Computational protocol: BZLF1 Governs CpG-Methylated Chromatin of Epstein-Barr Virus Reversing Epigenetic Repression

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Protocol publication

[…] Bisulfite modification of DNA was carried out using the EZ Methylation Gold Kit (Zymo). 100 nanogram each of 60 PCR products of bisulfite-modified DNA with an average product size of 463 bps were pooled for deep sequencing on a Roche Genome Sequencer FLX (Microsynth GmbH, Switzerland). Alignment of sequences was done using the CLC genomics workbench software with parameters for high throughput sequencing in the menu “reference assembly”. The local alignment was performed with parameters for long run reads (mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.5 and similarity of 0.9). Sequence reads were aligned to a modified version of the B95.8 wildtype sequence, in which we converted all cytosines in a non-CpG context to thymines. Cytosines of CpG dinucleotides were kept unconverted. The parameters of the sequence alignment were set to tolerate RY-mismatches in the context of CpG dinucleotides, allowing mismatches of purines and pyrimidines, which are expected if cytosines are unmethylated. Non-specific matches were placed randomly. Methylation analysis was carried out using the BiQ analyzer HT 0.9/beta-test version . Data were graphically analyzed and visualized with the Prism software package. […]

Pipeline specifications

Software tools CLC Genomics Workbench, BiQ Analyzer HT
Application BS-seq analysis
Organisms Homo sapiens
Diseases Epstein-Barr Virus Infections