Computational protocol: HHV-8-unrelated primary effusion-like lymphoma associated with clonal loss of inherited chromosomally-integrated human herpesvirus-6A from the telomere of chromosome 19q

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Protocol publication

[…] The iciHHV-6A genome in each DNA sample was amplified using 32 pairs of overlapping primers (Sup. Table 1). Each 10 μl PCR reaction contained 7.5 ng genomic DNA, 0.9 μl PCR buffer [final concentration: 1 mM each dNTP, 45 mM Tris-HCl (pH 8.8), 11 mM (NH4)2SO4, 113 μg/ml bovine serum albumin, 4.5 mM MgCl2, 6.7 mM 2-mercaptoethanol and 4.4 μM EDTA (pH 8.0)], 0.3 μM each primer (forward and reverse), 0.1 μM Tris and 0.1 μl of an 8:1 ratio of Taq polymerase (5 U/μl, Kapa Biosystems) and Pwo polymerase (2.5 U/μl, Genaxxon Bioscience). Thermal cycling conditions were 96 °C for 2 minutes, followed by 35 cycles (32 cycles for DR) of 96 °C for 10 s, 52–62 °C for 30 s and 68 °C for 4–10 minutes, and then a final extension step at 68 °C for 10 minutes.The 32 amplicons from each DNA sample were pooled and used to prepare barcoded libraries and templates according to the manufacturer’s protocols for sequencing on a MiSeq sequencer (Illumina) or an IonTorrent personal genome sequencer (Life Technologies). The MiSeq read data for 1501-Bl were assembled de novo into contigs by using ABySS v. 1.5.2. A draft sequence was generated using scaffold builder to order the contigs against the reference HHV-6A strain U1102 sequence (NC_001664). Gaps were closed by using Megamerger, GapFiller v. 1–11 and custom Perl scripts, or by manual Sanger sequencing. The integrity of the resulting sequence was verified by aligning it against the read data by using BWA v. 0.6.2-r126 and visualizing the alignment by using Tablet v. Data for 1500-Bl and 1500-T were generated on both the MiSeq and IonTorrent platforms, and were aligned against the 1501-Bl data by using BWA and visualized by using Tablet. […]

Pipeline specifications

Software tools ABySS, scaffold_builder, EMBOSS, GapFiller, BWA, Tablet
Application De novo sequencing analysis
Organisms Human betaherpesvirus 6A, Homo sapiens, Human herpesvirus 6
Diseases Exanthema Subitum, Hepatitis B, Immunologic Deficiency Syndromes, Lymphoma, Neoplasms, Epstein-Barr Virus Infections, Lymphoma, Primary Effusion