Evaluation of oligonucleotide sequence capture arrays and comparison of next-generation sequencing platforms for use in molecular diagnostics.

BACKGROUND: Next-generation DNA sequencing (NGS) techniques have the potential to revolutionize molecular diagnostics; however, a thorough evaluation of these technologies is necessary to ensure their performance meets or exceeds that of current clinical sequencing methods. METHODS: We evaluated the NimbleGen Sequence Capture 385K Human Custom Arrays for enrichment of 22 genes. We sequenced each sample on both the Roche 454 Genome Sequencer FLX (GS-FLX) and the Illumina Genome Analyzer II (GAII) to compare platform performance. RESULTS: Although the sequence capture method allowed us to rapidly develop a large number of sequencing assays, we encountered difficulty enriching G+C-rich regions. Although a high proportion of reads consistently mapped outside of the targeted regions, >80% of targeted bases for the GAII and >30% of bases for the GS-FLX were covered by a read depth of > or =20, and > 90% of bases for the GAII and > 80% of bases for the GS-FLX were covered by a read depth of > or =5. We observed discrepancies among sequence variants identified by the different platforms. CONCLUSIONS: Although oligonucleotide arrays are quick and easy to develop, some problematic regions may evade capture, necessitating sequential redesigning for complete optimization. Neither sequencing technology was able to detect every variant identified by Sanger sequencing because of well-known drawbacks of the NGS technologies. The rapidly decreasing error rates and costs of these technologies, however, coupled with advancing bioinformatic capabilities, make them an attractive option for molecular diagnostics in the very near future.

Analysis of hMLH1 and hMSH2 gene dosage alterations in hereditary nonpolyposis colorectal cancer patients by novel methods.

A significant fraction of hereditary nonpolyposis colorectal cancer cases with defective mismatch repair (ie, Lynch syndrome) have large genomic deletions or duplications in the mismatch repair genes, hMLH1 and hMSH2, which can be challenging to detect by traditional methods. For this study, we developed and validated a novel Southern blot analysis method that allows for ascertainment of the extent of the dosage alterations on an exon-by-exon basis and compared this method to a second novel technique, multiplex ligation-dependent probe amplification (MLPA). From a total of 254 patients referred for Lynch syndrome testing, 20 of the 118 MLH1 cases and 42 of the 136 MSH2 cases had large genomic alterations, as detected by Southern blot. MLPA and Southern blot results were concordant with the exception of three major discrepancies: one because of a lack of MLPA probes for the region altered, another because of a point mutation near the MLPA probe ligation site, and another that was unexplained. Compared to Southern blot, MLPA has a shorter turn-around time, the analysis is less costly, less time-consuming, and less labor-intensive, and results are generally clear and unambiguous. However, concerns with MLPA include the presence of false-negatives and -positives because of positioning of probes and DNA variants near the probe ligation site. Overall, both Southern blot and MLPA provide important tools for the complete evaluation of patients with Lynch syndrome.

Trisomy 20 mosaicism caused by a maternal meiosis II error is associated with normal intellect but multiple congenital anomalies.

Mosaicism for trisomy 20 is generally ascertained following prenatal sampling and rarely is associated with significant phenotypic abnormalities. Uniparental disomy for chromosome 20 in the euploid lines has been found in several cases, which showed relatively mild clinical features, primarily growth delay. Here we report on a case of mosaic trisomy 20 in a child with normal neurologic development who was ascertained because of multiple physical anomalies including spinal segmentation anomalies and altered skin pigmentation. Trisomic cells were found in buccal epithelial cells and in cultured skin fibroblasts but not in peripheral blood. Molecular analysis of blood cells, fibroblasts, and parental cells gave evidence of a maternal meiosis II error as the cause of the trisomy. Disomic cells presumably arose through trisomy rescue, and no evidence was found for uniparental disomy in these cells.