Development Of TCGA Splicing Variants Analysis Web-tools And Analysis Of Colorectal Cancer Liver Metastasis Driving Mutation

With the rapid development and cost reduction of NGS(Next-generation Sequencing),it is more frequently being used in cancer reaserch.By applying Math,Information Science,and Computer Science,Bioinformatics is crucial to analyse and interpret massive NGS data in cancer research.In this study,we conducted in-depth analysis of the cancer genome and transcriptome sequencing results,by using innovative bioinformatics tools and analytic strategyPart1:TSVdb:a web-tool for TCGA splicing variants analysisBackground:Collaborative projects such as The Cancer Genome Atlas(TCGA)have generated various-omics and clinical data on cancer.Many computational tools have been developed to facilitate the study of the molecular characterization of tumors based on TCGA.Alternative splicing of a gene produces splicing variants,and accumulating evidences have revealed its essential role in cancer-related processes,implying the urgent need to discover tumor-specific isoforms and uncover their potential functions in tumorigenesis.Results:We developed TSVdb,a web-based tool,to explore alternative splicing based on TCGA samples with 30 clinical variables from 33 tumors.TSVdb has an integrated interface for visualization of the clinical data,gene expression,usage of Exons/Junctions and splicing patterns.By using TSVdb,researchers can investigate the isoform expression variations between or across clinical subgroups and the relationships between isoforms and patient survival.TSVdb is available at http://www.tsvdb.com.Part2:Complrehensive analysis the primary-metastasis paired colorectal cancer NGS dataObjective:The liver is the most common distal metastatic organ of colorectal cancer.It is known that the development of colorectal cancer is driven by the accumulation of mutations.This study aims to discover the metastasis-driven gene by comparing the mutations profile of colorectal primary tumor and its matched liver metastases.In addition,the colorectal liver metastasis may be attributed to the cancer cell clusters but not the single cell.However,this hypothesis has not been directly confirmed by clinical samples.We tried to estimate the number of cells involved in colorectal cancer metastasis using primary-metastsis paired WES(Whole-exon sequencing)data.Material and methods:In this study,18 pairs of colorectal cancer primary tumors and their matched liver metastasis were subjected to WES,and the KRAS hotspot mutations were also evaluated by Sanger sequencing.In addition,this study collected 20 pairs of public primary-metastatic matched samples WES raw data and 79 targeted sequencing data.By pool analysis,we compared the mutation frequency and the variant allele frequency(VAF)in the primary-metastatic matched samples.This study also used VAP(Variant assurence pipeline)to analyze the distribution of primary and metastatic mutation VAF.A computer model was used to simulate the metastasis process involving different numbers of cells,and the number of cells involved in metastasis among the real-world sample was inferred.Results:In the NGS data of 18 pairs of primary-metastatic matched samples,6 pairs of them had consistent KRAS mutations between primary and metastatic tumor,while Sanger sequencing only detected mutations in 4 unmatched samples.In the NGS results,high-frequency mutations such as APC,TP53,KRAS,BRAF,PIK3CA were concordant between primary and metastasis,and these genes were concordant between matched samples of metastasis treated patients.However,TP53 mutation VAF elevated in metastasis comparing to primary tumor;CTNNBI mutations enriched in metastasis treated patients and primary-metastasis nonsynchronous resected patients.In addition,computer model showed that the single cell derived metastasis has metastasis private mutation VAF increasement,which leads to different VAF distributions compring to multi-cells derived metastasis.The mutation VAF distribution characteristics of clinical primary-metastatis was different with the simulation result of 1 or 10 cells metastasis.Conclusion:No metastasis driving mutations were observed,and the recurrent gene mutations were consistent between primary and metastasis and were not affected by chemotherapy.However,TP53 mutation VAF increased in metastasis.In addition,colorectal cancer liver metastasis origins from more than 10 cells.