Errors in published sequences of human cytomegalovirus primers and probes: do we need more quality control?
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ABSTRACT:
SUBMITTER: Habbal W
PROVIDER: S-EPMC1248504 | biostudies-literature | 2005 Oct
REPOSITORIES: biostudies-literature
Publications
Errors in published sequences of human cytomegalovirus primers and probes: do we need more quality control?
Habbal Wafa W Monem Fawza F Gärtner Barbara Christine BC
Journal of clinical microbiology 20051001 10
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| S-EPMC4229319 | biostudies-literature
Project description:The purpose of this study was to perform a 16S sequence-based quality control of two Leptospira strain collections. 16S rRNA gene sequencing was used to verify two Leptospira reference collections provided by the World Health Organization and maintained at a reference laboratory for leptospirosis in Brazil. Among the 89 serovars evaluated, four conflicting strains were identified in one of the collections. Although 16S rRNA gene sequencing cannot identify Leptospira beyond the species level, it is suitable for the identification of contamination and quality control of leptospiral reference collections. This study highlights the importance of the availability of high-quality 16S rRNA sequences in public databases. In addition, it emphasizes the need for periodical verifications and quality control of Leptospira reference collections.
| S-EPMC2803514 | biostudies-literature
Project description:ObjectivesThe aims of this study were to determine the effectiveness of local compression in patients presenting to the emergency room with intraoral bleeding and to identify when complex haemostatic measures may be required.Materials and methodsFive hundred forty patients who had experienced intraoral haemorrhage were retrospectively reviewed. The outcome variable was the haemostasis method used, i.e., simple (local compression with gauze) or complex (an alternative method after local compression has failed). Predictor variables were sex, age, American Society of Anesthesiologists (ASA) class, hepatic cirrhosis, bleeding disorder, use of antithrombotic agents, and site/cause of haemorrhage.ResultsThe mean patient age was 48.9±23.9 years, 53.5% were male, 42.8% were ASA class II or higher, and 23.7% were taking antithrombotic agents. Local compression was used most often (68.1%), followed by local haemostatic agents, sutures, systemic tranexamic acid or blood products, and electrocautery. The most common site of bleeding was the gingiva (91.7%), and the most common cause was tooth extraction (45.7%). Risk factors for needing a complex haemostasis method were use of antithrombotic agents (odds ratio 2.047, P=0.009) and minor oral surgery (excluding extraction and implant procedures; odds ratio 6.081, P=0.001).ConclusionA haemostasis method other than local compression may be needed in patients taking antithrombotic agents or having undergone minor oral surgery.
| S-EPMC6955419 | biostudies-literature
Project description:Two different qualities of pumpkin, cultivars G1519 and G1511, were grown in the same environment under identical management. However, their qualities, such as the contents of total soluble solids, starch, protein, and vitamin C, were significantly different. Do rhizospheric microbes contribute to pumpkin quality? To answer this question, this study investigated the soil microbial compositions in the rhizospheres of different quality pumpkin cultivars to determine the differences in these soil microbial compositions and thus determine how soil microbes may affect pumpkin quality. Firstly, a randomized complete block design with two pumpkin cultivars and three replications was performed in this study. The soil microbial compositions and structures in the rhizospheres of the two pumpkin cultivars were analyzed using a high-throughput sequencing technique. In comparison with the low-quality pumpkin cultivar (G1519), higher microbial diversity and richness could be found in the rhizospheres of the high-quality pumpkin cultivar (G1511). The results showed that there were significant differences in the soil bacterial and fungal community compositions in the rhizospheres of the high- and low-quality pumpkin cultivars. Although the compositions and proportions of microorganisms were similar in the rhizospheres of the two pumpkin cultivars, the proportions of Basidiomycota and Micropsalliota in the G1519 rhizosphere were much higher than those in the G1511 rhizosphere. Furthermore, the fungal phylum and genus Rozellomycota and Unclassified_p__Rozellomycota were unique in the rhizosphere of the high-quality pumpkin cultivar (G1511). All the above results indicate that soil microbes were enriched differentially in the rhizospheres of the low- and high-quality pumpkin cultivars. In other words, more abundant soil microbes were recruited in the rhizosphere of the high-quality pumpkin cultivar as compared to that of the low-quality cultivar. Rozellomycota and Unclassified_p__Rozellomycota may be functional microorganisms relating to pumpkin quality.
| S-EPMC9698040 | biostudies-literature
Project description:BackgroundThe use of preoperative beta-blockers has been accepted as a quality standard for patients undergoing coronary artery bypass graft (CABG) surgery. However, conflicting results from recent studies have raised questions concerning the effectiveness of this quality metric. We sought to determine the influence of preoperative beta-blocker administration before CABG in patients with left ventricular dysfunction.MethodsThe authors analyzed all cases of isolated CABGs in patients with left ventricular ejection fraction less than 50%, performed between 2012 January and 2017 June, at 94 centres recorded in the China Heart Failure Surgery Registry database. In addition to the use of multivariate regression models, a 1-1 propensity scores matched analysis was performed.ResultsOf 6116 eligible patients, 61.7% received a preoperative beta-blocker. No difference in operative mortality was found between two cohorts (3.7% for the non-beta-blockers group vs. 3.0% for the beta-blocker group; adjusted odds ratio [OR] 0.82 [95% CI 0.58-1.15]). Few differences in the incidence of other postoperative clinical end points were observed as a function of preoperative beta-blockers except in stroke (0.7% for the non-beta-blocker group vs. 0.3 for the beta-blocker group; adjusted OR 0.39 [95% CI 0.16-0.96]). Results of propensity-matched analyses were broadly consistent.ConclusionsIn this study, the administration of beta-blockers before CABG was not associated with improved operative mortality and complications except the incidence of postoperative stroke in patients with left ventricular dysfunction. A more granular quality metric which would guide the use of beta-blockers should be developed.
| S-EPMC8603532 | biostudies-literature
Project description:BackgroundPairing up primers to amplify desired targets and avoid undesired cross reactions can be a combinatorial challenge. Effective prediction of specificity and inclusivity from multiplexed primers and TaqMan®/Luminex® probes is a critical step in PCR design.ResultsCode is described to identify all primer and probe combinations from a list of unpaired, unordered candidates that should produce a product. It predicts and extracts all amplicon sequences in a large sequence database from a list of primers and probes, allowing degenerate bases and user-specified levels of primer-target mismatch tolerance. Amplicons hit by TaqMan®/Luminex® probes are indicated, and products may be annotated with gene information from NCBI. Fragment length distributions are calculated to predict electrophoretic gel banding patterns.ConclusionsSimulate_PCR is the only freely available software that can be run from the command line for high throughput applications which can calculate all products from large lists of primers and probes compared to a large sequence database such as nt. It requires no prior knowledge of how primers should be paired. Degenerate bases are allowed and entire amplicon sequences are extracted and annotated with gene information. Examples are provided for sets of TaqMan®/Luminex® PCR signatures predicted to amplify all HIV-1 genomes, all Coronaviridae genomes, and a group of antibiotic resistance genes. The software is a command line perl script freely available as open source.
| S-EPMC4226945 | biostudies-literature
Project description:The ribosomal DNA comprised of the ITS1-5.8S-ITS2 regions is widely used as a fungal marker in molecular ecology and systematics but cannot be aligned with confidence across genetically distant taxa. In order to study the diversity of Agaricomycotina in forest soils, we designed primers targeting the more alignable 28S (LSU) gene, which should be more useful for phylogenetic analyses of the detected taxa. This paper compares the performance of the established ITS1F/4B primer pair, which targets basidiomycetes, to that of two new pairs. Key factors in the comparison were the diversity covered, off-target amplification, rarefaction at different Operational Taxonomic Unit (OTU) cutoff levels, sensitivity of the method used to process the alignment to missing data and insecure positional homology, and the congruence of monophyletic clades with OTU assignments and BLAST-derived OTU names. The ITS primer pair yielded no off-target amplification but also exhibited the least fidelity to the expected phylogenetic groups. The LSU primers give complementary pictures of diversity, but were more sensitive to modifications of the alignment such as the removal of difficult-to align stretches. The LSU primers also yielded greater numbers of singletons but also had a greater tendency to produce OTUs containing sequences from a wider variety of species as judged by BLAST similarity. We introduced some new parameters to describe alignment heterogeneity based on Shannon entropy and the extent and contents of the OTUs in a phylogenetic tree space. Our results suggest that ITS should not be used when calculating phylogenetic trees from genetically distant sequences obtained from environmental DNA extractions and that it is inadvisable to define OTUs on the basis of very heterogeneous alignments.
| S-EPMC3283731 | biostudies-literature
Project description:BackgroundThe highly heterogenic characteristic of viruses is the major obstacle to efficient DNA amplification. Taking advantage of the large number of virus DNA sequences in public databases to select conserved sites for primer design is an optimal way to tackle the difficulties in virus genome amplification.ResultsHere we use hepatitis B virus as an example to introduce a simple and efficient way for virus primer design. Based on the alignment of HBV sequences in public databases and a program BxB in Perl script, our method selected several optimal sites for HBV primer design. Polymerase chain reaction showed that compared with the success rate of the most popular primers for whole genome amplification of HBV, one set of primers for full length genome amplification and four sets of walking primers showed significant improvement. These newly designed primers are suitable for most subtypes of HBV.ConclusionResearchers can extend the method described here to design universal or subtype specific primers for various types of viruses. The BxB program based on multiple sequence alignment not only can be used as a separate tool but also can be integrated in any open source primer design software to select conserved regions for primer design.
| S-EPMC2099425 | biostudies-literature
Project description:Although the technical and analytic complexity of whole genome sequencing is generally appreciated, best practices for data cleaning and quality control have not been defined. Family based data can be used to guide the standardization of specific quality control metrics in nonfamily based data. Given the low mutation rate, Mendelian inheritance errors are likely as a result of erroneous genotype calls. Thus, our goal was to identify the characteristics that determine Mendelian inheritance errors. To accomplish this, we used chromosome 3 whole genome sequencing family based data from the Genetic Analysis Workshop 18. Mendelian inheritance errors were provided as part of the GAW18 data set. Additionally, for binary variants we calculated Mendelian inheritance errors using PLINK. Based on our analysis, nonbinary single-nucleotide variants have an inherently high number of Mendelian inheritance errors. Furthermore, in binary variants, Mendelian inheritance errors are not randomly distributed. Indeed, we identified 3 Mendelian inheritance error peaks that were enriched with repetitive elements. However, these peaks can be lessened with the inclusion of a single filter from the sequencing file. In summary, we demonstrated that erroneous sequencing calls are nonrandomly distributed across the genome and quality control metrics can dramatically reduce the number of mendelian inheritance errors. Appropriate quality control will allow optimal use of genetic data to realize the full potential of whole genome sequencing.
| S-EPMC4144465 | biostudies-literature
Project description:MotivationDesigning PCR primers to target a specific selection of whole genome sequenced strains can be a long, arduous and sometimes impractical task. Such tasks would benefit greatly from an automated tool to both identify unique targets, and to validate the vast number of potential primer pairs for the targets in silico.ResultsHere we present RUCS, a program that will find PCR primer pairs and probes for the unique core sequences of a positive genome dataset complement to a negative genome dataset. The resulting primer pairs and probes are in addition to simple selection also validated through a complex in silico PCR simulation. We compared our method, which identifies the unique core sequences, against an existing tool called ssGeneFinder, and found that our method was 6.5-20 times more sensitive. We used RUCS to design primer pairs that would target a set of genomes known to contain the mcr-1 colistin resistance gene. Three of the predicted pairs were chosen for experimental validation using PCR and gel electrophoresis. All three pairs successfully produced an amplicon with the target length for the samples containing mcr-1 and no amplification products were produced for the negative samples. The novel methods presented in this manuscript can reduce the time needed to identify target sequences, and provide a quick virtual PCR validation to eliminate time wasted on ambiguously binding primers.Availability and implementationSource code is freely available on https://bitbucket.org/genomicepidemiology/rucs. Web service is freely available on https://cge.cbs.dtu.dk/services/RUCS.Contactmcft@cbs.dtu.dk.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC5860091 | biostudies-literature