Abstract
BackgroundPatient-Derived Tumour Xenografts (PDTXs) have emerged as the pre-clinical models that best represent clinical tumour diversity and intra-tumour heterogeneity. The molecular characterization of PDTXs using High-Throughput Sequencing (HTS) is essential; however, the presence of mouse stroma is challenging for HTS data analysis. Indeed, the high homology between the two genomes results in a proportion of mouse reads being mapped as human.ResultsIn this study we generated Whole Exome Sequencing (WES), Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing (RNA-seq) data from samples with known mixtures of mouse and human DNA or RNA and from a cohort of human breast cancers and their derived PDTXs. We show that using an In silico Combined human-mouse Reference Genome (ICRG) for alignment discriminates between human and mouse reads with up to 99.9% accuracy and decreases the number of false positive somatic mutations caused by misalignment by >99.9%. We also derived a model to estimate the human DNA content in independent PDTX samples. For RNA-seq and RRBS data analysis, the use of the ICRG allows dissecting computationally the transcriptome and methylome of human tumour cells and mouse stroma. In a direct comparison with previously reported approaches, our method showed similar or higher accuracy while requiring significantly less computing time.ConclusionsThe computational pipeline we describe here is a valuable tool for the molecular analysis of PDTXs as well as any other mixture of DNA or RNA species.
Highlights
Patient-Derived Tumour Xenografts (PDTXs) have emerged as the pre-clinical models that best represent clinical tumour diversity and intra-tumour heterogeneity
Optimizing sequence alignment using the In silico Combined human-mouse Reference Genome (ICRG) In PDTX samples, a proportion of High-Throughput Sequencing (HTS) reads originated from mouse DNA could have high enough homology to be aligned to the Human Reference Genome (HRG)
First we evaluated the impact of the reference genome using the Human Reference RNA (HRR) and Mouse Reference RNA (MRR) samples for which RNA sequencing (RNA-seq) data were obtained in triplicate (Table 3)
Summary
Patient-Derived Tumour Xenografts (PDTXs) have emerged as the pre-clinical models that best represent clinical tumour diversity and intra-tumour heterogeneity. Patient-Derived Tumour Xenografts (PDTXs) are emerging as the pre-clinical models that best represent the diversity of clinical tumours and intratumour heterogeneity [1,2,3]. We and others have observed that after implanting human cancer tissue fragments into immuno-compromised mice, the human stroma is rapidly lost and replaced by mouse stromal cells [2, 9, 10]. This results in an unknown proportion of mouse cells incorporated into the xenograft. Given the high homology of human and mouse genomes, mouse reads can be wrongly aligned to the human genome, hampering downstream analyses and data interpretation
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