دانلود رایگان مقاله تشخیص غیر تهاجمی و بسیار حساس جهش سرطان روده بزرگ

Abstract

Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective preventive screening procedure to detect adenomatous polyps, the precursors to CRC, is colonoscopy. Since every colorectal cancer starts as a polyp, detecting all polyps and removing them is crucial. By exactly doing that, colonoscopy reduces CRC incidence by 80%, however it is an invasive procedure that might have unpleasant and, in rare occasions, dangerous side effects. Despite numerous efforts over the past two decades, a non-invasive screening method for the general population with detection rates for adenomas and CRC similar to that of colonoscopy has not yet been established. Recent advances in next generation sequencing technologies have yet to be successfully applied to this problem, because the detection of rare mutations has been hindered by the systematic biases due to sequencing context and the base calling quality of NGS. We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS) to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

4. Discussion

The study presented here is the first attempt at determining the feasibility of using single molecule, third generation sequencing for the detection of CRC driving mutations in stool samples. The first step was to show that mutations present at low levels (1.5%) could be detected above background using a sequencing-based approach capable of detecting any mutation present in the amplicon. The raw error rate of the sequence data is critical in this respect because for genes such as APC, the cancer causing mutations can be found at any position in the amplicon and often consist of micro insertions and deletions, so that an error rate as low as 0.7% (the estimated error rate for the Illumina platform) will produce far too many false positives when scanning a test sequence of 1116 bp, which is the size of the MCR of the APC gene. Circular Consensus Sequencing of small amplicons, on the other hand, generates a highly accurate consensus sequence where the background is low enough to confidently call mutations at the 1.5% level