Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE)

<p>Abstract</p> <p>Background</p> <p>Rapid means to discover and enumerate unknown mutations in the exons of human genes on a pangenomic scale are needed to discover the genes carrying inherited risk for common diseases or the genes in which somatic mutations are required for clonal diseases such as atherosclerosis and cancers. The method of constant denaturing capillary electrophoresis (CDCE) permitted sensitive detection and enumeration of unknown point mutations but labor-intensive optimization procedures for each exonic sequence made it impractical for application at a pangenomic scale.</p> <p>Results</p> <p>A variant denaturing capillary electrophoresis protocol, cycling temperature capillary electrophoresis (CTCE), has eliminated the need for the laboratory optimization of separation conditions for each target sequence. Here are reported the separation of wild type mutant homoduplexes from wild type/mutant heteroduplexes for 27 randomly chosen target sequences without any laboratory optimization steps. Calculation of the equilibrium melting map of each target sequence attached to a high melting domain (clamp) was sufficient to design the analyte sequence and predict the expected degree of resolution.</p> <p>Conclusion</p> <p>CTCE provides practical means for economical pangenomic detection and enumeration of point mutations in large-scale human case/control cohort studies. We estimate that the combined reagent, instrumentation and labor costs for scanning the ~250,000 exons and splice sites of the ~25,000 human protein-coding genes using automated CTCE instruments in 100 case cohorts of 10,000 individuals each are now less than U.S. $500 million, less than U.S.$500 per person.</p

The Quandary of DNA-Based Treatment Assessment in De Novo Metastatic Prostate Cancer in the Era of Precision Oncology.

Guidelines for genetic testing have been established for multiple tumor types, frequently indicating the most confident molecularly targeted treatment options. However, considering the often-complex presentation of individual cancer patients, in addition to the combinatorial complexity and inherent uncertainties of molecular findings, deriving optimal treatment strategies frequently becomes very challenging. Here, we report a comprehensive analysis of a 68-year-old male with metastatic prostate cancer, encompassing pathology and MRI findings, transcriptomic results, and key genomics findings from whole-exome sequencing, both somatic aberrations and germline variants. We identify multiple somatic aberrations that are known to be enriched in prostate cancer, including a deletion of PTEN and a fusion transcript involving BRCA2. The gene expression patterns in the tumor biopsy were also strikingly similar to prostate tumor samples from TCGA. Furthermore, we detected multiple lines of evidence for homologous recombination repair deficiency (HRD), including a dominant contribution by mutational signature SBS3, which is specifically attributed to HRD. On the basis of the genomic and transcriptomic findings, and in light of the clinical case presentation, we discussed the personalized treatment options that exist for this patient and the various challenges that one faces in the process of translating high-throughput sequencing data towards treatment regimens

Molecular detection (k-ras) of exfoliated tumour cells in the pelvis is a prognostic factor after resection of rectal cancer?

<p>Abstract</p> <p>Background</p> <p>After total mesorectal excision (TME) for rectal cancer around 10% of patients develops local recurrences within the pelvis. One reason for recurrence might be spillage of cancer cells during surgery. This pilot study was conducted to investigate the incidence of remnant cancer cells in pelvic lavage after resection of rectal cancer. DNA from cells obtained by lavage, were analysed by denaturing capillary electrophoresis with respect to mutations in hotspots of the <it>k-ras </it>gene, which are frequently mutated in colorectal cancer.</p> <p>Results</p> <p>Of the 237 rectal cancer patients analyzed, 19 had positive lavage fluid. There was a significant survival difference (p = 0.006) between patients with <it>k-ras </it>positive and negative lavage fluid.</p> <p>Conclusion</p> <p>Patients with <it>k-ras </it>mutated cells in the lavage immediately after surgery have a reduced life expectation. Detection of exfoliated cells in the abdominal cavity may be a useful diagnostic tool to improve the staging and eventually characterize patients who may benefit from aggressive multimodal treatment of rectal cancer.</p

Automated amplicon design suitable for analysis of DNA variants by melting techniques

Background The technological development of DNA analysis has had tremendous development in recent years, and the present deep sequencing techniques present unprecedented opportunities for detailed and high-throughput DNA variant detection. Although DNA sequencing has had an exponential decrease in cost per base pair analyzed, focused and target-specific methods are however still much in use for analysis of DNA variants. With increasing capacity in the analytical procedures, an equal demand in automated amplicon and primer design has emerged. Results We have constructed a web-based tool that is able to batch design DNA variant assay suitable for analysis by denaturing gel/capillary electrophoresis and high resolution melting. The tool is developed as a computational workflow that implements one of the most widely used primer design tools, followed by validation of primer specificity, as well as calculation and visualization of the melting properties of the resulting amplicon, with or without an artificial high melting domain attached. The tool will be useful for scientists applying DNA melting techniques in analysis of DNA variations. The tool is freely available at http://meltprimer.ous-research.no/ . Conclusion Herein, we demonstrate a novel tool with respect to covering the whole amplicon design workflow necessary for groups that use melting equilibrium techniques to separate DNA variants

Differences in the CTCE migration times of all peaks relative to the most thermally stable homoduplex for target sequences 1–12

<p><b>Copyright information:</b></p><p>Taken from "Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE)"</p><p>http://www.biomedcentral.com/1471-2156/8/54</p><p>BMC Genetics 2007;8():54-54.</p><p>Published online 14 Aug 2007</p><p>PMCID:PMC2042502.</p><p></p> Five four-minute cycles (20 min) were employed with an amplitude of 12°C (47°C – 59°C). The results are illustrated as the average migration time difference +/- 1 standard deviation, n = 8. A representative electropherogram obtained from fragment #6 is incorporated to illustrate peak positions

The melting profile of three target sequences 6, 14 and 26 calculated with WinMelt illustrating a well-defined target of a single isomelting domain (6), a target with two isomelting domains (26) and a target with an irregular melting profile (16)

<p><b>Copyright information:</b></p><p>Taken from "Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE)"</p><p>http://www.biomedcentral.com/1471-2156/8/54</p><p>BMC Genetics 2007;8():54-54.</p><p>Published online 14 Aug 2007</p><p>PMCID:PMC2042502.</p><p></p> The symbols mark the position of the sequence differences AT>GC (#6), TA>CG (#14) and TA>CG (#26) in each target wild type>mutant pair in separation trials. The GC-clamp (~94°C) is not shown but was incorporated in the melting calculations attached to the higher melting temperature end of each target sequence

The quandary of dna-based treatment assessment in de novo metastatic prostate cancer in the era of precision oncology

Guidelines for genetic testing have been established for multiple tumor types, frequently indicating the most confident molecularly targeted treatment options. However, considering the often-complex presentation of individual cancer patients, in addition to the combinatorial complexity and inherent uncertainties of molecular findings, deriving optimal treatment strategies frequently becomes very challenging. Here, we report a comprehensive analysis of a 68-year-old male with metastatic prostate cancer, encompassing pathology and MRI findings, transcriptomic results, and key genomics findings from whole-exome sequencing, both somatic aberrations and germline variants. We identify multiple somatic aberrations that are known to be enriched in prostate cancer, including a deletion of PTEN and a fusion transcript involving BRCA2. The gene expression patterns in the tumor biopsy were also strikingly similar to prostate tumor samples from TCGA. Furthermore, we detected multiple lines of evidence for homologous recombination repair deficiency (HRD), including a dominant contribution by mutational signature SBS3, which is specifically attributed to HRD. On the basis of the genomic and transcriptomic findings, and in light of the clinical case presentation, we discussed the personalized treatment options that exist for this patient and the various challenges that one faces in the process of translating high-throughput sequencing data towards treatment regimens