Harshlight

Harshlight is a software tool that has been developed to aid in the automatic detection and masking of blemishes in HDONA microarray chips. As microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc., microarray scans show similar artifacts that might affect the subsequent analysis. However, all but the starkest blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs). Few tools are available to help detect those defects.

Harshlight uses statistical and image processing methods to identify three types of defects: localized blemishes affecting a few probes, diffuse defects affecting larger areas, and extended defects that may invalidate an entire chip. The tool is highly effective in detecting and eliminating large-scale artifacts, which can materially improve the analysis of HDONA chips, especially for experiments with subtle changes between samples.

For the widely used MAS5 algorithm, it has been shown that compact blemishes cause an average of 8 gene expression values per chip to change by more than 50%, two of them by more than twofold. However, the masking algorithm of Harshlight can restore about two-thirds of this damage, making the tool an essential resource for researchers who want to achieve the most accurate analysis of HDONA chips.