sanger seq of HPV
The virus was isolated from 32 samples using the DNeasy Blood & Tissue Kitt (Qiagen, USA) following the manufacturers instructions. After isolation, the concentration of the viral genomes was measured using a NanoDrop spectrophotometer (NanoDrop, Thermofisher, USA). PCR was performed to amplify the target region of the viral genome using specific primers as previously reported (5). The reaction conditions were optimized for specificity and efficiency and perform initial denaturation at 95C for 2 minutes, followed by 25-35 cycles of denaturation at 95C for 30 seconds, annealing at 57C for 30 seconds, and extension at 72C for 1 minute per kb of target DNA, with a final extension at 72C for 5-10 minutes. PCR products were qualified using a 1.5% agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen, USA) according to manufacturers instructions. Purified PCR products were sent to Ramaciotti Centre for Genomics at UNSW (Sydney, Australia) for bidirectional sequencing using the Sanger method. Raw sequence reads from both forward and reverse primers were assembled and trimmed to generate consensus sequences for each samples using BioEdit software. Final sequences were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Consensus sequences were compared with published sequences in the NCBI database using BLAST. The most similar sequences were selected for phylogenetic analysis. A phylogenetic tree was constructed using Mega X software to visualize the evolutionary relationships.
SUB14931252
sample14