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opencc-zeroDec 2015View details →

Data from: A phylogenomic approach based on PCR target enrichment and high throughput sequencing: resolving the diversity within the South American species of Bartsia l. (Orobanchaceae)

Advances in high-throughput sequencing (HTS) have allowed researchers to obtain large amounts of biological sequence information at speeds and costs unimaginable only a decade ago. Phylogenetics, and the study of evolution in general, is quickly migrating towards using HTS to generate larger and more complex molecular datasets. In this paper, we present a method that utilizes microfluidic PCR and HTS to generate large amounts of sequence data suitable for phylogenetic analyses. The approach uses the Fluidigm Access Array System (Fluidigm, San Francisco, CA, USA) and two sets of PCR primers to simultaneously amplify 48 target regions across 48 samples, incorporating sample-specific barcodes and HTS adapters (2,304 unique amplicons per Access Array). The final product is a pooled set of amplicons ready to be sequenced, and thus, there is no need to construct separate, costly genomic libraries for each sample. Further, we present a bioinformatics pipeline to process the raw HTS reads to either generate consensus sequences (with or without ambiguities) for every locus in every sample or—more importantly—recover the separate alleles from heterozygous target regions in each sample. This is important because it adds allelic information that is well suited for coalescent-based phylogenetic analyses that are becoming very common in conservation and evolutionary biology. To test our approach and bioinformatics pipeline, we sequenced 576 samples across 96 target regions belonging to the South American clade of the genus Bartsia L. in the plant family Orobanchaceae. After sequencing cleanup and alignment, the experiment resulted in ~25,300bp across 486 samples for a set of 48 primer pairs targeting the plastome, and ~13,500bp for 363 samples for a set of primers targeting regions in the nuclear genome. Finally, we constructed a combined concatenated matrix from all 96 primer combinations, resulting in a combined aligned length of ~40,500bp for 349 samples.

Data from: Degenerate adaptor sequences for detecting PCR duplicates in reduced representation sequencing data improve genotype calling accuracy

RAD-tag is a powerful tool for high-throughput genotyping. It relies on PCR amplification of the starting material, following enzymatic digestion and sequencing adaptor ligation. Amplification introduces duplicate reads into the data, which arise from the same template molecule and are statistically nonindependent, potentially introducing errors into genotype calling. In shotgun sequencing, data duplicates are removed by filtering reads starting at the same position in the alignment. However, restriction enzymes target specific locations within the genome, causing reads to start in the same place, and making it difficult to estimate the extent of PCR duplication. Here, we introduce a slight change to the Illumina sequencing adaptor chemistry, appending a unique four-base tag to the first index read, which allows duplicate discrimination in aligned data. This approach was validated on the Illumina MiSeq platform, using double-digest libraries of ants (Wasmannia auropunctata) and yeast (Saccharomyces cerevisiae) with known genotypes, producing modest though statistically significant gains in the odds of calling a genotype accurately. More importantly, removing duplicates also corrected for strong sample-to-sample variability of genotype calling accuracy seen in the ant samples. For libraries prepared from low-input degraded museum bird samples (Mixornis gularis), which had low complexity, having been generated from relatively few starting molecules, adaptor tags show that virtually all of the genotypes were called with inflated confidence as a result of PCR duplicates. Quantification of library complexity by adaptor tagging does not significantly increase the difficulty of the overall workflow or its cost, but corrects for differences in quality between samples and permits analysis of low-input material.

opencc-zeroApr 2020View details →

Selection and exploitation of prevalent, tandemly repeated genomic targets for improved real-time PCR-based detection of Wuchereria bancrofti and Plasmodium falciparum in mosquitoes

Background Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both next-generation sequencing (NGS) technologies and bioinformatics-based analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent. Methodology/Principal Findings Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number. Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms. For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay. Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome's most abundant tandem repeat. While resulting cycle quantification values comparing a qPCR assay targeting this ribosomal sequence and a commonly targeted repetitive DNA sequence from the literature supported our finding that this ribosomal sequence was the most prevalent tandemly repeated target in the W. bancrofti genome, the resulting assay did not significantly improve detection sensitivity in conjunction with field sample testing. Conclusions/Significance Examination of pathogen genomes facilitates the development of PCR-based diagnostics targeting the most abundant and specific genomic elements. While in some instances currently available tools may deliver equal or superior performance, systematic analysis of potential targets provides confidence that the selected assays represent the most advantageous options available and that informed assay selection is occurring in the context of a particular study's objectives.

opencc-zeroDec 2012View details →

Data from: Distribution of MICB diversity in the Zhejiang Han population: PCR sequence-based typing for exons 2–6 and identification of five novel MICB alleles

The polymorphism of major histocompatibility complex class I chain-related gene B (MICB) and variations in MICB alleles in a variety of populations have been characterized using several genotyping approaches. In the present study, a novel polymerase chain reaction sequence-based typing (PCR-SBT) method was established for the genotyping of MICB exons 2–6, and the allelic frequency of MICB in the Zhejiang Han population was investigated. Among 400 unrelated healthy Han individuals from Zhejiang Province, China, a total of 20 MICB alleles were identified, of which MICB*005:02:01, MICB*002:01:01, and MICB*004:01:01 were the most predominant alleles, with frequencies of 0.57375, 0.1225, and 0.08375, respectively. Nine MICB alleles were detected on only one occasion, giving a frequency of 0.00125. Of the 118 distinct MICB ∼ HLA-B haplotypes identified, 42 showed significant linkage disequilibrium (P < 0.05). Haplotypes MICB*005:02:01 ∼ B*46:01, MICB*005:02:01 ∼ B*40:01, and MICB*008 ∼ B*58:01 were the most common haplotypes, with frequencies of 0.0978, 0.0761, and 0.0616, respectively. Five novel alleles, MICB*005:07, MICB*005:08, MICB*027, MICB*028, and MICB*029 were identified. Compared with the MICB*005:02:01 sequence, a G > A substitution was observed at nucleotide position 210 in MICB*005:07, and a 1,134 T > C substitution in MICB*005:08 and an 862 G > A substitution in MICB*027 were detected. In addition, it appears that MICB*028 probably arose from MICB*004:01:01 with an A to G substitution at position 1,147 in exon 6. MICB*029 had a G > T transversion at nucleotide position 730 in exon 4, compared with that of MICB*002:01:01. On the basis of the new PCR-SBT assay, these observed results demonstrated MICB allelic variations in the Zhejiang Han population.

Data from: PCR duplication: a one-step cloning-free method to generate duplicated chromosomal loci and interference-free expression reporters in yeast

Here, we report on a novel PCR targeting-based strategy called 'PCR duplication' that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning.

Data from: An examination of the accuracy of a sequential PCR and sequencing test used to detect the incursion of an invasive species: the case of the red fox in Tasmania

1. Polymerase Chain Reaction (PCR) diagnostic tests are increasingly applied to the identification of wildlife. Yet rigorous verification is rare and the estimation of test accuracy (the probability that true positive and true negative samples are correctly identified – test sensitivity and specificity, respectively), particularly in combination with sequencing, is uncommon. This is important because PCR-based tests are prone to contamination in sampling and the laboratory. 2. Here, we use an experimental case–control approach to estimate the sensitivity and specificity of a sequential PCR-based wildlife detection test used to identify incursions of red foxes into Tasmania from predator faeces (scats). 3. Our results show that the sensitivity of the fox test is high (~94%) for the PCR-based test on its own, but this decreases to ~84% when combined with the DNA sequencing step. In contrast, the specificity increases from ~96% in the PCR only test to ~99.6% after inclusion of the DNA sequencing step. 4. The intense public scrutiny of the fox eradication program in Tasmania, has undoubtedly influenced the application of a sequential PCR test that maximises specificity at the expense of sensitivity and so increases the risk that scats containing fox DNA would not be detected. This could lead to the establishment of foxes in Tasmania as a consequence. 5. Synthesis and applications. Importantly, the estimation of the sensitivity and specificity of sequential tests enables decisions about the risk associated with mistaken identification (i.e. false negatives vs false positives) to be quantified for decision makers. The cost of false negative errors should be balanced against the costs of false positive errors, which could include the expenditure incurred in the application of unnecessary management actions were foxes not in fact present. Understanding the risks and costs associated with both false negative and false positive errors is therefore a key component to the decision making process for the management of the Tasmanian fox incursion.

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: PCR-Free enrichment of mitochondrial DNA from human blood and cell lines for high quality next-generation DNA sequencing

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.

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.

Data from: Sequence Capture using PCR-generated Probes (SCPP): a cost-effective method of targeted high-throughput sequencing for non-model organisms

Recent advances in high-throughput sequencing library preparation and subgenomic enrichment methods have opened new avenues for population genetics and phylogenetics of non-model organisms. To multiplex large numbers of indexed samples while sequencing predominantly orthologous, targeted regions of the genome, we propose modifications to an existing, in-solution capture that utilizes PCR products as target probes to enrich library pools for the genomic subset of interest. The sequence capture using PCR-generated probes (SCPP) protocol requires no specialized equipment, is highly flexible, and significantly reduces experimental costs for projects where a modest scale of genetic data is optimal (25-100 genomic loci). Our alterations enable application of this method across a wider phylogenetic range of taxa and result in higher capture efficiencies and coverage at each locus. Efficient and consistent capture over multiple SCPP experiments and at various phylogenetic distances is demonstrated, extending the utility of this method to both phylogeographic and phylogenomic studies.

Data from: A long PCR based approach for DNA enrichment prior to next-generation sequencing for systematic studies

Premise of the study: We present an alternative approach for molecular systematic studies that combines long PCR and next-generation sequencing (NGS). Our approach can be used to generate templates from any DNA source for NGS. Here we test our approach by amplifying complete chloroplast genomes and we present a set of 58 potentially universal primers for angiosperms to do so. Additionally, this approach is likely to be particularly useful for nuclear regions. Methods and Results: Chloroplast genomes of 30 species across angiosperms were amplified to test our approach. Amplification success varied depending on whether PCR conditions were optimized for a given taxon. To further test our approach, some amplicons were sequenced on an Illumina HiSeq 2000. Conclusions: Although here we tested this approach by sequencing plastomes, long PCR amplicons could be generated using DNA from any genome, expanding the possibilities of this approach for molecular systematic studies.

opencc-zeroDec 2010View details →

Data from: Optimizing methods for PCR-based analysis of predation

Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116–612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons.

opencc-zeroDec 2015View details →

Data from: Residual eDNA detection sensitivity assessed by quantitative real-time PCR in a river ecosystem

Several studies have demonstrated that environmental DNA (eDNA) can be used to detect the presence of aquatic species, days to weeks after the target species has been removed. However, most studies used eDNA analysis in lentic systems (ponds or lakes), or in controlled laboratory experiments. While eDNA degrades rapidly in all aquatic systems, it also undergoes dilution effects and physical destruction in flowing systems, complicating detection in rivers. However, some eDNA (i.e. residual eDNA) can be retained in aquatic systems, even those subject to high flow regimes. Our goal was to determine residual eDNA detection sensitivity using quantitative real-time polymerase chain reaction (qRT-PCR), in a flowing, uncontrolled river after the eDNA source was removed from the system; we repeated the experiment over two years. Residual eDNA had the strongest signal strength at the original source site and was detectable there up to 11.5 hours after eDNA source removal. Residual eDNA signal strength decreased as sampling distance downstream from the eDNA source site increased, and was no longer detectable at the source site 48 hours after the eDNA source water was exhausted in both experiments. This experiment shows that residual eDNA sampled in surface water can be mapped quantitatively using qRT-PCR, which allows a more accurate spatial identification of the target species location in lotic systems, and relative residual eDNA signal strength may allow the determination of the timing of the presence of target species.

FIGURE 7 in Description of a new Fridericia species (Oligochaeta: Enchytraeidae) and its molecular comparison with two morphologically similar species by PCRRFLP

FIGURE 7. The gel image TaqI of restriction digestion.

FIGURE 2 in Description of a new Fridericia species (Oligochaeta: Enchytraeidae) and its molecular comparison with two morphologically similar species by PCRRFLP

FIGURE 2. Fridericia crassiductata sp.n. A = oesophageal appendage, B = spermatheca.

FIGURE 9. The gel image Hin6I in Description of a new Fridericia species (Oligochaeta: Enchytraeidae) and its molecular comparison with two morphologically similar species by PCRRFLP

FIGURE 9. The gel image Hin6I of restriction digestion.

FIGURE 8 in Description of a new Fridericia species (Oligochaeta: Enchytraeidae) and its molecular comparison with two morphologically similar species by PCRRFLP

FIGURE 8. The gel image AluI of restriction digestion.

FIGURE 10. The gel image Csp6I in Description of a new Fridericia species (Oligochaeta: Enchytraeidae) and its molecular comparison with two morphologically similar species by PCRRFLP

FIGURE 10. The gel image Csp6I of restriction digestion.

opencc-by-4.0Nov 2021View details →

Oligonucleotide primers used for RT-PCR

Oligonucleotide primers used for RT-PCR