Abi prism 3100 genetic analyzer

Eight overlapping polymerase chain reaction (PCR) fragments covering the entire genome were amplified using the Platinum PCR SuperMix (Invitrogen, Carlsbad, CA, USA) following previously described protocols^{20 (link)}. Primers for specific HAdV-C PCR amplification were synthesized as previously reported^{20 (link)}. Following the PCR amplification of the full-length genome, the amplified DNA was used as template for sequencing using Sanger chemistry using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA). Sequence ladders were generated on the ABI Prism 3100 Genetic Analyzer (Life Technologies, Japan). Sequences were assembled and edited using Sequencher 5.0 (Genecodes Corp., Ann Arbor, MI, USA). To obtain high-quality data, we used a minimum threefold coverage for both directions across the genomes. In addition, any questionable sites identified during the sequence assembly and genome annotation were re-sequenced to clarify the ambiguities. Genome annotation was performed using Artemis software version 16.0.0 (Sanger, UK) and HAdV-2 prototype strain (NC_001405) was used as the template for the genomic comparative analysis.

Six VNTRs, (VNTR1, VNTR3a, VNTR3b, VNTR4, VNTR5 and VNTR6) were PCR-amplified by using forward primers 5´-labelled with 6-carboxyfluorescein and unlabelled reverse primers as reported [17 (link)] with bacterial DNA isolated from B. pertussis infected patients serving as template. The amplification products were analysed with an ABI Prism 3100 Genetic Analyzer (Life Technologies) at VBC Biotech, Vienna, Austria. The number of repeats found at the six VNTR loci translates into a distinct MLVA type (www.mlva.net/).

To generate promoter deletion constructs, primary monocyte genomic DNA was extracted from the interphase and phenol layer using TRIzol reagent (Life Technologies), according to the manufacturer's instructions. To obtain 5′ end promoter deletion products, primers (Supplementary Table S1) flanking the desired promoter regions were used for PCR amplification, using the purified genomic DNA as template. PCR was performed using iProof High Fidelity DNA Polymerase (Bio-rad) under the following parameters: initial denaturation at 98°C; 40 cycles of amplification, denaturation at 98°C for 10 s, annealing at Tm of primer pair +3°C for 30 s, elongation at 72°C for 2.5 min; followed by a final extension at 72°C.
The isolated promoter lengths were separately cloned into pGL4.20 vector (Promega) using standard molecular cloning techniques. The restriction enzymes used were XhoI and HindIII (Thermo Fisher Scientific). T4 DNA ligase was from Roche. For small-scale purification of plasmids, AxyPrep Plasmid Miniprep kit (Axygen Biosciences) was used. Large-scale purification of plasmids intended for transfection was carried out using PureLink HiPure Plasmid Filter Purification kit (Invitrogen). The full-length sequence of each promoter construct was confirmed by sequencing using Big Dye Terminator cycle sequencing kit and ABI Prism 3100 Genetic Analyzer (Life Technologies).

The viral genome of isolate 4p168 was sequenced as follows: the VHSV isolate was concentrated and sucrose gradient purified as described by Nishizawa et al. [30 (link)]. Viral RNA was extracted from the purified virus using the ISOGEN-LS^® (NIPPON GENE), and subjected to RT-PCR amplification with primers designed according to the sequence of strain FA281107 (GIII) (GenBank accession no. EU481506) for sequencing of each gene (Additional file 1). The entire nucleotide sequence of the coding region (from N to L gene) of the genome was determined by direct sequencing of each RT-PCR products. The 3′ termini was cloned using primer 5′-CTC GAT GAT GAT GAT GAT CTC-3′ [31 (link)] and the TOPO^® TA Cloning^® Kit (Life Technologies) with SURE 2 SuperCompetent Cells (Agilent Technologies). The 5′ termini was cloned using SMARTer™ RACE cDNA Amplification Kit (Clontech) and pGEM^®-T Easy Vector Systems (Promega). For all nucleotide sequencing, BigDye^® Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and ABI PRISM^® 3100 Genetic Analyzer (Life Technologies) were used.

Genomic DNA was extracted using the DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. And the extracted DNA was stored at -80°C until it was analyzed. The HBV DNA concentration was quantified as copies per microgram of genomic DNA by real-time PCR performed using the ABI 7300 TaqMan platform (Life Technologies, Carlsbad, CA, USA); multiplex PCR was used to determine the HBV genotype[26 (link)]. The DNA sequences flanking the polymerase region (nucleotides 2307–3215, 1–1623) were amplified with the primer pairs listed in Table 1, according to the polymerase region identified by the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/genome/5536). It was very difficult to obtain PCR products for some samples; therefore, we redesigned the primer pairs with decreased flanking length for successful PCR amplification in such cases. Nested PCR was also performed for a few samples.
Cyclic sequencing was performed with a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies), and the products were separated using an ABI PRISM 3100 Genetic Analyzer (Life Technologies). Mutations were confirmed by repeating the analysis on both strands.