Pyrosequencing: Methods and Protocols by Ulrich Lehmann, Jorg Tost - ivytivihed.ml
A high level of safety has been reported in both pediatric and adult populations receiving the Russian LAIV . Recently the Russian LAIVs were licensed to the WHO for the subsequent transfer of the technology to developing country manufacturers who could then provide influenza vaccines to the public royalty-free . LAIV produced by co-infection results in a pool of reassortant viruses with a random combination of the eight RNA genomes from the two parental viruses which then are subjected to passages under selective pressure in the presence of serum to MDV and at low temperature.
However, during preparation of LAIV candidates, even under selective pressure, variability in genes donated by the ca donor of both influenza A and B was reported  — . As a result, a sensitive genotyping method is required for the rapid identification and isolation of the reassortant clone containing the desired gene composition to enable vaccines to be manufactured in a timely manner. Several methods have been described and used for the screening and genotyping of reassortant influenza viruses, such as analysis of restriction fragment length polymorphism RFLP of viral genes produced by reverse-transcription polymerase chain reaction RT-PCR  —  , and multiplex RT-PCR techniques  — .
However, none of these techniques provide genetic sequencing data. Sometimes, in the case of high sequence homology between the genes of viruses used in reassortment the genotyping by RT-PCR-RFLP cannot be performed because the lack of suitable restriction sites in the genome . Pyrosequencing has previously been used for genotyping herpes simplex, hepatitis C viruses  ,  It has also been used for diagnostic applications, identification and subtyping of emerging influenza A viruses and influenza A reassortants including LAIV candidate viruses  , for monitoring of drug resistance in seasonal influenza A viruses  —  and for detection and differentiation of currently circulating human B-lineage viruses .
Pyrosequencing differs from other sequencing technologies in its ability to generate quantitative data from the nucleotide incorporation which allows for an accurate analysis and detection of minor variants in a population  — . In addition, pyrosequencing is a rapid technique which allows screening of a large number of samples. The proposed approach can be used as a rapid and reliable genotyping technique for the preparation of LAIV candidates and expedite the generation of vaccine.
Petersburg, Russia. The HI assay was used to determine the origin of hemagglutinin HA genes of reassortant virus clones. HI assays were performed in well V-microtiter plates using 0. Reassortant influenza viruses that possess the internal genes of MDV and the surface antigen genes of wt viruses used were prepared according to the method developed by IEM, St.
Petersburg, Russia  — . Each clone was analyzed for genome composition using the pyrosequencing assay.
- Proceedings of the Dynamic Flow Conference 1978 on Dynamic Measurements in Unsteady Flows;
- Three Approaches to Data Analysis: Test Theory, Rough Sets and Logical Analysis of Data.
- Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS).
- Methods and Protocols.
- Schaums Outlines of Vector Analysis (2nd Edition) (Schaums Outlines Series);
- Supplementary files.
The targets with signature nucleotides which were strain-specific for each gene were selected using BioEdit Sequence Alignment Editor. Primers were used at a final concentration of 0. A negative control water was used to determine the level of background associated with the primers. Nucleotide dispensations for the pyrosequencing assay were customized to improve the detection of strain-specific nucleotide differences . A final wash in 10 mM Tris-acetate, pH 7. The sequencing primer nM in annealing buffer 20 mM Tris-acetate, pH 7. Sequence results were obtained in the form of pyrograms and analyzed using visual interpretation and the PyroMark Q96 software Qiagen.
Raw pyrogram data obtained during preparation of influenza virus reassortants - candidates for live attenuated vaccines. The unique strain-specific nucleotides are highlighted in red. RT-PCR products analyzed on an agarose gel, showed a clear, single band without non-specific products or primer-dimers. The pyrosequencing runs of these RT-PCR fragments showed that all the developed assays were specific for wt and ca viruses. Strain-specific nucleotide peaks were detected with little to no background noise, allowing discrimination between the parental strains of viruses used in reassortment.
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The examples of obtained pyrograms for MDVs and wt viruses are shown on Fig. RNAs were analyzed by pyrosequencing of NS gene. The signature nucleotides of wt are shown in red, MDV — in green. The pyrosequencing assay for the NS gene was used to evaluate the limit of detection of minor viral RNA in mixtures. The peak heights in the pyrograms are proportional to the number of each nucleotide incorporated and also to the percentage of each RNA species present in the mixture. The pyrosequencing assays were utilized for the screening of reassortant clones produced during LAIV preparation.
Reassortant progeny which were inhibited by serum to wt virus in HI assay see Materials and Methods , i. The 96 well format of pyrosequencing allowed us to analyze the origin of three genes in 30 clones in one run. The analysis showed that all reassortants had the NA gene of the desired wt origin. Only one clone was detected number 4. Since only one candidate clone 4. The pyrosequencing analysis revealed that the rest of the internal genes of this clone had the correct MDV origin Table 7.
This clone was then subjected to the second round of cloning by a limiting dilution. Forty-six clones from the second cloning were analyzed by pyrosequencing for the NS and M genes. Forty-five of these clones had the M gene of the desired MDV origin, and only one clone had the wt M gene.
Pyrosequencing analysis confirmed that the clone The signature nucleotides of desired genes in the reassortants circled in green, non-desired — in red. At the first cloning stage, all clones had a correct origin of NA gene but all other genes were present in a mixed population. Clone 4. The five eggs infected at selective conditions were then blindly passaged and genotyped for the origin of NA, NS and M genes.
The pyrosequencing analysis showed that two eggs 2. Progeny from egg 2. Forty five clones derived from egg 2. The pyrosequencing analysis showed that only two clones from the highest limited dilution The pyrosequencing analysis of these clones identified that the rest of internal genes were of a desired MDV origin Table 7.
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Both assays verified the origin of all RNA segments and the accuracy of pyrosequencing assay described here. The tailed primer forward or reverse should not be longer than 20 bp. The sequencing primer should be designed between one and five nucleotides of the target SNP.
Select those primers with the highest scores assigned by the software according to their quality for PCR and Pyrosequencing. Even if other DNA source is considered e. PCR product , a minimum of 50 ng should be used as well. At this step, biotin is being incorporated into the PCR product, as described by Schuelke for fluorescent labelling of PCR fragments. Check the presence of a single and strong DNA fragment with the expected size.
If there is any unspecific product, which is highly unusual if the primer design was performed accurately, a HotStart Taq polymerase might be used with the same PCR mix described above, except for MgCl 2 2 mM , dNTPs 0. Before starting the pyrosequencing assay, let the buffers reach room temperature.
Seal the plate and shake the PCR plate constantly at ca. Place the vacuum prep tool VPT in the water trough and switch on the vacuum pump Figure 1. Figure 1. Vacuum workstation with the vacuum prep tool VPT and the different troughs Flush the filter probes for 20 sec. Denaturation solution for 5 sec. Washing buffer for 5 sec. Switch off the vacuum pump and return the VPT the horizontal position. Shake the VPT gently and let it rest for a few seconds on the bottom of the wells. Move the VPT to the water trough to wash the filters by agitating for 10 sec.
Cool the PSQ plate at room temperature for at least 5 min to allow the annealing of the sequencing primer. Load the cartridge with the estimated amount of PSQ reagents. Place the plate in the PSQ 96 instrument and insert the cartridge carefully. Comprehensive and adaptable, Pyrosequencing: Methods and Protocols, Second Edition will greatly aid doctorial students, postdoctoral investigators and research scientists studying different aspects of genetics and cellular and molecular biology.
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