evidence of positive selection acting specifically upon peptide-binding codons across birds, and we estimate the strength of diversifying selection in songbirds to be about twice that in non-passerines. Analysis using comparative methods suggest weaker evidence for a higher GC content in the 3rd codon position of exon 3 in non-passerine birds, a pattern that contrasts with among-clade GC patterns found in other avian studies and may suggests different mutational mechanisms. Our primers represent a useful toolfor the characterization of functional and evolutionarily relevant MHC variation across the hyperdiverse songbirds.
Data from: Major histocompatibility complex class I evolution in songbirds: universal primers, rapid evolution and base compositional shifts in exon 3
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Data from: Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods
The quantification of the biological diversity in environmental samples using high-throughput DNA sequencing is hindered by the PCR bias caused by variable primer–template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator-specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high-throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer–template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer–template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region.
Data from: Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity
The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses, and it has been recommended as a core plant DNA barcode. Despite this popularity, the universality and specificity of PCR primers for the ITS region are not satisfactory, resulting in amplification and sequencing difficulties. By thoroughly surveying and analysing the 18S, 5.8S and 26S sequences of Plantae and Fungi from GenBank, we designed new universal and plant-specific PCR primers for amplifying the whole ITS region and a part of it (ITS1 or ITS2) of plants. In silico analyses of the new and the existing ITS primers based on these highly representative data sets indicated that (i) the newly designed universal primers are suitable for over 95% of plants in most groups; and (ii) the plant-specific primers are suitable for over 85% of plants in most groups without amplification of fungi. A total of 335 samples from 219 angiosperm families, 11 gymnosperm families, 24 fern and lycophyte families, 16 moss families and 17 fungus families were used to test the performances of these primers. In vitro PCR produced similar results to those from the in silico analyses. Our new primer pairs gave PCR improvements up to 30% compared with common-used ones. The new universal ITS primers will find wide application in both plant and fungal biology, and the new plant-specific ITS primers will, by eliminating PCR amplification of nonplant templates, significantly improve the quality of ITS sequence information collections in plant molecular systematics and DNA barcoding.
Data from: MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species
We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding environmental DNA (eDNA) from fishes. Primers were designed using aligned whole mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163–185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi Aquarium with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing of the region using high-throughput next-generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera. These fishes are not only taxonomically diverse, ranging from sharks and rays to higher teleosts, but are also greatly varied in their ecology, including both pelagic and benthic species living in shallow coastal to deep waters. We also sampled natural seawaters around coral reefs near the aquarium and detected 93 fish species using this approach. Of the 93 species, 64 were not detected in the four aquarium tanks, rendering the total number of species detected to 232 (from 70 families and 152 genera). The metabarcoding approach presented here is non-invasive, more efficient, more cost-effective and more sensitive than the traditional survey methods. It has the potential to serve as an alternative (or complementary) tool for biodiversity monitoring that revolutionizes natural resource management and ecological studies of fish communities on larger spatial and temporal scales.
Supplementary material 4 from: Hintikka S, Carlsson JE, Carlsson J (2022) The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind. Metabarcoding and Metagenomics 6: e80416. https://doi.org/10.3897/mbmg.6.80416
COI library ASV tax
Supplementary material 1 from: Hintikka S, Carlsson JE, Carlsson J (2022) The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind. Metabarcoding and Metagenomics 6: e80416. https://doi.org/10.3897/mbmg.6.80416
File S1
Supplementary material 3 from: Hintikka S, Carlsson JE, Carlsson J (2022) The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind. Metabarcoding and Metagenomics 6: e80416. https://doi.org/10.3897/mbmg.6.80416
16S library ASV tax
Supplementary material 5 from: Hintikka S, Carlsson JE, Carlsson J (2022) The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind. Metabarcoding and Metagenomics 6: e80416. https://doi.org/10.3897/mbmg.6.80416
Unassigned COIASVs krona
Supplementary material 2 from: Hintikka S, Carlsson JE, Carlsson J (2022) The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind. Metabarcoding and Metagenomics 6: e80416. https://doi.org/10.3897/mbmg.6.80416
Metadata all
Data from: Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods
Open the record for dataset details and reuse information.
Data from: MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species
Open the record for dataset details and reuse information.
Data from: Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity
Open the record for dataset details and reuse information.
Data from: The choice of universal primers and the characteristics of the species mixture determines when DNA metabarcoding can be quantitative.
Open the record for dataset details and reuse information.
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