ABSTRACT
Next-Generation Sequencing is a rapidly advancing technology that has research and clinical applications. For many cancers, it is important to know the precise mutation(s) present, as specific mutations could indicate or contra-indicate certain treatments as well as be indicative of prognosis. Using the Ion Torrent Personal Genome Machine and the AmpliSeq Cancer Hotspot panel v2, we sequenced two pancreatic cancer cell lines, BxPC-3 and HPAF-II, alone or in mixtures, to determine the error rate, sensitivity, and reproducibility of this system. The system resulted in coverage averaging 2000× across the various amplicons and was able to reliably and reproducibly identify mutations present at a rate of 5%. Identification of mutations present at a lower rate was possible by altering the parameters by which calls were made, but with an increase in erroneous, low-level calls. The panel was able to identify known mutations in these cell lines that are present in the COSMIC database. In addition, other, novel mutations were also identified that may prove clinically useful. The system was assessed for systematic errors such as homopolymer effects, end of amplicon effects and patterns in NO CALL sequence. Overall, the system is adequate at identifying the known, targeted mutations in the panel.
Subject(s)
Biomarkers, Tumor/genetics , DNA Mutational Analysis , Gene Expression Profiling , Genome, Human , Genomics/methods , High-Throughput Nucleotide Sequencing , Mutation , Pancreatic Neoplasms/genetics , Cell Line, Tumor , Computational Biology , Databases, Genetic , Genetic Predisposition to Disease , Humans , Oligonucleotide Array Sequence Analysis , Pancreatic Neoplasms/pathology , Phenotype , Reproducibility of Results , SoftwareABSTRACT
Plus-strand RNA viruses serve as templates for translation and then transcription by newly synthesized RdRp. A ribosome-binding tRNA-shaped structure (TSS) and upstream hairpin H4 in the 3' UTR of Turnip crinkle virus (TCV) play key roles in translation and transcription. Second-site mutations generated to compensate for altering the critical asymmetric internal loop of H4 included a three- to two-base alteration in the terminal loop of a 3' proximal hairpin (Pr) located downstream of the TSS. Unlike the non-deleterious three-base alteration, single mutations in Pr loop were detrimental for RdRp transcription while enhancing translation and RdRp binding. One deleterious mutation in the Pr loop altered the structures of both the TSS and H4. These complex interactions in the 3' UTR support a compact structural arrangement likely permitting RdRp access to a number of residues within a 195-base region including the 3' end that are necessary for efficient transcription initiation.