L.S. Rector 1, N.A. Yamada 2, M.E. Aston 1, M.C. Sederberg 1 R.A. Ach 2, P. Tsang 2, E. Carr 2, A. Scheffer-Wong 2, N. Sampas 2, B. Peter 2, S. Laderman 2, L. Bruhn 2, A.R. Brothman 1,3 1 ARUP Laboratories, 500 Chipeta Way, Salt Lake City, Utah 2 Agilent Laboratories, Agilent Technologies, Santa Clara, CA 3 Departments of Pediatrics, Human Genetics and Pathology, University of Utah School of Medicine, Salt Lake City, Utah Objectives of Study Develop a robust method for generating FISH probes especially those less than 50kb to characterize abnormalities detected by aCGH Evaluate modified in silico selected probe design Characterize and validate these probes with a rigorous objective criteria Abstract Array comparative genomic hybridization (aCGH) is now an accepted methodology for detection of genomic copy number changes. FISH is often used to confirm and further characterize abnormalities detected by aCGH. It is not unusual for abnormalities detected by aCGH to be in a genomic region or of a genomic size which prohibits traditional FISH using large probes such as bacterial artificial chromosome (BAC) vectors. Our goal was to establish a robust method for visualizing FISH probes on metaphase chromosomes to further characterize small abnormalities which would not be readily seen by conventional FISH techniques. Oligonucleotide FISH (oFISH) is an alternative to BAC FISH because it allows for higher resolution visualization of smaller loci. In this study, libraries of long oligonucleotides (194mers) with high complexity (~>150 oligonucleotides per locus) were chemically synthesized. Probe generation involved PCR amplification, followed by the introduction of fluorescent labels by chemical modification. By directing oFISH probes at the most informative elements of the region under investigation, we achieved high specificity and eliminated the need for Cot-1 or other suppressive hybridization reagents. Upon the initial evaluation of 14 oFISH probes, 12 of these (86%) gave strong signals with good specificity and sensitivity. The two oFISH probes which did not hybridize well initially included regions at distal 4p and Xq which both contain a high frequency of repeat sequences. When a modified in silico oligo selection method was used to enhance signal while maintaining specificity, easily detectable signal was achieved at the expected sites in the 4p and Xq regions even though as little as 10% (6.5 Kb) of the initial region was included in the probe sequences. Ongoing evaluation of additional oFISH probes indicates that this method will be powerful for routine analysis of previously difficult genomic regions. The ease, reproducibility and high level accuracy of this technique suggest that it may prove invaluable to the human genetics community. Discussion By using the oFISH method, we have been able to visualize small probes or those which were either unavailable or unacceptable using traditional FISH methods. The in silico selected design displays an overall better performance over unselected sequences while maintaining specificity. This methodology for probe design should be considered for future applications. As research in the field of molecular genomics advances, we are likely to see a greater number of small regions which will require characterization in whole cells; the oFISH method is well suited to play a significant role in that endeavor. Discussion By using the oFISH method, we have been able to visualize small probes or those which were either unavailable or unacceptable using traditional FISH methods. The in silico selected design displays an overall better performance over unselected sequences while maintaining specificity. This methodology for probe design should be considered for future applications. As research in the field of molecular genomics advances, we are likely to see a greater number of small regions which will require characterization in whole cells; the oFISH method is well suited to play a significant role in that endeavor. Customized oligonucleotide FISH probes offer increased sensitivity in characterizing abnormalities detected by array CGH An enterprise of the University of Utah and its Department of Pathology Methods Abnormality detected by aCGH (uArray, ISCA Consortium design Agilent) Probe design based on genomic coordinates Probe generation by chemical synthesis, involving PCR and fluorescent labeling Co-denaturation/hybridization of probes and slides using standard FISH methods Analysis of probes Each probe was tested for within- and between-run reproducibility, in addition to a specificity and sensitivity study resulting in 6 separate slides evaluated per probe Analysis Criteria: Rationale: Introduction FISH is one of the most common methods used to confirm and further define array findings. Regions for which commercial probes are not available, regions that are difficult to detect by using FISH, or regions smaller than 50 kb make it challenging to see many abnormalities identified by aCGH. Introduction FISH is one of the most common methods used to confirm and further define array findings. Regions for which commercial probes are not available, regions that are difficult to detect by using FISH, or regions smaller than 50 kb make it challenging to see many abnormalities identified by aCGH. Results Conclusion oFISH is highly reproducible and produces robust signals in 83% (15/17) of the initial probes studied and confirmed aCGH findings in 100% of samples tested. Difficult genomic regions not previously detectable by traditional FISH methods are visible using oFISH, such as the NPHP1 region at 2q13. (see poster PB#1092) In silico selected probes appear to improve signals over simple tiling probes (5/6). Conclusion oFISH is highly reproducible and produces robust signals in 83% (15/17) of the initial probes studied and confirmed aCGH findings in 100% of samples tested. Difficult genomic regions not previously detectable by traditional FISH methods are visible using oFISH, such as the NPHP1 region at 2q13. (see poster PB#1092) In silico selected probes appear to improve signals over simple tiling probes (5/6). Representative Examples I = Intensity; Sp = Specificity; Se = Sensitivity; Rep = Reproducibility *In silico probes available **Cross hybridized to 10q *** This locus represents a known polymorphic site and was shown to hybridize to two separate regions 1 Representative Examples 97 kb NPHP1 (2q13) deletion seen by aCGH 79.5 kb 9q34 duplication seen by aCGH CategoryScoreInterpretation Signal Intensity 1Non-existent 2Poor 3Average 4Fair 5Good Specificity 1<50% % % % 5>90% Sensitivity 1<50% % % % 5>90% Reproducibility 120% 240% 360% 480% 5100% Sensitivity = # of signals seen at correct location # of expected signals Specificity = # of signals seen at correct location Total # of signals # of slides with same Reproducibility = intensity, sensitivity, and specificity Total # of slides scored In cases where no signal was detected, there were no values for any category Value = sum of individual scores In Silico Selected LocusSize (kb)ISpSeRepValue 6p p p p p Xq Comparison of Simple Tiling Values with In Silico Selected Values LocusSize (kb)Simple TilingIn Silico 6p p p p p Xq aCGH Samples LocusArray FindingsoFISH Findings 2q13Deletion 2q37.3Deletion 4p16.3Duplication 4p16.3Deletion 5p15.3Duplication 5p13.3Duplication 6p25.3Duplication 9p24.3Duplication 9q34.3Duplication 10q26.3Duplication References Bailey, Kidd, Eichler Human copy number polymorphic genes. Cytogenet. Genome Res. 123: , 2008 Baldwin et al. Genet Med 10: , 2008 International Standard Cytogenomic Array Consortium (ISCA, Wolff, DJ et al. Guidance for Fluorescence in Situ Hybridization Testing in Hematologic Disorders. Journal of Molecular Diagnostics 2007, 9: References Bailey, Kidd, Eichler Human copy number polymorphic genes. Cytogenet. Genome Res. 123: , 2008 Baldwin et al. Genet Med 10: , 2008 International Standard Cytogenomic Array Consortium (ISCA, Wolff, DJ et al. Guidance for Fluorescence in Situ Hybridization Testing in Hematologic Disorders. Journal of Molecular Diagnostics 2007, 9: Simple Tiling Probes Locus size (kb) Tiled (kb)ISpSeRepValues Xq27.1* q q37.3* **5515 4p16.3* p p p p22.2* p25.3*** ***5516 8q p q q p13.3* p13.3* p13.3* q FISH and inverted DAPI banding of metaphase cell confirms NPHP1 deletion (arrow indicates deleted chromosome) 16p13.3 oFISH probe and inverted DAPI banding (15.6 kb ) Metaphase cell identified location of probe at 9q34 (arrow indicates likely duplication) Interphase cell confirmed duplication 6p22 oFISH probe and inverted DAPI banding (6.7kb)