Following the detection of papillomavirus-like contigs in two DNA-Seq datasets, we attempted to obtain additional papillomavirus sequence from DNA-Seq reads by extending Megahit contigs with GapFiller (v1.1.1; Nadalin et al., 2012 (link)) and then further scaffolding these contigs with SSPACE (v3.0; Boetzer et al., 2011 (link)). Primers were designed to amplify the unknown sequence between the lengthened contigs based on the typical papillomavirus gene order (Supplementary Table S2 ). Prior to PCR, DNA extracts from infected samples were enriched for circular DNA fragments using rolling circle amplification (RCA). RCA was performed in a 20μl reaction with 1.25U Phi29 DNA Polymerase (Thermo Fisher), 50μM exonuclease-resistant random hexamers (Thermo Fisher), 5μg BSA, 450μM dNTPs, and 2μl template DNA (<400ng). Reaction mixtures were incubated at 30°C for 18h followed by a 10min denaturation step at 65°C. RCA products were diluted 1/500 and used as PCR templates, with primers combined to amplify different portions of the circular genome. Lengthened contigs and Sanger sequencing reads were assembled de novo in Geneious (v10.2) to obtain the final circular papillomavirus genome sequence.
Exonuclease resistant random hexamers
Manufactured by Thermo Fisher Scientific
Exonuclease resistant random hexamers are short DNA sequences composed of six randomly selected nucleotides. These hexamers are designed to be resistant to degradation by exonuclease enzymes, which can break down DNA from the ends. This property makes them useful for various molecular biology applications where DNA integrity is important.
Automatically generated - may contain errors
3 protocols using exonuclease resistant random hexamers
1
Papillomavirus Genome Reconstruction from DNA-Seq Data
2
Whole Genome Amplification and Size Selection
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Circularized DNA was split into five aliquots of 10 μL, and each aliquot was amplified in its own 50-μL reaction containing Phi29 polymerase (NEB) and exonuclease resistant random hexamers (Thermo) [5 μL of 10× Phi29 Buffer, 2.5 μL of 10 uM (each) dNTPs, 2.5 μL random hexamers (10 uM), 10 μL of DNA, 29 μL ultrapure water, 1 μL of Phi29]. Reactions were incubated at 30 °C overnight. All reactions were pooled, and volume was adjusted to 300 μL with ultrapure water. DNA was extracted using SPRI beads with a size cutoff to eliminate DNA <2,000 bp (0.5 beads:1 sample). A mix of 90 μL of ultrapure water, 10 μL NEB buffer 2, and 5 μL T7 Endonuclease was added to the beads to elute and debranch the DNA. Beads were incubated for 2 h on a thermal shaker at 37 °C under constant agitation. The tubes containing the beads were then placed on magnets, and supernatant was recovered. The DNA in the supernatant was then extracted again using SPRI beads with a size cutoff to eliminate DNA <2,000 bp (0.5 beads:1 sample) and eluted in 15 μL of ultrapure water.
3
Whole Genome Amplification via Phi29
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Circularized DNA was split into four aliquots of 10 μL, and each aliquot was amplified in its own 50-μL reaction containing Phi29 polymerase (NEB) and exonuclease resistant random hexamers (Thermo Fisher Scientific) [5 μL of 10× Phi29 Buffer, 2.5 μL of 10 mM (each) dNTPs, 2.5 μL random hexamers (10 µM), 10 μL of DNA, 29 μL ultrapure water, 1 μL of Phi29]. Reactions were incubated overnight at 30°C. T7 Endonuclease was added to each reaction and then incubated for 2 h at 37°C with occasional agitation. The debranched DNA was then extracted using SPRI beads at a 0.5:1 ratio and eluted in 50 μL of H2O.
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