Optimization Of Next-generation Sequencing Technology Using Liquid Biopsy Multitemplate(CTC、cfDNA、WBC)

According to the current data of cancer,cancer has become the main cause of human death,and no matter the incidence or the mortality of cancer in china account for 50% of the world’s cancer incidence and mortality.Since the rapid development of next-generation sequencing technology,The use of liquid tumor biopsy is becoming more and more popular,and the strategy of liquid biopsy combined with next-generation sequencing brings new solutions to cancer detection ways and cancer treatment.This stady base on our sanmed biotech company’s muti-sample(cfDNA\CTC\WBC)next-generation sequencing technology,and the main content of the research is to optimize the operation process.In this stady,I first use positive control cell line to test the optimized protocol,Next step,using patient sample to test the new protocol,and analysis the sequencing data comepare with the old protocol and the optimized protocol.Our final goal is to improve our sequencing result and the real mutation detection by optimize our protocol.In the experiment,each blood sample will go through our liquid biopsy system to capture our CTC,and we also need to get the plasma for extracting cfDNA,and take out part of blood sample(400μl)as each sample’s control.To deal with this three component sample using ThermoFisher’s library contraction kit and Ion torrent sequencing platform to do the library sequencing,Analysing the data using our company’s targetcall software system and get the sample’s taget gene mutations.Results:(1)Using positive cell line to test the the optimized process,first we find the yield of cfDNA was improved obviously after we optimized extraction protocol.Second we elininated the PCR inhibitors after we update our capture chip process and using un-extraction cell lysis protocol to deal with our CTC sample,we can 100% detect positive cell line’s mutations by using optimized protocol,but for the old protocol,the persentege is about 50%.(2)In 153 patient sample cases,compare with the optimized protcol,old protocol have deal with 69 patient sample,and both cfDNA and CTC component in this 69 patient samples have detected the same mutation patient rate is 21.74%,both component detected mutations but don’t have the same mutation patient rate is 21.74%,only cfDNA sample have mutation patient rate is 15.94%,and the only CTC sample detection rate is 15.94%,all in all detected mutation rate is up to 75.36%,still have 24.64% of petient sample detected nothing in both component.And the update protocol,the detection rate is 21.43%、44.05%、17.86%,detection rate is up to 92.86%,only7.14%of petient sample detected nothing in both component。Optimized protocl actually improved mutations detection.At the same time,We found no matter witch kind of protocol we used,still some mutation only detected only in cfDNA sample or CTC sample.We also found the overlapped sites rates detected by cfDNA and CTC is about 45.2% and 33.3% by using optimized protocol or without.(3)We choosed 17 patient sample’s sequecing data to verify the mutation using PCR ways and sanger sequencing,and we found that the consistency of this three ways is up to 88%.(4)Exploratory studies were conducted on the detection of unconventional samples(pleural fluid samples).No mutations were detected in the cfDNA component of whole blood,but the mutation sites were detected in CTC,while the subsequent continuous detection of pleural fluid samples all detected the same mutation,this results show high consistency in the detection.Conclusions:(1)The optimized protocol significantly improved the quality of next-generation sequencing and mutation,both in cell line validation and case analysis.Besides,the optimized protocol increased the proportion of overlap sites rates between cfDNA and CTC components(25.3% before optimization,45.2% after optimization),which indicated the necessity to test both cfDNA and CTC,and multi-template next-generation sequencing test could get more information in a single detection.(2)The results of multi-template next-generation sequencing were consistent with the validation data of PCR method and first-generation sequencing method(triple consistent rate is 88%),which verified the reliability of multi-template next-generation sequencing technology and provided an other chose to detecting mutation.(3)Exploratory studies were conducted on the detection of unconventional samples(pleural fluid samples).hydrothorax samples’ s results show high consistency in the detection.that indicated this application of multi-template sequencing technology for liquid biopsy may be extended to other types of body fluids,such as hydrothorax.This will extended the application use.