Computational protocol: A comparison of foamy and lentiviral vector genotoxicity in SCID-repopulating cells shows foamy vectors are less prone to clonal dominance

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Protocol publication

[…] Genomic DNA was extracted and processed according to the manufacturer’s directions using the Gentra Puregene Cell Kit (Qiagen, Valencia, CA). RIS from in-vivo datasets were identified and analyzed as previously described. VISA was used with the modified setting of a minimum alignment score of 100. RIS within repeats that had the same first 16 genomic nucleotides adjacent to the LTR, but aligned to different chromosomal loci, were further analyzed as follows to confirm they were in fact distinct RIS: RIS were aligned to the human genome using BLAT. RIS were excluded if percent identity was less than 97%, or could be aligned to multiple chromosomal loci with identical confidence (percent identity is greater than or equal to 97%, query length is equal to the span of the alignment). The Database for Annotation, Visualization, and Integrated Discovery 6.7 (refs. ,) was used for enrichment analysis as previously described. For hotspot analysis, FV and random RIS were normalized and matched to the smallest dataset (LV) at 460 RIS. First, all 825 FV RIS were randomized by assigning a random number to each RIS in Excel and then ordered from largest random number to smallest random number. Since there were only a total of 460 LV RIS, we analyzed the first 460 of the randomized FV RIS to normalize the datasets for hotspot analysis. Statistical significance was determined using chi-square analysis.Analysis of vector copy number was performed through a multiplexed quantitative PCR assay using primers and probes targeting an EGFP transgene and RNase P as an internal control. The EGFP/EYFP assay consisted of a custom TaqMan Probe (Applied Biosystems, Grand Island, NY) containing a 5’ FAM reporter dye and a 3’ minor groove binder/nonfluorescent quencher, with primers/probe sequences as previously reported by Zhout et al. The RNase P assay (Applied Biosystems) contained a probe with a 5’ Hex reporter dye. The standard curve was generated with genomic DNA extracted from HT1080 cells transduced with a single vector containing EYFP. Reactions were run in triplicate with TaqMan Genotyping Master Mix (Applied Biosystems) in a Bio-Rad CFX384 Touch under the following thermal cycling conditions: 95 °C 10 minutes + 40 × (95 °C 15 seconds + 60 °C 1 minute). […]

Pipeline specifications

Software tools VISA, BLAT
Application WGS analysis
Organisms Homo sapiens, Mus musculus, Spleen focus-forming virus
Diseases X-Linked Combined Immunodeficiency Diseases