Application Of Genetic Testing For Non-small Cell Lung Cancer With High-throughput Sequencing Technologies

Primary lung cancer is one of the fastest-growing malignant tumors with high morbidity and mortality.Its prevalence and mortality is the number one cancer in men and number two in women.The most common one is the non-small cell lung adenocarcinoma?NSCLC?.It is believed that driver gene mutations,such as EGFR,BRAF and KRAS,can lead to lung cancer.Approaches like Q-PCR,Fish and multiplex PCR are currently applied to detect the gene mutations in clinic,however,the disadvantages of those methods are either low-throughput or high cost.In this thesis,we developed a targeted deep sequencing?TDS?solution/panel including 284 frequent mutation genes in lung cancer.The TDS panel has four advantages to our knowledge: 1)The minimal input gDNA amount is 100 ng.2)We use an homogenous enzyme system instead of costly sonication equipment in the fragmentation step.3)By NGS,it can detect various types of mutations such as SNV,indel,fusion simultaneously.4)The whole working hours for library preparation reduced from 3 to 1,5 days.5)At the same time,we compared the BGISEQ-500 Sequence data with HISEQ-4000 s,the results show that they have high consistency.After all,we change BGISEQ-500 by low sequence cost.To validate the efficiency,85 clinical lung cancer samples were tested in this method and compared with other technique platforms such as Sanger sequencing,fluorescence PCR and IHC,and the NGS results of the EGFR L858 R,T790M mutation,E746-A750 deletion,KRAS G12 D mutation and EML4-ALK fusion gene of these samples were also compared to the result from.The consistency of the NGS method with other techniques respectively are 94.1%?96.5%?95.3%?98.8%?100%.Using standard samples?EGFR_L858R ? EGFR₁9exon_del ? KRAS_G12D ?ALK_Fusion?diluted in 10%,5%,3%,2%,1%,0.5% frequency to find out the detection limitation of the developed NGS method,which showed the minimum detectable mutation allele frequency was 0.5%.The application of the NGS method can also effectively reduce the clinical sample to report TAT time and the cost of clinical genetic testing.