Identification of a Novel Astrovirus (Astrovirus VA1) Associated with an Outbreak of Acute Gastroenteritis
Stacy R. FinkbeinerYan LiSusan RuoneChristina ConrardyNicole GregoricusDenise ToneyHerbert W. VirginLarry J. AndersonJan VinjéDavid WangSuxiang Tong
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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.Keywords:
Astrovirus
Sanger sequencing
Pyrosequencing
OBJECTIVE To evaluate the potential value of pyrosequencing method in the detection of HBV resistance genes through the comparison with PCR product direct sequencing method.METHODS The patients with chronic hepatitis B treated with lamivudine were selected,the sensitivity and specificity of two methods mentioned above were compared by detecting the variation of lamivudine resistance mutation site rt180aa.RESULTS Both pyrosequencing and Sanger sequencing could detect the dominant virus(90%) exactly.When the proportion of rt180 mutation ranged from 90% to 50%,which was detected by pyrosequencing,the detection value by Sanger sequencing were reduced to 76.92% and 50.00%;when the proportion value of rt180M was within 50%-10% determined by pyrosequencing,it would be reduced to 10.00%-15.38% by Sanger sequencing method;that means quasispecies less than 10% could be only detected by pyrosequencing rather than by Sanger sequencing.CONCLUSION As compared with the Sanger sequencing,pyrosequencing method is with better sensitivity in the detection of variation of hepatitis B virus,which can detect the quasispecies and its population drift during the antiviral therapy process,with great significance in research on the antiviral effect and the forecast of drug resistance.
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Viral quasispecies
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ABSTRACT Macrolide-resistant Mycoplasma pneumoniae (MRMP) is emerging worldwide and has been associated with treatment failure. In this study, we used pyrosequencing to detect low-frequency MRMP quasispecies in respiratory specimens, and we compared the findings with those obtained by Sanger sequencing and SimpleProbe PCR coupled with a melting curve analysis (SimpleProbe PCR). Sanger sequencing, SimpleProbe PCR, and pyrosequencing were successfully performed for 96.7% (88/91), 96.7% (88/91), and 93.4% (85/91) of the M. pneumoniae -positive specimens, respectively. The A-to-G transition at position 2063 was the only mutation identified. Pyrosequencing identified A2063G MRMP quasispecies populations in 78.8% (67/88) of the specimens. Only 38.8% (26/67) of these specimens with the A2063G quasispecies detected by pyrosequencing were found to be A2063G quasispecies by Sanger sequencing or SimpleProbe PCR. The specimens that could be detected by SimpleProbe PCR and Sanger sequencing had higher frequencies of MRMP quasispecies (51% to 100%) than those that could not be detected by those two methods (1% to 44%). SimpleProbe PCR correctly categorized all specimens that were identified as wild type or mutant by Sanger sequencing. The clinical characteristics of the patients were not significantly different when they were grouped by the presence or absence of MRMP quasispecies, while patients with MRMP identified by Sanger sequencing more often required a switch from macrolides to an alternative M. pneumoniae -targeted therapy. The clinical significance of mutant quasispecies should be investigated further with larger patient populations and with specimens obtained before and after macrolide therapy.
Viral quasispecies
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Pyrosequencing
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Objective
To investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical colorectal cancer tissues.
Methods
Genomic DNA, extracted from K-ras mutant cell lines SW480 (homozygous, c.35G>T), DLD-1 (heterozygous, c.38G>A) and wild-type HT-29, was first used as the sequencing template respectively to test the accuracy of pyrosequencing methodology.The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2%, 3%, 5%, 10%, 20%, 30% and 50%, the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples.The pyrosequencing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test.Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at codons 12 and 13 on DNA isolated from a panel of 30 colorectal cancer samples derived from clinical formalin-fixed and paraffin embedded (FFPE) tissues.
Results
Cancer cell lines with known K-ras mutations (SW480 and DLD-1) were readily detectable by pyrosequencing-based analysis.When the proportions of mutant colorectal cancer cell line DNA were 5% and 10% content, the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3% (4/12) and 58.3% (7/12) respectively, whereas the mutation rates detected by pyrosequencing-based assay were 91.7% (11/12) and 100% (12/12) respectively, there were significant differences between those two sequencing methodology (P A transitions ［50%（5/10）］, followed by G>T transversions ［30%（3/10）］.
Conclusion
The pyrosequencing assay provides an accurate and sensitive method for mutation screening of K-ras exon 2 codons 12 and 13 in routine diagnostic specimens, thereby allowing the selection of the cancer treatment in clinical individualized practice.(Chin J Lab Med,2012,35:585-592)
Key words:
Colorectal neoplasms; Genes，ras; Exons; Mutation; Sequence analysis，DNA
Pyrosequencing
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genomic DNA
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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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Problem statement: Pyrosequencing has the potential to rapidly and reliably sequence DNA taking advantages over traditional Sanger di-deoxy sequencing approach. Approach: A comprehensive review of the literature on the principles, applications, challenges and prospects of pyrosequencing was performed. Results: Pyrosequencing was a DNA sequencing technology based on the sequencing-by-synthesis principle. It employs a series of four enzymes to accurately detect nucleic acid sequences during the synthesis. Pyrosequencing had the potential advantages of accuracy, flexibility, parallel processing and could be easily automated. The technique dispenses with the need for labeled primers, labeled nucleotides and gel-electrophoresis. Pyrosequencing had opened up new possibilities for performing sequence-based DNA analysis. The method had been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Pyrosequencing had been successful for both confirmatory sequencing and de novo sequencing. By increasing the read length to higher scores and by shortening the sequence reaction time per base calling, pyrosequencing may take over many broad areas of DNA sequencing applications as the trend was directed to analysis of fewer amounts of specimens and large-scale settings, with higher throughput and lower cost. Conclusion/Recommendations: The Competitiveness of pyrosequencing with other sequencing methods can be improved in future."
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Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool of surveillance specimens to demonstrate that it is possible to determine TDR rates in HIV protease from a population of individuals.The protease region from 96 treatment naïve, HIV+ serum specimens was genotyped using standard Sanger sequencing method. The 462 bp protease amplicons from these specimens were pooled in equimolar concentrations and re-sequenced using the GS FLX Titanium system. The nucleotide (NT) and amino acid (AA) differences from the reference sequence, along with TDR mutations, detected by each method were compared. In the protease sequence, there were 212 nucleotide and 81 AA differences found using conventional sequencing and 345 nucleotide and 168 AA differences using pyrosequencing. All nucleotide and amino acid polymorphisms found at frequencies >/=5% in pyrosequencing were detected using both methods with the rates of variation highly correlated. Using Sanger sequencing, two TDR mutations, M46L and I84V, were each detected as mixtures at a frequency of 1.04% (1/96). These same TDR mutations were detected by pyrosequencing with a prevalence of 0.29% and 0.34% respectively. Phylogenetic analysis established that the detected low frequency mutations arose from the same single specimens that were found to contain TDR mutations by Sanger sequencing. Multiple clinical protease DR mutations present at higher frequencies were concordantly identified using both methods.We show that pyrosequencing pooled surveillance specimens can cost-competitively detect protease TDR mutations when compared with conventional methods. With few modifications, the method described here can be used to determine population rates of TDR in both protease and reverse transcriptase. Furthermore, this pooled pyrosequencing technique may be generalizable to other infectious agents where a survey of DR rates is required.
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Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.
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The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.
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