Similar protocols

Pipeline publication

[…] b'CR cycling conditions were as follows: initial denaturation at 95\xc2\xb0C for 5 minutes, 35 cycles of denaturation at 95\xc2\xb0C for 30 seconds, annealing at 56\xc2\xb0C for 1 minute, elongation at 72\xc2\xb0C for 2 minutes, and a final extension at 72\xc2\xb0C for 10 minutes. The PCR products were run on the agarose gel (1%). Following this, the amplicons were purified with the Invisorb Fragment Cleanup (Invitek) Kit and nucleotide sequences were obtained by means of fluorescent BigDyeTM Terminator Cycle Sequencing using v 3.1 Ready Reaction ABI PRISM (Applied Biosystems) to obtain both the forward and reverse sequences. PCR and sequencing were performed in duplicate., The obtained sequences were assembled by means of the Staden package []. They were then evaluated to determine the nucleotide divergence relative to the nucleotide sequences of HPV16 (K02718), HPV31 (J04353), and HPV58 (D90400). Sequence comparisons were carried out using the Basic Local Alignment Search Tool (BLAST) and multiple alignments were performed by using the CLUSTALW (Mega 5.2, Beta version) program []., The Neighbor-Joining algorithm and the Kimura 2-Parameter model trees, with 1000 bootstrapped replicates, were built by using the MEGA package, version 5.2 []. Phylogenetic analyses were performed with LCR sequences of HPV16, HPV31, and HPV58. The partial sequence of L1 and LCR genes of the HPV16, HPV31, and HPV58 was deposited in the NCBI GenBank database, under the following accession numbers: HPV16 L1 gene: KJ467225-467238; HPV16 LCR: KJ452220-452242; HPV31 L1 gene: KJ452216-452219; HPV31 LCR: KJ435060-435067; HPV58 L1 gene: KJ467239-477246; HPV58 LCR: KJ567247-467252. The references for the viral sequences used to construct the phylogenetic branches were collected from the GenBank sequence database and are listed in ., The putative impact of the HPV variants was estimated in silico by predicting the B-cell and T-ce' […]

Pipeline specifications

Software tools Staden, BLASTN, Clustal W, MEGA