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Most are well-aware of Nexus Copy Number’s strengths in detection of copy number from different technologies and this often overshadows the many other powerful features of Nexus Copy Number.  A recent customer publication nicely illustrates  the power of these additional analysis tools.  Here, I’m going to walk through a recent customer publication, highlighting the areas where the authors used these powerful research tools available within Nexus Copy Number.

The paper:
Hamad Ali,  Milad S. Bitar,  Ashraf Al Madhoun,  Makia Marafie,  Fahd Al-Mulla. Functionally-focused algorithmic analysis of high resolution microarray-CGH genomic landscapes demonstrates comparable genomic copy number aberrations in MSI and MSS sporadic colorectal cancer. PLoS One. 2017 Feb 23;12(2).

All images below are from the above referenced journal article.

Overview
Dr. Ali et al., Kuwait University, recently looked at genomic landscapes of both MSI and MSS sporadic colorectal cancer (CRC) using Nexus Copy Number.  The team identified CNAs in 116 well-defined colorectal cancers using high-density oligonucleotide-based microarrays (Agilent Technologies) and the FASST algorithm in Nexus Copy Number.  Array CGH profiles of the samples showed many chromosomal gains and losses consistent with other publications.  Using the tools within Nexus Copy Number, they were able to ascertain that there are substantial differences between the copy number profiles of the two CRC subtypes (MSI and MSS).  They found distinct patterns of genomic aberrations among the samples.  Though there are many unique copy number differences between MSI and MSS CRC, the authors found that functional MSI and MSS copy number changes are similar.

Simultaneous view of the aberration profiles of samples in a project
The multi-sample view in Nexus Copy Number displays the gain/loss patterns of each sample in a single view allowing easy visual inspection of the various profiles arising from the samples. The team identified three distinct profiles 1) small-sized and few gains and losses, 2) numerous small alternating gains/losses typically described in solid tumors as chromothripsis 3) large-sized gains and losses spanning chromosome arms or whole chromosomes (Figure 2 in paper). 

Hierarchical clustering analysis tooljournal.pone.0171690.g004-small.png 
Nexus Copy Number offers the following clustering algorithms: Single Linkage Hierarchical, Average Linkage Hierarchical, Complete Linkage Hierarchical, and K-means.

The team performed unsupervised complete linkage hierarchical clustering analysis using the clustering tool in Nexus Copy Number and found five distinct patterns of genomic aberrations.  The tool allows labeling of the samples with sample characteristics (e.g. BRAF mutation, KRAS mutation, tumor subtype, etc.) showing quick visual overview of any overrepresented characteristics in the clusters. 

Clinicopathological characteristics of the clusters – Factor Enrichment tool
To find if any aberrations were significantly associated with clinicopathological characteristics, the authors used the Factor Enrichment tool on the resulting clusters of samples.  This analysis revealed that cluster 1 was significantly associated with MSS CRC, wild type BRAF, and was left-sided with rectosigmoidal subsite enrichment whereas cluster two was significantly associated with MSI CRC and BRAF V600E mutation.

journal.pone.0171690.g005.png

Genomic aberrations in MSI vs. MSS CRC – Comparisons Tool
They also compared the results of the unsupervised clustering with the aberration profiles of MSI and MSS CRC and found them to be very similar to clusters 1 and 2, respectively. In addition, using the Comparison tool within Nexus Copy Number which highlights the significantly differing regions of CN change between two groups, they compared the two subtypes (MSI and MSS) and found there were extensive differences in the gains and losses between the two subtypes.

 

Identification of functionally relevant CN changes – Tools for frequency significance testing (GISTIC and STAC algorithms) and Enrichment Analysis tool

journal.pone.0171690.g006.pngThe group used the included STAC and GISTIC algorithms (two frequency significance tests offered in Nexus Copy Number) to identify functionally relevant copy number changes.  The GISTIC algorithm identifies regions of aberrations that are at a statistically significantly higher frequency than the “background” aberration with a greater weight given to those events with higher amplitude as these are less likely to be random.  As the algorithm was developed using a cancer data set, it is more applicable to cancer sets. The STAC algorithm tries to identify a set of aberrations that are stacked on top of each other such that they would not occur randomly. 

After statistically significant aberrant regions were identified via GISTIC, the authors performed gene enrichment analysis within Nexus Copy Number to identify functionally relevant genes in the top GO terms which included genes involved in cellular senescence, S-phase control of the cell cycle, and telomere maintenance among others.  With the two complementary methods for identifying statistically significant regions of CNAs the authors found a high degree of similarity between the functionally relevant copy number alterations in MSI and MSS CRC suggesting that shared genomic events may be key in the evolution of both types of CRCs. 

Integrating copy number with gene expression results
The authors acknowledge that the genomic aberrations in MSI and MSS CRCs may have a direct effect on gene expression (thought to be the driving force in the pathology of both MSI and MSS).  But they were limited by the FFPE samples and acknowledge that further studies in gene expression are warranted for a better understanding of the molecular pathology of CRC.  Though the authors were unable to incorporate gene expression results for this study, Nexus Copy Number can integrate gene expression data as aggregate as well as on a per sample basis so expression results could be incorporated into the study within the same software.

Hopefully this summary shows you the power of Nexus Copy Number as an easy-to-use tool for advanced research analysis.  Read the entire customer publication here to see in detail how the authors utilized several tools in Nexus Copy Number to uncover new findings in characterization of CRCs.

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