Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues

Abstract

Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at http://www.ebi.ac.uk/ena/data/view/PRJEB6630).We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at http://www.ncbi.nlm.nih.gov/geo/accession number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma.

Overview of recurrent chromosomal losses in retinoblastoma detected by low coverage next generation sequencing

“…This loss spans well beyond the retinoblastoma locus including more than 11 cytogenetic bands from 13q14.2 to 13q31.3 in pool 1. Other losses from 13q33.1 to 13q33.3previously reported associated to Rb were also identified (36). Thus, “windows” analysis confirmed results obtained with the integrity score, and revealed the cytogenetic location and extension of recurrent losses at the RB locus with higher accuracy.…”

“…Establishing upper and lower limits this graphic display allows visual analysis and association of losses or gains detected with the NGS data to the corresponding cytogenetic bands of each chromosome, facilitating a genome wide view of losses and gains. See Supplementary data at (36) for the individual view of each chromosome and corresponding genes distribution in the Maps File. With this approach we first focused the analysis on Chr13 to define the extent of losses we successfully detected with the integrity score mentioned before and results are presented in Figure 3.…”

“…Table 2 shows the chromosomes, arm and cytogenetic bands on which we found more consistency in this association. Figure 4 also illustrates Chr 7 from Rb pool 1 as the chromosomal element that display with more clarity the pattern gain-clear cytogenetic bands, and Chr11 with the pattern loss-dark cytogenetic bands (see also Maps File in (36)).…”

“…Another lost region shared between Mb and Rb is situated in Chr 19 at the cytogentic band 19q13.42. It goes from position 54,520,000 to 55,560,001 spanning 1 Mb of this chromosome and 66 genes are situated in this region (see Table 1 in (36)). This region has been previously reported to harbor a human leukocyte receptor cluster (41) and 10 of these 66 genes belong to the KIR gene family (Killer cell immunoglobulin-like receptors) these are also polymorphic and highly homologous genes, thus it is likely that these apparent losses are also an artifact of the mapping process mentioned before.…”

“…This region has been previously reported to harbor a human leukocyte receptor cluster (41) and 10 of these 66 genes belong to the KIR gene family (Killer cell immunoglobulin-like receptors) these are also polymorphic and highly homologous genes, thus it is likely that these apparent losses are also an artifact of the mapping process mentioned before. Other shared losses exist in Chr17q21.3, Chr19p13.11, Chr5q13.2, and although are of smaller magnitude it will be interesting a deeper analysis on them (See Table 1 in (36)).…”