Detection Of Rare Mutations Using Unique Barcode Combined Region Target Capture

Liquid biopsy, which can detect the circulation tumor DNA(ctDNA) in blood of other body fluid, make us discover the mutation in patients timely and repeatable, that is very important in the individualized therapy. The ctDNA is usually low in body fluid, and the tumor is highly heterogeneous. So we cannot get accurate information about the low rate mutation sites by the normal next-generation sequencing technology which error rate is about 1%. The advent of Duplex Sequencing is an ultra-sensitive sequencing methodology capable of increasing the accuracy by 1000 times. Therefore, six urine samples with known mutation frequency were used to verify the reliability of the methods of Duplex Sequencing combining Region Target Capture technology, and samples from bladder cancer patients before and after surgery were used to prove the feasibility of this method in liquid biopsy.Sensibility of methods: Added 1%, 0.1%, 0.01% 823 gastric cancer cell line DNA(90 known mutations) into the normal urine as the analog samples, detected the mutations using a combination of Duplex Sequencing with cancer mutation target capture.Feasibility of detecting bladder cancer mutation: Used Duplex Sequencing method combined Region Target Capture sequencing to detect DNA mutation in urine from one bladder cancer patients before and after operation, one normal urine, compared the gene mutations of three samples.The results of the test of sensibility showed that mutations can be detected in the three gradients with 4000 x sequencing depth and the higher the depth we got, the more mutations we found. When it reached 8000 x, detection rate of real mutations is more than 66% in the samples containing 1% mutation, the accuracy rate is more than 93%. In the feasibility test, capture rate was about 60%; 39, 1 and 4 mutations were detected in patients before and after surgery, and normal controls respectively.In this research, Duplex sequencing is a precise approach to detect the low-frequency mutation, and is capable of binding to capture chip to catch the urine-related gene mutations. Further work has to be focused on the library construction process, data analysis, and application.