Total RNA Sequencing Analysis of DCIS Progressing to Invasive Breast Cancer
Technical Report,01 Sep 2016,31 Aug 2017
Johns Hopkins University Baltimore United States
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This project is designed to complement a multi-institutional, NIH-funded study of genetic and epigenetic alterations of pre-invasive DCIS that did or did not progress to invasive breast cancer, with an in-depth analysis of expression data on the entire range of informative RNA categories. During the current reporting period, we have further analyzed an Affymetrix HTA 2.0 array-based comprehensive transcriptome assay of samples from 5 collaborating institutions. Despite promising results from a smaller pilot experiment, and initially promising data reported in the prior report, further analysis lead us to conclude that much of the signal from these arrays have to be attributed to technical variation, and we are limited in the depth of biological information we can obtain from these arrays. In collaboration with Affymetrix scientists, we were able to determine QC measures that are predictive of subsequent array data quality, but at the cost of losing a large number of irreplaceable samples in our cohort. We have therefore continued our collaboration with Dr. C. Perou at UNC to maximize the possibility of a successful RNA Sequencing effort, and have confirmed the encouraging pilot results using the new Illumina TruSeq RNA Access Library Preparation kit followed by RNA sequencing performed using the Illumina NextSeq500. We have now completed the initial batch of 48 study samples, of which 75 yielded good read counts. We describe additional optimization steps we are implementing to complete the analysis of this unique sample cohort.
- Medicine and Medical Research
- Genetic Engineering and Molecular Biology