Blood Sample Preparation For Sequencing And Applied In The Research Of Hepatic Carcinoma

Next generation sequencing technology(NGS)is the most commonly used high-throughput sequencing technology.Its process is complex,so that every step can make mistake and eventually lead to the inconsistencies of sequencing results between different detection units.Therefore,it is very necessary to make the flow of the NGS stability and standardization,which is important for clinical promotion.The detection process of NGS can be divided into three key parts: sample preparation,sequencing and biological information analysis.Among them,most research and medical units are only responsible for the part of sample preparation.Otherwise,compared to tissue sample,blood is the most ideal sample for sequencing with the advantages of small risk of trauma,high patient compliance,low price and easy availability.In view of these facts,we conducted this study to make the blood sequencing sample preparation process stability and standardization,and then apply it to the gene differential expression analysis of the hepatic carcinoma.At the same time,we established a new technology for sequencing sample preparation,which laid a foundation for further improving the efficiency of sequencing.The research contents are as follows: 1.Establishment of Storage Scheme for Blood Sequencing SampleThe storage of blood sample is not only the first step in the sample preparation,but also the first step in the NGS.RNA is unstable and easily degradable,so it is most affected by the sample storage conditions.However,the storage scheme for blood samples used for RNA extraction is not uniform now.Especially,the study of the blood short storage scheme which is more suitable for sample transportation or clinical work is rare.Therefore,the aim of the second part was to study of the blood short storage scheme which was applicable to RNA sequencing(RNA-seq)and systematically analyzed the differences among the blood samples which were placed for different storage time or with different RIN values.The results were shown that the blood samples stored at 4°C within 7 days with RNA integrity number(RIN)values ranging from 5.3 to 9.0 were available for RNA-seq.2.BIEA: Breathing-based Isothermal Emulsion Amplification MethodMonoclonal cluster construction is one of the key parts of library preparation,which still relys on PCR techniques.Among them,the emulsion PCR(emPCR)and bridge PCR are used widely.Especially emPCR has more extensive application,but there are still some technical defects that make it poor stability and amplification efficiency.At present,the stability of emulsion system caused by heat cycle is the key technical bottleneck of emPCR.Focusing on this point,we applied the ?DNA breathing? in emulsion amplification to establish an isothermal emulsion amplification scheme(BIEA).Then we tried to optimize its reaction temperature,reaction time,temperature-controlling equipment and magnetic beads.Finally,the quality control analysis(QC)analysis of high-throughput sequencing platform was used to evaluate its monoclonicity.The results were showed that the BIEA with stable emulsion system,simplified temperature-controlling device,and decreased investment would be a highly streamlined and inexpensive option for future single-molecular amplification based researches.3.Differential Expression Genes in PBMCs for Hepatic CarcinomaThe peripheral blood mononuclear cells(PBMCs)in the blood not only can monitor tumor progression througt reflectting the patients' cellular immune function,but also can reduce tumor heterogeneity.Therefore,it is regarded as the ideal sample type for the gene differential expression analysis of cancer.In this part,we applied the designed blood storage scheme in the study of hepatic carcinoma.The RNAs of which RIN values were in the safety range were used for subsequently sequencing,differential expression gene(DEG)analysis and RT-qPCR validation.The results showed that 5 candidate indicators of metastatic hepatic carcinoma gene diagnosis were selected in this study,and the number of DEGs and the expression level of IL1 B were found to have the potential to supervise the progression of liver cancer.In conclusion,the blood storage scheme and BIEA method established in this paper will help stabilize and standardize the sample preparation of NGS,thus reducing the inconsistency of the results between different sequencing platforms.On this basis,we used easily available and minimally invasive PBMCs as experimental materials to identify 5 DEGs,which would be expected to become the candidate biomarkers for the diagnosis of hepatic carcinoma with metastasis,and the number of identified DEGs and the expression level of IL1 B have the potential to supervise progression of hepatic carcinoma.It will be helpful for understanding the mechanism of tumor metastasis and providing a new perspective for the future studies about clinical diagnosis and treatment of hepatic carcinoma.