Custom Array Comparative Genomic Hybridization: the Importance of DNA Quality, an Expert Eye, and Variant Validation

Abstract

Abstract:The presence of false positive and false negative results in the Array Comparative Genomic Hybridization (aCGH) design is poorly addressed in literature reports. We took advantage of a custom aCGH recently carried out to analyze its design performance, the use of several Agilent aberrations detection algorithms, and the presence of false results. Our study provides a confirmation that the high density design does not generate more noise than standard designs and, might reach a good resolution. We noticed a not negligible presence of false negative and false positive results in the imbalances call performed by the Agilent software. The Aberration Detection Method 2 (ADM-2) algorithm with a threshold of 6 performed quite well, and the array design proved to be reliable, provided that some additional filters are applied, such as considering only intervals with average absolute log 2 ratio above 0.3. We also propose an additional filter that takes into account the proportion of probes with log 2 ratio exceeding suggestive values for gain or loss. In addition, the quality of samples was confirmed to be a crucial parameter. Finally, this work raises the importance of evaluating the samples profiles by eye and the necessity of validating the imbalances detected.

Copy number variations in candidate genomic regions confirm genetic heterogeneity and parental bias in Hirschsprung disease

“…Several of them looked unlikely at the visual inspection and, as a matter of fact, mainly not confirmed on the replicate, when available. Conversely, most of the variants classified as likely or possible at the visual inspection were also replicated [30] (Additional file 1).…”

“…The quality of the experiments was evaluated on the basis of the QC metrics generated by the Genomic Workbench 5.0.14 software (Agilent Technologies), such as the DLRSpread (derivative log ratio spread), a measure of the log ratio noise for each sample. DLRSs and the other sample metrics are detailed elsewhere [30].…”

“…Results obtained with the custom aCGH, together with the concordance degree among the replicates on the same design array, showed that the replication rate was not very high, and that the visual inspection outperformed the mere software call [30]. However, a high false positive rate is not surprising as a few studies have shown a not infrequent presence of false positive and false negative results from aCGH [51–54].…”

Total colonic aganglionosis and cleft palate in a newborn with Janus-cysteine 618 mutation of RET proto-oncogene: a case report

“…Deoxyribonucleic acid (DNA) extraction from lymphocytes (QIAamp DNA blood Midi Kit, Qiagen) was followed by an array comparative genomic hybridization (a-CGH) using the whole genome 8x60K. Scanned images of the arrays were processed and analyzed using Feature Extraction software and Genomic Workbench software (Agilent Technologies) with the statistical algorithm Aberration detection method-2 (ADM-2) and a sensitivity threshold of 6.0 as recently benchmarked [ 7 ]. This genetic analysis did not reveal cryptic chromosomal anomalies associated with HSCR or cleft palate.…”

“…Chromosomal loci with risks of Hirschsprung disease, modified[6,7] (Continued) C8orf37 Chromosome 8 open reading frame 37, C10orf 27 Chromosome 10 open reading frame 27, DENND3 DENN domain-containing protein 3 gene, DHCR7 7dehydrocholesterol reductase gene, DR Digenic recessive, EDNRB Endothelin receptor type b gene, EDN3 Endothelin 3 gene, GDNF Glial cell line-derived neurotrophic factor gene, GFRA1 GDNF family receptor alpha-1, HSCR Hirschsprung disease, IFT Intraflagellar transport, IKBKAP Inhibitor of kappa light polypeptide gene enhancer gene, KIAA1279 Kinesin binding protein, LZTFL1 Leucine zipper transcription factor like 1, L1CAM L1 cell adhesion molecule gene, MKKS McKusick-Kaufman syndrome gene, MKS1 Meckel syndrome-1 gene, MR Mental retardation, NCLN Nicalin, NF1 Neurofibromin 1 gene, NKX2-1 NK1 Homebox 1, NRG1 Neuregulin 1, NRTN Neurturin, NUP98 Nucleoporin 98, PHOX2B Paired-like homeobox 2B gene, PTHB1 Parathyroid hormon-responsive B1 gene, RET Rearranged during transfection protooncogene, RMRP RNAse mitochondrial RNA processing gene, SDCCAG8 Serologically defined colon cancer antigen 8 gene, SEMA…”