Characteristics of the ISKS cohort.

*<p>some probands have >1 sarcoma; SD, standard deviation; LPS, liposarcoma.</p

Genetic events detected in the peripheral blood of ISKS probands.

*<p>Database version R16 November, 2012; del: deleterious; neut: neutral; FS: frame shift; LOH: loss of heterozygosity; NA: not applicable. The assignment of pathogenicity was performed as outlined in supplementary Figure 3.</p

Next-Generation Sequence Analysis of Cancer Xenograft Models

<div><p>Next-generation sequencing (NGS) studies in cancer are limited by the amount, quality and purity of tissue samples. In this situation, primary xenografts have proven useful preclinical models. However, the presence of mouse-derived stromal cells represents a technical challenge to their use in NGS studies. We examined this problem in an established primary xenograft model of small cell lung cancer (SCLC), a malignancy often diagnosed from small biopsy or needle aspirate samples. Using an <i>in silico</i> strategy that assign reads according to species-of-origin, we prospectively compared NGS data from primary xenograft models with matched cell lines and with published datasets. We show here that low-coverage whole-genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. Exome capture sequencing revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene expression experiments, whereas mouse-specific transcript profiles correlated with published datasets from human cancer stroma. We conclude that primary xenografts represent a useful platform for complex NGS analysis in cancer research for tumours with limited sample resources, or those with prominent stromal cell populations.</p></div

Comprehensive correlation analysis between the RNA-Seq and Affymetrix expression array platforms.

<p>(<b>A</b>) Comparison of gene expression detected by RNA-Seq and Affymetrix expression array platforms for identical SCLC samples (mean, n = 3, P&lt;0.01). (<b>B</b>) Comparison of the gene expression between SCLC primary tumours <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074432#pone.0074432-Peifer1" target="_blank">[34]</a> (Y axis, mean, n = 15) and primary xenografts (X axis, mean, n = 3) (P&lt;0.01). (<b>C</b>) Comparison of gene expression detected by Affymetrix array of micro-dissected human cancer stroma <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074432#pone.0074432-Casey1" target="_blank">[19]</a> (Y axis, mean, n = 28) and mouse-specific RNA-Seq expression data in the SCLC xenograft models (X axis, mean, n = 3) (P&lt;0.01).</p

Summary of the results produced by the proposed strategy to isolate and identify species-specific NGS reads in human xenografts.

<p>For each read category, the proportion (%) of the total number of reads is specified.</p

Overview of the steps followed to identify and isolate common and species-specific sequence reads, including gene identification and pathway analysis.

<p>The software components utilized in each step are also specified. Solid lines represent the principal analytical path followed and dashed lines represent auxiliary steps.</p

Copy number variations, inter and intra-chromosomal rearrangements and B allele frequencies of NCI-H209 cell line and a xenograft tumour derived from it.

<p>(A) Circos plot representing copy number variations, inter and intra-chromosomal rearrangements of NCI-H209 cell line and a xenograft tumour derived from it. Copy number variations (red, gain; green, loss) were calculated based on coverage using the correspondent peripheral blood as control. Inter and intra-chromosomal rearrangements are represented in blue (inter-chromosomal) and dark blue (intra-chromosomal). (B, C) Detailed profile of copy number variations and B-allele frequencies of chromosome 1 from the analysed cell line and xenograft. As described above, the correspondent peripheral blood was used as control for both type of analysis. Copy number profiles are shown in red (gain), green (loss) and grey (no change). LOH are shown light blue.</p