Abstract
Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system. Hematopoietic cells originating from genetically modified stem cells will harbor insertions in specific genomic positions called integration sites, which represent unique genetic marks of clonal identity. Therefore, the analysis of vector integration sites present in the genomic DNA of circulating cells allows to determine the number of clones in the blood ecosystem. Shannon diversity index is adopted to evaluate the heterogeneity of the transduced population of gene corrected cells. However, this measure can be affected by several technical variables such as the DNA amount used and the sequencing depth of the library analyzed and therefore the comparison across samples may be affected by these confounding factors. We developed an advanced spline-regression approach that leverages on confounding effects to provide a normalized entropy index. Our proposed method was first validated and compared with two state of the art approaches in a specifically designed in vitro assay. Subsequently our approach allowed to observe the expected impact of vector genotoxicity on entropy level decay in an in vivo model of hematopoietic stem cell gene therapy based on tumor prone mice.
Highlights
Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system
We have shown that the Shannon entropy index, a widely used measure of genetic variability, is strongly affected by the variability of technical factors
In particular we have shown that our approach can remove confounding effects from the Shannon entropy index
Summary
Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system. Shannon diversity index is adopted to evaluate the heterogeneity of the transduced population of gene corrected cells This measure can be affected by several technical variables such as the DNA amount used and the sequencing depth of the library analyzed and the comparison across samples may be affected by these confounding factors. The analysis of vector IS from DNA of blood cells harvested at specific time points after transplant from GT patients provide information on number of hundreds to thousands of clones present in circulation and their relative abundance. in[16] it was highlighted that estimates of species diversity in sites/habitats at local scale, namely the α-diversity[17], for rarefied microbiome count data may be strongly biased This is mainly due to the rare species which may be over- (or under-) represented in the samples that have been normalized to a smaller depth by rarefaction. Simple scaling does not remove the effect provided from the library depth neither on species richness nor on species diversity. Beule[18] introduces a novel normalization method for species count data called scaling with ranked subsampling (SRS) and the authors demonstrate its suitability for the analysis of microbial communities
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