Partial Sanger sequencing of HIV-1 pol-1 gene(Pr/RT regions) in Tunisia
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PROVIDER: PRJEB71896 | ENA |
REPOSITORIES: ENA
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Project description:Pistacia vera isolate:Tunisia Genome sequencing
| PRJNA527814 | ENA
Project description:In an effort to evaluate the accuracy of HIV-1 phylogenies based on genomes of increasing length, we developed a comprehensive near-full-length HIV-1 genome RT-PCR assay and performed a comparative evaluation via phylogenetic analyses. To this end, we conducted comparative analyses of HIV-1 phylogenies derived based on HIV-1 PR/RT (2253-3359 in the HXB2 genome) and pol region (2253-5250 in the HXB2 genome) sequences isolated from 134 HIV-1-infected patients in Cyprus (2017-2019). The HIV-1 genotypic subtypes determined using six subtyping tools (REGA 3.0, COMET 2.3, jpHMM, SCUEAL, Stanford, and Geno2pheno) were compared to investigate the discrepancies generated among different tools. To evaluate the accuracy of defined HIV-1 phylogenies, the samples exhibiting at least one discrepant subtyping result among different subtyping tools in both PR/RT and pol regions or only in the pol region (n = 38) were selected for near-full-length HIV-1 genome (790-8795 in HXB2 genome) sequencing using a newly developed RT-PCR/sequencing assay. The obtained sequences were employed for HIV-1 genotypic subtype determination and subjected to comparative phylogenetic-based analyses. It was observed that 39.6% of the 134 samples presented discrepancies in the PR/RT region, while 28.4% presented discrepancies in the pol region. REGA 3.0 produced the fewest discrepancies collectively in both regions and was selected for subsequent subtyping and comparative phylogenetic analyses of near-full-length HIV-1 genome sequences. The analyses of near-full-length HIV-1 genome sequences identified 68.4% of the 38 'discrepant samples' (n = 26) as belonging to uncharacterized recombinant HIV-1 strains, while 21.1% were circulating recombinant forms (CRFs) (n = 8) and 10.5% belonged to pure group M subtypes (n = 4). The findings demonstrated a significant reduction of 11.2% in discrepancies when pol region sequences were used compared to PR/RT region sequences, indicating that increased nucleotide sequence lengths are directly correlated with more consistent subtype classification. The results also revealed that if the discrepancy in pol region subtyping results persists, then there is a high likelihood (89.5%) that the query sequence is a recombinant HIV-1 strain, 68.4% of which belong to uncharacterized recombinant HIV-1 strains. The results of this study showed that REGA 3.0 presented the best performance in subtyping recombinant HIV-1 strains, while Stanford performed better in defining phylogenies of pure group M subtypes. The study highlights that, especially in populations with polyphyletic HIV-1 epidemics resulting in a high prevalence of recombinant HIV-1 strains, neither PR/RT nor pol region sequences are reliable for the determination of HIV-1 genotypic subtypes in samples showing discrepancies among different subtyping tools, and only near-full-length or full-length HIV-1 genome sequences are sufficiently accurate.
| S-EPMC9608988 | biostudies-literature
Project description:Punica granatum isolate:Tunisia-2019 Genome sequencing and assembly
| PRJNA324150 | ENA
Project description:Escherichia coli strain:KPC-Tunisia Genome sequencing and assembly
| PRJNA406824 | ENA
Project description:Punica granatum isolate:Tunisia-2019 Genome sequencing and assembly
| PRJNA565884 | ENA
Project description:To investigate the biological differences between HIV-, HIV+/ART-experienced and HIV+/ART-naive diffuse large B-cell lymphoma, we performed RNA sequencing of 70 pre-treatment formalin-fixed paraffin-embedded (FFPE) whole lymph node biopsies of diffuse large B-cell lymphoma.
2024-06-01 | GSE262621 | GEO
Project description:A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences.The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out.Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships.Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage.The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution.
| S-EPMC5719533 | biostudies-literature
Project description:BackgroundHead-to-head comparison of BeadChip and WGS/WES genotyping techniques for their precision is far from straightforward. A tool for validation of high-throughput genotyping calls such as Sanger sequencing is neither scalable nor practical for large-scale DNA processing. Here we report a cross-validation analysis of genotyping calls obtained via Illumina GSA BeadChip and WGS (Illumina HiSeq X Ten) techniques.ResultsWhen compared to each other, the average precision and accuracy of BeadChip and WGS genotyping techniques exceeded 0.991 and 0.997, respectively. The average fraction of discordant variants for both platforms was found to be 0.639%. A sliding window approach was utilized to explore genomic regions not exceeding 500 bp encompassing a maximal amount of discordant variants for further validation by Sanger sequencing. Notably, 12 variants out of 26 located within eight identified regions were consistently discordant in related calls made by WGS and BeadChip. When Sanger sequenced, a total of 16 of these genotypes were successfully resolved, indicating that a precision of WGS and BeadChip genotyping for this genotype subset was at 0.81 and 0.5, respectively, with accuracy values of 0.87 and 0.61.ConclusionsWe conclude that WGS genotype calling exhibits higher overall precision within the selected variety of discordantly genotyped variants, though the amount of validated variants remained insufficient.
| S-EPMC7488117 | biostudies-literature
Project description:In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.
| S-EPMC8402755 | biostudies-literature