Big dye terminator sequencing method

After purification of the PCR products by the MinElute PCR Purification Kit (QIAGEN, Milan, Italy), sequencing was performed in a dedicated facility (Bio-Fab Research, Roma, Italy). Sequences were generated using the Big Dye Terminator Sequencing method (Life Technologies, Carlstadt, CA, USA) on the ABI 3730 sequencer (Life Technologies), and analyzed with the Sequencing Analysis 5.2 software (Life Technologies). The obtained sequences were compared to reference sequences deposited in GenBank. Sequence alignments were performed with ClustalW2 at the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) website using default parameters [27 ].

MCPyV-positive DNA samples were subjected to nested PCR for the amplification of NCCR region. Two sets of primers, ORIF1/ORIR1 (nucleotide positions 4832–4853 and nucleotide positions 5334–5314) and ORIF2/ORIR2 (nucleotide positions 5077–5100 and nucleotide positions 5280–5261), were employed to generate an NCCR fragment of 504 and 203 base pair (bp). Numbering of nucleotides was based on the sequence of MCC350, a strain of North American origin (GenBank: EU375803) [2 (link)]. In detail, PCR reactions were carried out following a published protocol [13 (link)]. PCR products were analyzed on 2% agarose gels by ethidium bromide staining. The positive PCR products were purified using the MinElute PCR Purification Kit (QIAGEN, Italy) and confirmed by sequencing, using sense and antisense primers, by a dedicated facility (Bio-Fab research s.r.l., Rome, Italy). Sequences were generated using the Big Dye Terminator Sequencing method (Life Technologies) on the ABI 3730 sequencer (Life Technologies, Monza, Italy), and analyzed with the Sequencing Analysis 5.2 software (Life Technologies).

Samples simultaneously positive to MCPyV DNA detection in urine and plasma were arbitrarily selected and used for MCPyV VP1 amplification and sequencing. The MCPyV VP1 gene was amplified through a nested PCR using two different primer sets generating two fragments of 494 bp and 307 bp, respectively [55 (link)]. The 307 bp resulting fragments were analyzed on 2% agarose gels by ethidium bromide staining, purified using the MinElute PCR Purification Kit (QIAGEN, Milan, Italy) and confirmed by sequencing, using sense and antisense primers, by a dedicated facility (Bio-Fab Research, Roma, Italy). Sequences were generated using the Big Dye Terminator Sequencing method (Life Technologies, Carlstad, CA, USA) on the ABI 3730 sequencer (Life Technologies), and analyzed with the Sequencing Analysis 5.2 software (Life Technologies). Sequence alignments were conducted with ClustalW2 at the EMBL-EBI website using default parameters [56 ].