Journal articles: 'Sanger/pyrosequencing'

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Tewari, Deepanker, Stephen Cieply, and Julia Livengood. "Identification of bacteria recovered from animals using the 16S ribosomal RNA gene with pyrosequencing and Sanger sequencing." Journal of Veterinary Diagnostic Investigation 23, no. 6 (2011): 1104–8. http://dx.doi.org/10.1177/1040638711425583.

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Bacterial identification using genetic sequencing is fast becoming a confirmatory tool for microbiologists. Its application in veterinary diagnostic laboratories is still growing. In addition to availability of Sanger sequencing, pyrosequencing has recently emerged as a unique method for short-read DNA sequencing for bacterial identifications. Its ease of use makes it possible to diagnose infections rapidly at a low cost even in smaller laboratories. In the current study, pyrosequencing was compared with Sanger sequencing for identification of the bacterial organisms. Fifty-four bacterial isolates spanning 23 different bacterial families encountered in veterinary diagnostic microbiology laboratories were sequenced using 16S ribosomal RNA gene with pyrosequencing and Sanger sequencing. Pyrosequencing was able to identify 80% of isolates to the genus level, and 43% isolates to the species level. Sanger sequencing with approximately 500 bp performed better for both genus (100%) and species (87%) identification. Use of different sequence databases to identify bacteria isolated from animals showed relative importance of public databases compared to a validated commercial library. A time and limited cost comparison between pyrosequencing and genetic sequencing of 500 bp showed pyrosequencing was not only faster but also comparable in cost, making it a viable alternative for use in classifying bacteria isolated from animals.

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Novak-Frazer, Lilyann, Samuel P. Anees-Hill, Darin Hassan, et al. "Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens." Journal of Antimicrobial Chemotherapy 75, no. 12 (2020): 3501–9. http://dx.doi.org/10.1093/jac/dkaa357.

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Abstract Background Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. Objectives To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. Methods Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017–March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. Results The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. Conclusions The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

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Fakruddin, Md, and Abhijit Chowdhury. "Pyrosequencing-An Alternative to Traditional Sanger Sequencing." American Journal of Biochemistry and Biotechnology 8, no. 1 (2012): 14–20. http://dx.doi.org/10.3844/ajbbsp.2012.14.20.

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Schildgen, V., J. Lusebrink, C. Schulz, et al. "EGFR mutations: Comparison of Sanger- and pyrosequencing." Journal of Clinical Oncology 29, no. 15_suppl (2011): e21019-e21019. http://dx.doi.org/10.1200/jco.2011.29.15_suppl.e21019.

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Prigigallo, Maria I., Ahmed Abdelfattah, Santa O. Cacciola, et al. "Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing." Phytopathology® 106, no. 3 (2016): 305–13. http://dx.doi.org/10.1094/phyto-07-15-0167-r.

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A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

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Wiederhold, Nathan P., Jodi L. Grabinski, Guillermo Garcia-Effron, David S. Perlin, and Samuel A. Lee. "Pyrosequencing To Detect Mutations in FKS1 That Confer Reduced Echinocandin Susceptibility in Candida albicans." Antimicrobial Agents and Chemotherapy 52, no. 11 (2008): 4145–48. http://dx.doi.org/10.1128/aac.00959-08.

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ABSTRACT Pyrosequencing was compared to Sanger dideoxy sequencing to detect mutations in FKS1 responsible for reduced echinocandin susceptibility in Candida albicans. These methods were in complete agreement for 10 of 12 clinical isolates with elevated echinocandin MICs, supporting the potential feasibility of pyrosequencing to detect mutations within diploid fungi.

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Lüke, Claudia, and Peter Frenzel. "Potential ofpmoAAmplicon Pyrosequencing for Methanotroph Diversity Studies." Applied and Environmental Microbiology 77, no. 17 (2011): 6305–9. http://dx.doi.org/10.1128/aem.05355-11.

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ABSTRACTWe analyzed the potential ofpmoAamplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogeneticpmoAlineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.

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Poulet, Axel, Maud Privat, Flora Ponelle, et al. "Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software." BioMed Research International 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/5623089.

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Screening forBRCAmutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of theBRCAgenes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.

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Harrington, Colleen T., Elaine I. Lin, Matthew T. Olson, and James R. Eshleman. "Fundamentals of Pyrosequencing." Archives of Pathology & Laboratory Medicine 137, no. 9 (2013): 1296–303. http://dx.doi.org/10.5858/arpa.2012-0463-ra.

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Context.—DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined. Objective.—To demonstrate the fundamental principles of pyrosequencing. Data Sources.—Salient features of pyrosequencing are demonstrated using the free software program Pyromaker (http://pyromaker.pathology.jhmi.edu), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results. Conclusions.—We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.

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Cardus, Beatrix, Richard Colling, Angela Hamblin, and Elizabeth Soilleux. "Comparison of methodologies for the detection of BRAF mutations in bone marrow trephine specimens." Journal of Clinical Pathology 72, no. 6 (2019): 406–11. http://dx.doi.org/10.1136/jclinpath-2019-205734.

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AimsBRAF V600E detection assists in the diagnosis of hairy cell leukaemia (HCL); however, testing practices vary. We evaluated the clinical utility of 5 BRAF mutation testing strategies for use on bone marrow trephines (BMT).Methods11 HCL, 5 HCL ‘mimic’, 2 treated HCL and 10 normal BMT specimens were tested for mutant BRAF, comparing Sanger sequencing, pyrosequencing, amplicon-based next generation sequencing (NGS), automated (Idylla) PCR and immunohistochemistry (IHC).ResultsPCR and IHC were cheaper and identified V600E in 100 % of HCL cases. Pyrosequencing detected the mutation in 91%, NGS in 55% of cases and Sanger sequencing in 27%. All assays gave wild-type BRAF results in HCL mimics and normal BMT samples.ConclusionsPCR and IHC were most sensitive and cost-effective, but these have limited scope for multiplexing and are likely to be replaced by NGS gene panels or whole genome sequencing in the medium to long term.

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Wang, Liu, and Pengfeng Xiao. "Haplotype-Contained PCR Products Analysis by Sequencing with Selective Restriction of Primer Extension." BioMed Research International 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/1397902.

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We develop a strategy for haplotype analysis of PCR products that contained two adjacent heterozygous loci using sequencing with specific primers, allele-specific primers, and ddNTP-blocked primers. To validate its feasibility, two sets of PCR products, including two adjacent heterozygous SNPs, UGT1A1⁎6 (rs4148323) and UGT1A1⁎28 (rs8175347), and two adjacent heterozygous SNPs, K1637K (rs11176013) and S1647T (rs11564148), were analyzed. Haplotypes of PCR products, including UGT1A1⁎6 and UGT1A1⁎28, were successfully analyzed by Sanger sequencing with allele-specific primers. Also, haplotypes of PCR products, including K1637K and S1647T, could not be determined by Sanger sequencing with allele-specific primers but were successfully analyzed by pyrosequencing with ddNTP-blocked primers. As a result, this method is able to effectively haplotype two adjacent heterozygous PCR products. It is simple, fast, and irrespective of short read length of pyrosequencing. Overall, we fully hope it will provide a new promising technology to identify haplotypes of conventional PCR products in clinical samples.

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Cheung, Foo, Joe Win, Jillian M. Lang, et al. "Analysis of the Pythium ultimum transcriptome using Sanger and Pyrosequencing approaches." BMC Genomics 9, no. 1 (2008): 542. http://dx.doi.org/10.1186/1471-2164-9-542.

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Debeljak, Marija, Stacy Riel, Ming-Tseh Lin, James R. Eshleman, and Channing J. Paller. "Analytical Validation of SOD2 Genotyping." Methods and Protocols 6, no. 1 (2022): 4. http://dx.doi.org/10.3390/mps6010004.

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Manganese superoxide dismutase-2 (SOD2) plays a crucial role in cells’ protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline SOD2 SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples (13 homozygous Ala, 12 heterozygous Ala/Val, and 12 homozygous Val) were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate.

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Normanno, Nicola, Carmine Pinto, Gian Luigi Taddei, et al. "Frequency and clinical correlations of epidermal growth factor receptor (EGFR) mutations in a large cohort of Italian non-small cell lung cancer (NSCLC) patients (pts) within the EGFR FASTnet program." Journal of Clinical Oncology 30, no. 15_suppl (2012): e18021-e18021. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e18021.

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e18021 Background: Gefitinib was approved in Italy for treatment of pts with advanced NSCLC carrying mutant EGFR in May 2010. Methods: The EGFR FASTnet program was designed to facilitate the exchange of biological material, clinico-pathological data and reports between medical oncologists, primary pathologists and referral laboratories. EGFR mutational analysis was carried by Sanger sequencing, Real Time PCR, Pyrosequencing, Fragment Analysis and High resolution melting. The Italian Association of Medical Oncology (AIOM) and the Italian Society of Surgical Pathology and Cytopathology (SIAPEC-IAP) have full access to the anonymous EGFR FASTnet database. Results: As of December 31, 2011, 503 oncologists, 135 pathologists and 38 referral laboratories joined the EGFR FASTnet program. The enrolled cohort of 3819 pts with advanced NSCLC was significantly enriched for adenocarcinoma histology (3172 [83%]), female sex (1361 [36%]) and smoking history (never smoker 911 [24%], former smoker>15 yrs 880 [23%], light smoker 194 [5%]). Mutational analysis was feasible in 3567 pts (93%). At registration, 72% of the pts had not received yet treatment for advanced disease. Mutational analysis was carried by Sanger sequencing in 2021 cases (57%), Real Time PCR in 174 (5%), Pyrosequencing in 636 (18%) and other techniques in 736 (21%). EGFR mutations were found in 520 cases (14.6%): 334 in exon 19 (9.4%), 163 in exon 21 (4.6%), 7 in exon 18 (0.2%) and 16 in exon 20 (0.4%). Proportion of mutated cases was slightly higher with Real time PCR compared to other techniques: Sanger 14.8%, Real time PCR 21.3%, Pyrosequencing 13.5%, other 13.3% (p = 0.049). A higher mutation rate was found in never smokers (32.0%), light smokers (18.7%) and former smokers >15 yrs (12.4%), as well as in adenocarcinoma (15.7%) and females (25.2%). Conclusions: The pts for EGFR mutational screening are spontaneously selected by medical oncologists according to known predictive factors. The results of the mutational analysis from clinical practice in Italy are consistent with data from literature.

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MOLINS, Arántzazu, Patricia MOYA, Francisco J. GARCÍA-BREIJO, José REIG-ARMIÑANA, and Eva BARRENO. "A multi-tool approach to assess microalgal diversity in lichens: isolation, Sanger sequencing, HTS and ultrastructural correlations." Lichenologist 50, no. 1 (2018): 123–38. http://dx.doi.org/10.1017/s0024282917000664.

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AbstractLichen thalli represent the most conspicuous examples of fungal-algal interactions. Studies that describe phycobiont diversity within entire thalli are based mainly on Sanger sequencing. In some lichen species, this technique could underestimate the intrathalline coexistence of multiple microalgae. In this study different multi-tool approaches were applied to two lichen taxa, Circinaria hispida and Flavoparmelia soredians, to detect algal coexistence. Here, we combined Sanger sequencing, a specific polymerase chain reaction (PCR) primer, 454-pyrosequencing, phycobiont isolation and ultrastructural characterization. Furthermore, we compared pyrenoid ultrastructural features of lichenized phycobionts with microalgae isolated in culture. An improved methodology was used to isolate and propagate phycobionts which, in combination with fast genetic identification, resulted in a considerable reduction in time and cost to complete the process. This isolation method, coupled with a specific PCR primer, allowed for the detection of coexisting algae in C. hispida (four Trebouxia lineages). 454-pyrosequencing detected only a fraction of such diversity, while Sanger sequencing identified only the primary phycobiont. Ultrastructural features of the isolated algae were observed by transmission electron microscopy; the maintenance of the pyrenoid characteristics suggested the existence of different Trebouxia lineages. In F. soredians a single Trebouxia lineage was identified using all these approaches.In cases of lichens with algal coexistence, a combination of different molecular and ultrastructural approaches may be required to reveal the underlying algal diversity within a single thallus. The approach proposed in this study provides information about the relationship between molecular and ultrastructural data, and represents an improvement in the delimitation of taxonomic features which is needed to recognize intrathalline Trebouxia diversity.

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Moder, Karen-Anja, Franziska Layer, Wolfgang König, and Brigitte König. "Rapid screening of clarithromycin resistance in Helicobacter pylori by pyrosequencing." Journal of Medical Microbiology 56, no. 10 (2007): 1370–76. http://dx.doi.org/10.1099/jmm.0.47371-0.

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Helicobacter pylori infections can be effectively treated with clarithromycin, a macrolide, in combination with other antibiotics, such as amoxicillin, tetracycline or metronidazole. The failure of H. pylori eradication is mainly associated with macrolide-resistant strains. Three point mutations (A2142G/C, A2143G, T2182C) in the peptidyltransferase region of domain V of the 23S rRNA have been described as being associated with clarithromycin resistance. Therefore, the determination of clarithromycin resistance by pyrosequencing was evaluated. H. pylori from 81 gastric biopsies was cultured and clarithromycin resistance was determined by Etest, as well as by pyrosequencing technology (PSQ 96 system; Biotage). The respective mutations were set in relation to the MIC measured in μg ml−1 by Etest. In this study, point mutations in positions 2142 and 2143 were associated with clarithromycin resistance. Mutations in position 2182 did not contribute to clarithromycin resistance. In addition, from 22 out of the 81 biopsies, clarithromycin resistance was determined directly without culturing H. pylori to save additional time. Identical results were obtained as compared to resistance testing with pure H. pylori strains. All results obtained by pyrosequencing were evaluated by Sanger sequencing. The data show that pyrosequencing to detect point mutation is a fast and reliable method for determining clarithromycin resistance in H. pylori, and provides the same results as the Etest.

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Gulecal-Pektas, Yasemin. "Bacterial Diversity and Composition in Oylat Cave (Turkey) with Combined Sanger/Pyrosequencing Approach." Polish Journal of Microbiology 65, no. 1 (2016): 69–75. http://dx.doi.org/10.5604/17331331.1197277.

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Morroni, Marco, Mireille Jacquemond, and Mark Tepfer. "Deep Sequencing of Recombinant Virus Populations in Transgenic and Nontransgenic Plants Infected with Cucumber mosaic virus." Molecular Plant-Microbe Interactions® 26, no. 7 (2013): 801–11. http://dx.doi.org/10.1094/mpmi-02-13-0057-r.

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Recombination is a major source of virus variability, and the question of whether novel recombinant viruses would emerge in transgenic plants expressing viral sequences has been a biosafety issue. We describe the results of pyrosequencing the recombinant viral RNAs appearing in transgenic plants expressing the coat protein (CP) gene and 3′ noncoding region of Cucumber mosaic virus RNA3, as well as in nontransgenic controls. The populations of recombinants in both transgenic and nontransgenic plants were similar to those previously described from Sanger sequencing but many more recombinant types were observed, including a novel class of large deletions removing all or nearly the entire CP gene. These results show that populations of recombinant viral genomes arising de novo can be characterized in detail by pyrosequencing, and confirm that the transgenic plants did not harbor novel recombinants of biosafety concern.

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Jeong, Hae-Young, and Ji-Hyun F. Kim. "An Optimized Strategy for Genome Assembly of Sanger/pyrosequencing Hybrid Data using Available Software." Genomics & Informatics 6, no. 2 (2008): 87–90. http://dx.doi.org/10.5808/gi.2008.6.2.087.

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Gharizadeh, Baback, Zelek S. Herman, Robert G. Eason, Olufisayo Jejelowo, and Nader Pourmand. "Large-scale Pyrosequencing of synthetic DNA: A comparison with results from Sanger dideoxy sequencing." ELECTROPHORESIS 27, no. 15 (2006): 3042–47. http://dx.doi.org/10.1002/elps.200500834.

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Song, Ju Yeon, Haeyoung Jeong, Dong Su Yu, et al. "Draft Genome Sequence of Streptomyces clavuligerus NRRL 3585, a Producer of Diverse Secondary Metabolites." Journal of Bacteriology 192, no. 23 (2010): 6317–18. http://dx.doi.org/10.1128/jb.00859-10.

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ABSTRACT Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.

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Jost, Ted, Christophe Lacroix, Christian Braegger, and Christophe Chassard. "Assessment of bacterial diversity in breast milk using culture-dependent and culture-independent approaches." British Journal of Nutrition 110, no. 7 (2013): 1253–62. http://dx.doi.org/10.1017/s0007114513000597.

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Initial neonatal gut colonisation is a crucial stage for developing a healthy physiology, beneficially influenced by breast-feeding. Breast milk has been shown not only to provide nutrients and bioactive/immunological compounds, but also commensal bacteria, including gut-associated anaerobic Bifidobacterium spp. The aim of the present study was to investigate bacterial diversity in breast milk, with emphasis on identifying gut-associated obligate anaerobes. Breast milk collected from seven mothers at three sampling points (days 3–6, 9–14 and 25–30 postpartum) was analysed by combined culture-dependent and state-of-the-art, culture-independent methods (Sanger sequencing and 454-pyrosequencing). In addition to the predominance of facultative anaerobes such as Staphylococcus, Streptococcus and Propionibacterium (>90 % of isolated strains and 23·7 % relative abundance using pyrosequencing), significant populations of obligate anaerobes, including Bifidobacterium and Veillonella, were detected using pyrosequencing and confirmed by the isolation of viable strains (3·4 % of isolates and 1·4 % relative abundance). Pyrosequencing also revealed the presence of DNA of multiple major gut-associated obligate anaerobes (6·2 % relative abundance) such as Bacteroides and, for the first time, several members of the Clostridia, including butyrate producers, such as Faecalibacterium and Roseburia, which are important for colonic health. The present study suggests that breast milk may be a major source of bacterial diversity to the neonatal gut, including gut-associated obligate anaerobes, and may thus significantly influence gut colonisation and maturation of the immune system.

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Tsiatis, Athanasios C., Alexis Norris-Kirby, Roy G. Rich, et al. "Comparison of Sanger Sequencing, Pyrosequencing, and Melting Curve Analysis for the Detection of KRAS Mutations." Journal of Molecular Diagnostics 12, no. 4 (2010): 425–32. http://dx.doi.org/10.2353/jmoldx.2010.090188.

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Benedictis, Paola De, Cristian De Battisti, Laurent Dacheux, et al. "Lyssavirus Detection and Typing Using Pyrosequencing." Journal of Clinical Microbiology 49, no. 5 (2011): 1932–38. https://doi.org/10.5281/zenodo.13531243.

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(Uploaded by Plazi for the Bat Literature Project) Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3′ terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.

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Benedictis, Paola De, Cristian De Battisti, Laurent Dacheux, et al. "Lyssavirus Detection and Typing Using Pyrosequencing." Journal of Clinical Microbiology 49, no. 5 (2011): 1932–38. https://doi.org/10.5281/zenodo.13531243.

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(Uploaded by Plazi for the Bat Literature Project) Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3′ terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.

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Kautz, Stefanie, Benjamin E. R. Rubin, Jacob A. Russell, and Corrie S. Moreau. "Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing with Traditional Methods To Uncover Bacterial Diversity." Applied and Environmental Microbiology 79, no. 2 (2012): 525–34. http://dx.doi.org/10.1128/aem.03107-12.

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ABSTRACTWe are only beginning to understand the depth and breadth of microbial associations across the eukaryotic tree of life. Reliably assessing bacterial diversity is a key challenge, and next-generation sequencing approaches are facilitating this endeavor. In this study, we used 16S rRNA amplicon pyrosequencing to survey microbial diversity in ants. We compared 454 libraries with Sanger-sequenced clone libraries as well as cultivation of live bacteria. Pyrosequencing yielded 95,656 bacterial 16S rRNA reads from 19 samples derived from four colonies of one ant species. The most dominant bacterial orders in the microbiome of the turtle antCephalotes varianswereRhizobiales,Burkholderiales,Opitutales,Xanthomonadales, andCampylobacterales, as revealed through both 454 sequencing and cloning. Even after stringent quality filtering, pyrosequencing recovered 445 microbe operational taxonomic units (OTUs) not detected with traditional techniques. In comparing bacterial communities associated with specific tissues, we found that gut tissues had significantly higher diversity than nongut tissues, and many of the OTUs identified from these groups clustered within ant-specific lineages, indicating a deep coevolutionary history ofCephalotesants and their associated microbes. These lineages likely function as nutritional symbionts. One of four ant colonies investigated was infected with aSpiroplasmasp. (orderEntomoplasmatales), a potential ant pathogen. Our work shows that the microbiome associated withCephalotes variansis dominated by a few dozen bacterial lineages and that 454 sequencing is a cost-efficient tool to screen ant symbiont diversity.

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Margeridon-Thermet, Severine, Evguenia S. Svarovskaia, Farbod Babrzadeh, et al. "Low-Level Persistence of Drug Resistance Mutations in Hepatitis B Virus-Infected Subjects with a Past History of Lamivudine Treatment." Antimicrobial Agents and Chemotherapy 57, no. 1 (2012): 343–49. http://dx.doi.org/10.1128/aac.01601-12.

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ABSTRACTWe sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of ∼3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in ≥0.5% of UDPS reads in a sample. Sanger sequencing identified ≥1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%;P= 0.06). UDPS detected ≥1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%;P= 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P< 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.

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Scantamburlo, Giada, Konstantina Tziolia, Michaela Zopf, et al. "Allele Drop Out Conferred by a Frequent CYP2D6 Genetic Variation For Commonly Used CYP2D6*3 Genotyping Assays." Cellular Physiology and Biochemistry 43, no. 6 (2017): 2297–309. http://dx.doi.org/10.1159/000484380.

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Background/Aim: Accurate genotyping of CYP2D6 is challenging due to its inherent genetic variation, copy number variation (duplications and deletions) and hybrid formation with highly homologous pseudogenes. Because a relatively high percentage (∼25%) of clinically prescribed drugs are substrates for this enzyme, accurate determination of its genotype for phenotype prediction is essential. Methods: A cohort of 365 patient samples was genotyped for CYP2D6 using Sanger sequencing (as the gold standard), hydrolysis probe assays or pyrosequencing. Results: A discrepant result between the three genotyping methods for the loss of function CYP2D6*3 (g.2549delA, rs35742686) genetic variant was found in one of the samples. This sample also contained the CYP2D6 g.2470T>C (rs17002852) variation, which had an allele frequency of 2.47% in our cohort. Redesign of the CYP2D6*3 pyrosequencing and hydrolysis probe assays to avoid CYP2D6 g.2470 corrected the anomaly. Conclusion: To evidence allele drop out and increase the accuracy of genotyping, intra-patient validation of the same genetic variation with at least two separate methods should be considered.

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Anderson, Steven, Kenneth J. Bloom, Dino U. Vallera, et al. "Multisite Analytic Performance Studies of a Real-Time Polymerase Chain Reaction Assay for the Detection of BRAF V600E Mutations in Formalin-Fixed, Paraffin-Embedded Tissue Specimens of Malignant Melanoma." Archives of Pathology & Laboratory Medicine 136, no. 11 (2012): 1385–91. http://dx.doi.org/10.5858/arpa.2011-0505-oa.

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Context.—A polymerase chain reaction–based companion diagnostic (cobas 4800 BRAF V600 Mutation Test) was recently approved by the US Food and Drug Administration to select patients with BRAF-mutant metastatic melanoma for treatment with the BRAF inhibitor vemurafenib. Objectives.—(1) To compare the analytic performance of the cobas test to Sanger sequencing by using screening specimens from phase II and phase III trials of vemurafenib, and (2) to assess the reproducibility of the cobas test at different testing sites. Design.—Specimens from 477 patients were used to determine positive and negative percent agreements between the cobas test and Sanger sequencing for detecting V600E (1799T&gt;A) mutations. Specimens were evaluated with a massively parallel pyrosequencing method (454) to resolve discordances between polymerase chain reaction and Sanger results. Reproducibility of the cobas test was assessed at 3 sites by using 3 reagent lots and an 8-member panel of melanoma samples. Results.—A valid cobas result was obtained for all eligible patients. Sanger sequencing had a failure rate of 9.2% (44 of 477). For the remaining 433 specimens, positive percent agreement was 96.4% (215 of 223) and negative percent agreement, 80% (168 of 210). Among 42 cobas mutation-positive/Sanger V600E-negative specimens, 17 were V600E positive and 24 were V600K positive by 454. The cobas test detected 70% of V600K mutations. In the reproducibility study, a correct interpretation was made for 100% of wild-type specimens and specimens with greater than 5% mutant alleles; V600E mutations were detected in 90% of specimens with less than 5% mutant alleles. Conclusions.—The cobas test (1) had a lower assay failure rate than that of Sanger, (2) was more sensitive in detecting V600E mutations, (3) detected most V600K mutations, and (4) was highly reproducible.

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Schildgen, Verena, Carolin Schulz, Jessica Lüsebrink, et al. "Combination of Pyrosequencing® and Sanger sequencing reveals alleged novel mutation in exon 18 of EGFR." Personalized Medicine 10, no. 2 (2013): 201–9. http://dx.doi.org/10.2217/pme.12.122.

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Edgcomb, Virginia, William Orsi, John Bunge, et al. "Protistan microbial observatory in the Cariaco Basin, Caribbean. I. Pyrosequencing vs Sanger insights into species richness." ISME Journal 5, no. 8 (2011): 1344–56. http://dx.doi.org/10.1038/ismej.2011.6.

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López-Uribe, Margarita M., Christine K. Santiago, Steve M. Bogdanowicz, and Bryan N. Danforth. "Discovery and characterization of microsatellites for the solitary bee Colletes inaequalis using Sanger and 454 pyrosequencing." Apidologie 44, no. 2 (2012): 163–72. http://dx.doi.org/10.1007/s13592-012-0168-3.

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Yadav, Navneet Kumar, Pooja Shukla, Ankur Omer, Shruti Pareek, and R. K. Singh. "Next Generation Sequencing: Potential and Application in Drug Discovery." Scientific World Journal 2014 (February 5, 2014): 1–7. http://dx.doi.org/10.1155/2014/802437.

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The world has now entered into a new era of genomics because of the continued advancements in the next generation high throughput sequencing technologies, which includes sequencing by synthesis-fluorescent in situ sequencing (FISSEQ), pyrosequencing, sequencing by ligation using polony amplification, supported oligonucleotide detection (SOLiD), sequencing by hybridization along with sequencing by ligation, and nanopore technology. Great impacts of these methods can be seen for solving the genome related problems of plant and animal kingdom that will open the door of a new era of genomics. This may ultimately overcome the Sanger sequencing that ruled for 30 years. NGS is expected to advance and make the drug discovery process more rapid.

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Finkbeiner, Stacy R., Yan Li, Susan Ruone, et al. "Identification of a Novel Astrovirus (Astrovirus VA1) Associated with an Outbreak of Acute Gastroenteritis." Journal of Virology 83, no. 20 (2009): 10836–39. http://dx.doi.org/10.1128/jvi.00998-09.

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ABSTRACT The etiology of a large proportion of gastrointestinal illness is unknown. In this study, random Sanger sequencing and pyrosequencing approaches were used to analyze fecal specimens from a gastroenteritis outbreak of unknown etiology in a child care center. Multiple sequences with limited identity to known astroviruses were identified. Assembly of the sequences and subsequent reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends generated a complete genome of 6,586 nucleotides. Phylogenetic analysis demonstrated that this virus, named astrovirus VA1 (AstV-VA1), is highly divergent from all previously described astroviruses. Based on RT-PCR, specimens from multiple patients in this outbreak were unequivocally positive for Ast-VA1.

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Press, Richard D., and Fei Yang. "Comparison of Different Laboratory Methods for Quantification of BCR-ABL Kinase Domain Mutations In CML Patients Undergoing Tyrosine Kinase Inhibitor Therapy." Blood 116, no. 21 (2010): 2760. http://dx.doi.org/10.1182/blood.v116.21.2760.2760.

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Abstract Abstract 2760 Introduction: Although most CML patients treated with tyrosine kinase inhibitors (TKI's) achieve durable responses, some develop drug resistance that is usually due to a heterogeneous array of acquired mutations in the BCR-ABL kinase domain (KD). While many of these mutations confer resistance to imatinib, most (but not all) of these mutations will respond to a second-generation TKI (dasatinib or nilotinib). The qualitative detection of KD mutations, typically by direct DNA sequencing, is thus required for the optimal management of suspected drug resistance. Once a specific mutation is identified, however, a laboratory method to quantitatively monitor the mutation's subsequent response to the new therapy would be desirable. Toward that goal, we have developed and validated 2 different laboratory assays for the quantitative analysis of BCR-ABL KD mutations – pyrosequencing, and allele-specific PCR – and report their performance in the long-term serial monitoring of drug resistant CML patients. Methods: For pyrosequencing, sequencing primers were designed 1–28 nucleotides adjacent to the polymorphic sites of common KD variants T315I, M351T, Y253H/F, E255K, F359V, M244V, Q252H, and G250E, and quantitation of mutant allele burdens was accomplished with the SNP-AQ function of the PyroMark ID instrument (Qiagen). For allele-specific PCR, real-time PCR primers were designed that preferentially amplified the mutant allele of common variants T315I, M351T, Y253H, E255K, and F359V. The 17 patients included in this retrospective study were all of those from our institution with a known KD mutation at any of the 5 loci targeted by our allele-specific PCR assays and with at least 5 available archival samples (from each patient) with known Sanger sequence information. Results: Of the 17 patients (65% male, average age=51), 16 had CML (1 had Ph+-ALL), and all were treated with imatinib as the initial TKI. 11 of the 17 patients achieved a major molecular response on imatinib. The total follow-up duration, from the time of imatinib initiation, was 6.9 years [median (IQR 4.0–8.8)], during which samples for BCR-ABL RQ-PCR were drawn every 3.0 months [median (IQR 1.9–4.2)]. The second-generation TKI was dasatinib in 9 patients, nilotinib in 1 patient, and AP24534 in one patient. The spectrum of KD mutations included T315I (8 pts), M351T (3 pts), Y253H/F (4 pts), E255K (4 pts), F359V (4 pts), Q252H (2 pts), and G250E (2 pts). Eight patients had 2 different KD mutations, and one patient had 3 different mutations. The first detectable KD mutation was found after 1.7 years of TKI therapy [median (IQR 1.0–2.0)]. From these 17 patients, 269 archival samples were available for quantitation of the mutation burden by pyrosequencing and allele-specific PCR [median 17 samples per patient (IQR 8–27)]. For allele-specific PCR (AS-PCR), the lower limit of detection was 100 copies of mutant DNA per PCR reaction. For pyrosequencing (Pyro), the lowest BCR-ABL transcript level that reliably yielded a signal above background was ∼0.03% on the international scale, and a mutant allele burden below 5% could not be reliably detected. For Sanger sequencing, a mutant allele burden below ∼20% could not be reliably detected. Of the 217 samples for which readable data could be generated by both Pyro & AS-PCR, AS-PCR was slightly more sensitive for the detection of a KD mutation - yielding positive results in 84 samples, as compared to 79 mutations detectable with Pyro. In contrast, Sanger sequencing detected slightly fewer mutations than either Pyro or AS-PCR, consistent with its presumed lower detection sensitivity. In 12 patients, there were a total of 48 samples that had a KD mutation detectable by both Pyro and allele-specific PCR in both the analyzed sample and an immediately prior sample, allowing a “delta allele burden” value to be calculated. The change in mutant allele burden between consecutively drawn sample pairs was no different for allele burdens quantitated by pyrosequencing as compared to those quantitated by allele-specific PCR (average 0.05 log difference; P>0.8). Conclusions: Quantitative monitoring of the BCR-ABL kinase domain mutation allele burden can be accurately accomplished with either pyrosequencing or allele-specific PCR. Disclosures: No relevant conflicts of interest to declare.

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Lopez-Rios, Fernando, Barbara Angulo, Belen Gomez, et al. "Comparison of molecular testing methods for the detection of EGFR mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer." Journal of Clinical Pathology 66, no. 5 (2013): 381–85. http://dx.doi.org/10.1136/jclinpath-2012-201240.

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AimTo conduct a methods correlation study of three different assays for the detection of mutations at EGFR gene in human formalin-fixed paraffin-embedded tumour (FFPET) specimens of non-small cell lung carcinomas (NSCLC).MethodsWe conducted a 2-site method comparison study of two european conformity (CE) in vitro diagnostic (IVD)-marked assays, the cobas EGFR Mutation Test and the Therascreen EGFR29 Mutation Kit, and 2× bidirectional Sanger sequencing. We blind-tested 124 NSCLC FFPET specimens with all three methods; the cobas test was performed at both sites. Positive (PPA) and negative percent agreements (NPA) were determined for the cobas test versus each of the other two methods. Specimens yielding discordant test results between methods were further tested using quantitative massively parallel pyrosequencing (MPP).ResultsPPA between cobas and Sanger was 98.8%; NPA was 79.3%. Overall there were seven discordant results. MPP confirmed an exon 19 deletion in two cases and L858R mutation in four cases. PPA between cobas and Therascreen was 98.9% and NPA was 100%. There was one discordant result. Reproducibility of the cobas test between the two sites was 99.2%.ConclusionsThe invalid rates for the cobas test and Therascreen were lower than Sanger sequencing. The cobas and Therascreen assays showed a high degree of concordance, and both were more sensitive for the detection of exon 19 deletion and L858R mutations than Sanger. The cobas test was highly reproducible between the two testing sites, used the least amount of DNA input and was the only test with automated results reporting.

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Tedersoo, Leho, R. Henrik Nilsson, Kessy Abarenkov, et al. "454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases." New Phytologist 188, no. 1 (2010): 291–301. http://dx.doi.org/10.1111/j.1469-8137.2010.03373.x.

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Ueno, Saneyoshi, Grégoire Le Provost, Valérie Léger, et al. "Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak." BMC Genomics 11, no. 1 (2010): 650. http://dx.doi.org/10.1186/1471-2164-11-650.

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Goldberg, S. M. D., J. Johnson, D. Busam, et al. "A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes." Proceedings of the National Academy of Sciences 103, no. 30 (2006): 11240–45. http://dx.doi.org/10.1073/pnas.0604351103.

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Hahn, Kristen R., Timothy W. Janzen, Matthew C. Thomas, et al. "Single nucleotide repeat analysis of B. anthracis isolates in Canada through comparison of pyrosequencing and Sanger sequencing." Veterinary Microbiology 169, no. 3-4 (2014): 228–32. http://dx.doi.org/10.1016/j.vetmic.2013.12.020.

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Youssef, Noha, Brandi L. Steidley, and Mostafa S. Elshahed. "Novel High-Rank Phylogenetic Lineages within a Sulfur Spring (Zodletone Spring, Oklahoma), Revealed Using a Combined Pyrosequencing-Sanger Approach." Applied and Environmental Microbiology 78, no. 8 (2012): 2677–88. http://dx.doi.org/10.1128/aem.00002-12.

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ABSTRACTThe utilization of high-throughput sequencing technologies in 16S rRNA gene-based diversity surveys has indicated that within most ecosystems, a significant fraction of the community could not be assigned to known microbial phyla. Accurate determination of the phylogenetic affiliation of such sequences is difficult due to the short-read-length output of currently available high-throughput technologies. This fraction could harbor multiple novel phylogenetic lineages that have so far escaped detection. Here we describe our efforts in accurate assessment of the novelty and phylogenetic affiliation of selected unclassified lineages within a pyrosequencing data set generated from source sediments of Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma. Lineage-specific forward primers were designed for 78 putatively novel lineages identified within the pyrosequencing data set, and representative nearly full-length small-subunit (SSU) rRNA gene sequences were obtained by pairing those primers with reverse universal bacterial primers. Of the 78 lineages tested, amplifiable products were obtained for 52, 32 of which had at least one nearly full-length sequence that was representative of the lineage targeted. Analysis of phylogenetic affiliation of the obtained Sanger sequences identified 5 novel candidate phyla and 10 novel candidate classes (withinFibrobacteres,Planctomycetes, and candidate phyla BRC1, GN12, TM6, TM7, LD1, WS2, and GN06) in the data set, in addition to multiple novel orders and families. The discovery of multiple novel phyla within a pilot study of a single ecosystem clearly shows the potential of the approach in identifying novel diversities within the rare biosphere.

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Ruiz, Anna, Gemma Llort, Carmen Yagüe, et al. "Genetic Testing in Hereditary Breast and Ovarian Cancer Using Massive Parallel Sequencing." BioMed Research International 2014 (2014): 1–8. http://dx.doi.org/10.1155/2014/542541.

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High throughput methods such as next generation sequencing are increasingly used in molecular diagnosis. The aim of this study was to develop a workflow for the detection ofBRCA1andBRCA2mutations using massive parallel sequencing in a 454 GS Junior bench top sequencer. Our approach was first validated in a panel of 23 patients containing 62 unique variants that had been previously Sanger sequenced. Subsequently, 101 patients with familial breast and ovarian cancer were studied.BRCA1andBRCA2exon enrichment has been performed by PCR amplification using the BRCA MASTR kit (Multiplicom). Bioinformatic analysis of reads is performed with the AVA software v2.7 (Roche). In total, all 62 variants were detected resulting in a sensitivity of 100%. 71 false positives were called resulting in a specificity of 97.35%. All of them correspond to deletions located in homopolymeric stretches. The analysis of the homopolymers stretches of 6 bp or longer using the BRCA HP kit (Multiplicom) increased the specificity of the detection ofBRCA1andBRCA2mutations to 99.99%. We show here that massive parallel pyrosequencing can be used as a diagnostic strategy to test forBRCA1andBRCA2mutations meeting very stringent sensitivity and specificity parameters replacing traditional Sanger sequencing with a lower cost.

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Colombino, Maria, Carla Rozzo, Panagiotis Paliogiannis, et al. "Comparison of BRAF Mutation Screening Strategies in a Large Real-Life Series of Advanced Melanoma Patients." Journal of Clinical Medicine 9, no. 8 (2020): 2430. http://dx.doi.org/10.3390/jcm9082430.

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Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation status plays a predominant role in the management of MM patients. The aim of this study was to compare BRAF mutational testing performed by conventional nucleotide sequencing approaches with either real-time polymerase chain reaction (rtPCR) or next-generation sequencing (NGS) assays in a real-life, hospital-based series of advanced MM patients. Consecutive patients with AJCC (American Joint Committee on Cancer) stage IIIC and IV MM from Sardinia, Italy, who were referred for molecular testing, were enrolled into the study. Initial screening was performed to assess the mutational status of the BRAF and NRAS genes, using the conventional methodologies recognized by the nationwide guidelines, at the time of the molecular classification, required by clinicians: at the beginning, Sanger-based sequencing (SS) and, after, pyrosequencing. The present study was then focused on BRAF mutation detecting approaches only. BRAF wild-type cases with available tissue and adequate DNA were further tested with rtPCR (Idylla™) and NGS assays. Globally, 319 patients were included in the study; pathogenic BRAF mutations were found in 144 (45.1%) cases examined with initial screening. The rtPCR detected 11 (16.2%) and 3 (4.8%) additional BRAF mutations after SS and pyrosequencing, respectively. NGS detected one additional BRAF-mutated case (2.1%) among 48 wild-type cases previously tested with pyrosequencing and rtPCR. Our study evidenced that rtPCR and NGS were able to detect additional BRAF mutant cases in comparison with conventional sequencing methods; therefore, we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy of BRAF detection.

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Lowe, Christopher F., Linda Merrick, P. Richard Harrigan, Tony Mazzulli, Christopher H. Sherlock, and Gordon Ritchie. "Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory." Journal of Clinical Microbiology 54, no. 1 (2015): 127–33. http://dx.doi.org/10.1128/jcm.02229-15.

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Sanger sequencing or DNA hybridization have been the primary modalities for hepatitis B (HBV) resistance testing and genotyping; however, there are limitations, such as low sensitivity and the inability to detect novel mutations. Next-generation sequencing (NGS) for HBV can overcome these limitations, but there is limited guidance for clinical microbiology laboratories to validate this novel technology. In this study, we describe an approach to implementing deep pyrosequencing for HBV resistance testing and genotyping in a clinical virology laboratory. A nested PCR targeting thepolregion of HBV (codons 143 to 281) was developed, and the PCR product was sequenced by the 454 Junior (Roche). Interpretation was performed by ABL TherapyEdge based on European Association for the Study of the Liver (EASL) guidelines. Previously characterized HBV samples by INNO-LiPA (LiPA) were compared to NGS with discordant results arbitrated by Sanger sequencing. Genotyping of 105 distinct samples revealed a concordance of 95.2% (100/105), with Sanger sequencing confirming the NGS result. Resistance testing by NGS was concordant with LiPA in 85% (68/80) of previously characterized samples. Additional mutations were found in 8 samples, which related to the identification of low-level mutant subpopulations present at <10% (6/8). To balance the costs of testing for the validation study, reproducibility of the NGS was investigated through an analysis of sequence variants at loci not associated with resistance in a single patient sample. Our validation approach attempts to balance costs with efficient data acquisition.

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Cuartas, Jaison H., Juan F. Alzate, Claudia X. Moreno-Herrera, and Edna J. Marquez. "Metagenomic analysis of orange colored protrusions from the muscle of Queen ConchLobatus gigas(Linnaeus, 1758)." PeerJ 6 (February 15, 2018): e4307. http://dx.doi.org/10.7717/peerj.4307.

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The endangered marine gastropod,Lobatus gigas,is an important fishery resource in the Caribbean region. Microbiological and parasitological research of this species have been poorly addressed despite its role in ecological fitness, conservation status and prevention of potential pathogenic infections. This study identified taxonomic groups associated with orange colored protrusions in the muscle of queen conchs using histological analysis, 454 pyrosequencing, and a combination of PCR amplification and automated Sanger sequencing. The molecular approaches indicate that the etiological agent of the muscle protrusions is a parasite belonging to the subclass Digenea. Additionally, the scope of the molecular technique allowed the detection of bacterial and fungi clades in the assignment analysis. This is the first evidence of a digenean infection in the muscle of this valuable Caribbean resource.

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Lee, Ryan M., Jyothi Thimmapuram, Kate A. Thinglum, et al. "Sampling the Waterhemp (Amaranthus tuberculatus) Genome Using Pyrosequencing Technology." Weed Science 57, no. 5 (2009): 463–69. http://dx.doi.org/10.1614/ws-09-021.1.

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Recent advances in sequencing technologies (next-generation sequencing) offer dramatically increased sequencing throughput at a lower cost than traditional Sanger sequencing. This technology is changing genomics research by allowing large scale sequencing experiments in nonmodel systems. Waterhemp is an important weed in the midwestern United States with characteristics that makes it an interesting ecological model. However, very few genomic resources are available for this species. One half of a 70 by 75 picotiter plate of 454-pyrosequencing was performed on total DNA isolated from waterhemp, generating 158,015 reads of an average length of 271 bp, or a total of nearly 43 Mbp of sequence. Included in this sequence was a nearly complete sequence of the chloroplast genome, sequences of several important herbicide resistance genes, leads for simple sequence repeat (SSR) markers, and a sampling of the repeated elements (e.g., transposons) present in this species. Here we present the waterhemp genomic data gleaned from this sequencing experiment and illustrate the value of next-generation sequencing technology to weed science research.

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Camargo Barros-Filho, Mateus, Larissa Barreto Menezes de Lima, Mariana Bisarro dos Reis, et al. "PFKFB2 Promoter Hypomethylation as Recurrence Predictive Marker in Well-Differentiated Thyroid Carcinomas." International Journal of Molecular Sciences 20, no. 6 (2019): 1334. http://dx.doi.org/10.3390/ijms20061334.

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Despite the low mortality rates, well-differentiated thyroid carcinomas (WDTC) frequently relapse. BRAF and TERT mutations have been extensively related to prognosis in thyroid cancer. In this study, the methylation levels of selected CpGs (5-cytosine-phosphate-guanine-3) comprising a classifier, previously reported by our group, were assessed in combination with BRAF and TERT mutations. We evaluated 121 WDTC, three poorly-differentiated/anaplastic thyroid carcinomas (PDTC/ATC), 22 benign thyroid lesions (BTL), and 13 non-neoplastic thyroid (NT) tissues. BRAF (V600E) and TERT promoter (C228T and C250T) mutations were tested by pyrosequencing and Sanger sequencing, respectively. Three CpGs mapped in PFKFB2, ATP6V0C, and CXXC5 were evaluated by bisulfite pyrosequencing. ATP6V0C hypermethylation and PFKFB2 hypomethylation were detected in poor-prognosis (PDTC/ATC and relapsed WDTC) compared with good-prognosis (no relapsed WDTC) and non-malignant cases (NT/BTL). CXXC5 was hypomethylated in both poor and good-prognosis cases. Shorter disease-free survival was observed in WDTC patients presenting lower PFKFB2 methylation levels (p = 0.004). No association was observed on comparing BRAF (60.7%) and TERT (3.4%) mutations and prognosis. Lower PFKFB2 methylation levels was an independent factor of high relapse risk (Hazard Ratio = 3.2; CI95% = 1.1–9.5). PFKFB2 promoter methylation analysis has potential applicability to better stratify WDTC patients according to the recurrence risk, independently of BRAF and TERT mutations.

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Mark, Kristiina, Carolina Cornejo, Christine Keller, Daniela Flück, and Christoph Scheidegger. "Barcoding lichen-forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation." Genome 59, no. 9 (2016): 685–704. http://dx.doi.org/10.1139/gen-2015-0189.

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Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database—currently the most complete database for lichen-forming fungi—can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).

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Fu, Yong-Bi. "Genetic Relationships of Cultivated Flax and Its Wild Progenitor as Revealed by 454 Pyrosequencing, Sanger Resequencing and Microsatellite Data." Sci 6, no. 2 (2024): 35. http://dx.doi.org/10.3390/sci6020035.

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Flax (Linum usitatissimum L.), as the earliest oil and fiber crop, is a model plant for genetic inferences of plant domestication processes involving multiple domestication events. However, a puzzle has emerged from several genetic studies, as dehiscent cultivated flax is genetically more related to its progenitor pale flax (L. bienne Mill.), and winter cultivated flax is well mixed with oil or fiber cultivated flax, while capsular dehiscence and winter hardiness are the major characteristics of pale flax. For this, a comparative analysis was conducted with 16 Linum samples representing pale flax and four domestication groups of cultivated flax (oil, fiber, winter, and dehiscent) using 454 pyrosequencing, Sanger resequencing and microsatellite data. It was found that the genomic sampling of genetic variants from the three applied methods yielded similar genetic information on pale flax and four groups of cultivated flax. The revealed genetic relationships did not show significant departures from the previous findings, but instead supported an early, independent domestication of a primitive flax lineage for oil use, followed by a subsequent flax domestication process with multiple domestication events for capsular dehiscence, oil, fiber and winter hardiness. Domestication on capsular dehiscence occurred earlier than domestication on winter hardiness. Domestication on winter hardiness was more complicated than domestication on capsular dehiscence.

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Longstaff, Louise, Emily Porter, Victoria J. Crossley, Sophie E. Hayhow, Christopher R. Helps, and Séverine Tasker. "Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis." Journal of Feline Medicine and Surgery 19, no. 2 (2016): 240–45. http://dx.doi.org/10.1177/1098612x15606957.

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Objectives The aim of the study was to determine whether feline coronavirus (FCoV) RNA in effusion samples can be used as a diagnostic marker of feline infectious peritonitis (FIP); and in FCoV RNA-positive samples to examine amino acid codons in the FCoV spike protein at positions 1058 and 1060 where leucine and alanine, respectively, have been associated with systemic or virulent (FIP) FCoV infection. Methods Total RNA was extracted from effusion samples from 20 cats with confirmed FIP and 23 cats with other diseases. Feline coronavirus RNA was detected using a reverse transcriptase quantitative polymerase chain reaction assay (qRT-PCR), and positive samples underwent pyrosequencing of position 1058 with or without Sanger sequencing of position 1060 in the FCoV spike protein. Results Seventeen (85%) of the effusion samples from 20 cats with FIP were positive for FCoV RNA, whereas none of the 23 cats with other diseases were positive. Pyrosequencing of the 17 FCoV-positive samples showed that 11 (65%) of the cats had leucine and two (12%) had methionine at position 1058. Of the latter two samples with methionine, one had alanine at position 1060. Conclusions and relevance A positive FCoV qRT-PCR result on effusions appears specific for FIP and may be a useful diagnostic marker for FIP in cats with effusions. The majority of FCoVs contained amino acid changes previously associated with systemic spread or virulence (FIP) of the virus.

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Journal articles on the topic 'Sanger/pyrosequencing'

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