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==Method and Materials== ===Clinical sample and DNA collection=== *NSCLC samples and corresponding normal lung tissues for validation study in Chinese population were obtained from 150 patients who underwent pulmonary resection for primary NSCLC at Changhai Hospital, Shanghai, China. The study was approved by Fudan University and Changhai Hospital and Informed consents were obtained from the patients. Exclusion criteria included subjects with a family history of lung cancer, previous radiotherapy, and chemotherapy or adjuvant therapy before surgery. All tissues were immediately frozen at -80℃ after surgical resection. Histological examination and tumor-node-metastasis classification were conducted according to World Health Organization classification criteria (5) and AJCC Cancer Staging Manual, 7th Edition (6), respectively. Age, gender, smoking status, histology type, TNM stage and differentiation status were collected as the covariates when conducting association between DNA methylation and disease status. Smoking status was assigned to binary status: never and ever smoking. TNM stage was assigned to early stage (I and II) or late stage (III and IV) when it is necessary so that the sample size can be big enough to get the efficient statistic power. * Plasma ** 10 PC-P, Noi: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-6-9 *CTT-frozen and CTT-FFPE RRBS library and sequencing [http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-6-2] ** The aim of expriments on TT-frozen and CTT-FFPE * [[N37 samples]] with WGBS Bam files were stored in TSCC server: === Mapping, bisulfite conversion rate=== * First batch RRBS alignment statistic[http://genome-tech.ucsd.edu/LabNotes/index.php/File:150209_SN216_mapping_summary_statistics.xlsx] * Second batch RRBS alignment statistic[http://genome-tech.ucsd.edu/LabNotes/index.php/File:150209_SN216_mapping_summary_statistics.xlsx] === Average Methyaltion Level for Specific Genomic Regions=== Obviously, we need compare the effect of MHL and average methylation level, especially in the same genomic regions. Here is how to get average methylation level for some genomic postions. PileOMeth extract -r chr10:123-456 genome.fa alignments.bam ===Pairwise R2 (LD) calcualtion within the methylation block from haploinfo files=== * Calculate the pairwise R2 with perl script [[Shicheng:MONOD:perlr2argapr2|code]] # prepare script to calculate LD R2 by genomic region [[Shicheng:haploinfo2LDR2.pl]] # collect WGBS(WB,ES,Roadmap) haploinfo files. # R script for LD block plot [[Shicheng:MONOD:methLDblockplot.R|code]] # relationship between R2 and distance (absolute and relative) [[Shicheng:MONOD:r2argapr2|code]] # [[Haplotype and MHL]] === Number of MHBs with different R2 cut-off=== cd /home/shg047/oasis/monod/hapinfo/WGBS [[qsub hapinfo2mhb.job]] ===[[Umi-BSPP and Methylation Haplotype Loading (MHL)]]=== ===Bioinformatics and Statistics=== We need to look into each cancer individually on the list, maybe rank the cancer samples from most preferable to least preferable. Once we get optimistic results, we can further ask for samples from other centers. We cannot realistically ask specifically for each stage of cancer, although having samples from multiple stages might be better. prevalence treatment options for early detection (stage 1) current diagnostic methods We need to ask for buffy coat (1 tube) and matched serum samples (2 tubes) per patient We need to ask for primary tumor samples, they might be Formalin-fixed paraffin-embedded (FFPE) samples check Blueprint data to see how successful we were at capturing DNA purified from FFPE samples look into the kit for fixing degraded DNA Noi said we successfully captured with 50 ng converted DNA before, and it is possible to obtain this much from 2 tubes of serum samples, however, we can also look into the amplified DNA from Illumina (can we perform capture on these samples?) RRBS option Dinh need to check the bayes classification algorithm Can it be used on RRBS data? We can downsample high coverage data to lower the effect of clonal reads on the analysis. Noi to check the input requirement for performing RRBS, can we perform RRBS in parallel on the test samples. In the biomarker discover stage, the biomarker whose methylation were 100% un-methylated in normal plasmas were enrolled while any regions which were detected to be methylated in normal plasmas were filter out from the candidates. In the procedure of differential methylation test, regions whose variance in total samples were stage at lowest 30% quantile were removed to decrease the burden of multi-test correction. For the random forest prediction, optimal parameters were tuned before the tanning of the model the best number of tries and tress were determined by the grid search method with lowest out of beg prediction error. Intersection analysis of the genome position were conducted by BEDTOOLS and the regions whose distance less than 25bp were taken as the same biomarker region. R packages of IRanges, Biostrings, stringr, randomForest, impute, rpart, e1071, biclust were used in the the statistic and bioinformatics analysis. Potential biomarker based on published papers from NCBI were extracted for lung cancer (74 papers), colon cancer (8 papers) and pancreatic cancer (13 papers) with the strategy of title including “methylation” and corresponding cancer symbols as well as abstract including “Diagnosis”. ===Gene Ontology Analysis=== [[MONOD:Gene ontology P-value plot R code| R code]] === Cluster and Classification Analysis === [[1360 tissue specific MHL in tissue cluster]] === LD in molecular level and pearson correlation in population level === [[LDvsCor.R]] [[File:1572.tm.png|400px]] === Cancer specific methylation haplotype === [[Cancer specific methylation haplotype|Analysis Details]] Dr. Zhang hope to visualize some explicit cancer specific methylation haplotype. I checked the wiki page for his previous anlaysis and conducted similar analysis based on paired cancer tissues, cancer plasma and normal plasma. wiki page:[[http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/MONOD/2014-7-6#Inspect_individual_regions]] ===Prepare SRA submission=== cd /home/shg047/oasis/monod/hapinfo/SRA cp ../*-P-* ./ cp ../*-T* ./ cp ../*N37* ./
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