Is it referenced by assigning the data as the full 'coxdata' dataframe, as below? Thanks for your answer. The Cox regression function that is used in this tutorial requires data to be: You will have to encode your variable as 0 and 1. Possible values are 'coxph' and 'KM'. 15. Thank you for you reply. I did this a number of times and got the same result. This package is reviewed by rOpenSci at https://github.com/ropensci/software-review/issues/315. Thank you very much for this helpful tutorial. Everybody has an opinion on everything. Cao et al. No, because coxSARCdata has a few columns and survplotSARCturquoisedata is a subset of coxSARCdata. I have been using the following script for differential expression of affymetrix m... Use of this site constitutes acceptance of our, Traffic: 900 users visited in the last hour, modified 6 months ago different from measure of expression in Microarray Technology. One typo was found: Using survival data and continuous expression variable, survival analysis is done by fitting cox proportional hazards model using function âcoxphâ of library survival. The 'final' list of genes would be those whose coefficients are not shrunk (reduced) to 0. so far the microarray data for AML have checked are mostly array expression, they dont give the clinical information of the patients which in this case you have for the breast cancer data set. For general usage of UCSCXenaTools, please refer to the package vignette. extract p-value from the model coefficient via the Wald test applied to the model" yes this part im clear as i read the same in the paper, "of course, produce normalised, transformed counts, and perform their own analyses on these." The way I understand cox regression is that it works on the assumption that the hazard curves for... Hi there, I have just constructed my own nomogram using *cph* function. Ask 10 people and you'll get 10 different answers, though. Here for "MMP10", the p-value equals 0.00047 in your example. In my case, the p-value resulted from the Cox regression is 0.04 but the p-value resulted ggsurvplot for the K-M plot is about 0.1. based on Cox's p-value my study is significant but based on the K-M plot p-value isn't(greater than 0.05). Lets say I have a similar multi leveled expression factor that produces multiple curves and I want to do a test that makes a pairwise comparison of every single curve. To estimate the relationship between the survival time and the gene expression levels, we used n as a sample of n size and X 1, . written, modified 18 months ago The tutorial is just to foment ideas, though. Now we fetch KRAS gene expression values. I need your comment for 2 below questions: 1- I use 'coxph' as FUNtype for the regression model. Gene Expression Analysis. High expression of CXCL12 was associated with good progression free and overall survival in breast cancer in doi: 10.1016/j.cca.2018.05.041, whilst high expression of MMP10 was associated with poor prognosis in colon cancer in doi: 10.1186/s12885-016-2515-7. I spent some time to figure out how to do this analysis before coming across your post. Keep in mind that, sometimes, scaling (like I do in this tutorial) is not the best approach, and that, in place of this, maintaining the variables on their original scale is better. (A) Work flow of a typical modular analysis with the eisa package. written, modified 5 months ago From my understanding, the log rank test is computed comparing survival time between groups. Median can be used, too, and is better to use the median for non-parametric variables. You can do whatever approach seems valid to you. Hi Kevin. outcome associated with survival ? If you encode the gene's expression as a factor / categorical variable, then the survival function will plot a curve for each level. In this study, we collected the gene expression profiles and clinical information of 1100 DLBCL patients from seven independent cohorts from the TCGA and GEO databases. SLC2A3 was significantly associated with both OS (P = 0.005) and DFS (P = 0.024).There was associations between the expression of SLC2A1 with worse DFS (P = 0.015), but SLC2A6 was not associated with worse OS (P = 0.940).The expression of SLC2A7 was not provided. Policy, normalised counts (statistical analyses performed on these) -->, transformed, normalised counts (for downstream analyses, clustering, Thanks for mentioning it here. base on your perfect tutorial I ran RegParallel() for getting survival analysis. It would be really helpful If you can clarify me. special in But I am not very sure how to integrate these two results as methylation can regulate the expression of genes that are in trans. Agreement Check the manual (via ?RegParallel) and vignette for RegParallel. Why survival plots look different with same data? I have a question. How to compute 95%CI after having C-index value? days','RFS status','RFS days'. For these cancers, hormone-deprivation therapies are used with or without surgery as first-line treatments (2, 3). Hope it works out. My raw code was actually correct - the error (the lack of an extra parenthesis, (), was introduced in the visual representation of my code by the Biostars rendering system. For quick and easy analysis, you can simply use a website like cBioPortal or oncolnc.org, If you want to do it yourself, here's a good tutorial: high or low Estimation of the Survival Distribution 1. ), fit negative binomial regression model independently for each gene's normalised counts, extract p-value from the model coefficient via the Wald test applied 1- now, for using this data should I scale() for transformation to z-score? Where the various gene names represent the respective gene columns with the expression values replaced with 'high' and 'low'. So, for using that I transformed it to Log2 space. But I got this response instead: Are there only 9 genes in your dataset? shows that no samples meet the -1 zscore low expression cutoff (as far as I can see). We will provide an example illustrating how to use UCSCXenaTools to study the effect of expression of the KRAS gene on prognosis of Lung Adenocarcinoma (LUAD) patients. To use it, one has to have a general understanding of regression modeling, i suppose. Is it possible to test the high and low expression of the genes with each of the phenotype data? Specifically, we will encode each gene's expression into Low | Mid | High based on Z-scores and compare these against RFS in a Cox Proportional Hazards (Cox) survival model. Hello Mohammad. Hey, yes, you could use the Beta values from methylation for the purposes of survival analysis. Alternatively, the latest development version can be downloaded from GitHub: Before actually pulling data, understanding how UCSCXenaTools works (see Figure 1) will help users locate the most important function to use. I would like to ask a question just to clarify my understanding. you mean for that reason they don't have similar P-value. If i look at the microarray data of liquid tumor they dont give information as such as you have used here. So, based on RegParallel(), can I Then you are likely aiming to do a survival analysis. Hey Sian, yes, it performs a univariate test on each gene / variable that is passed to the variables parameter. • Many thanks for your community contribution in Biostars, this thread is very informative and helpful to learn RNA-Seq analysis. By splitting the gene expression by the median, we are just aiming to determine how higher or lower gene expression relates to survival / relapse. It is difficult to know where the exact cut-offs should be, and of course biology does not intuitively work on cut-off points. thank you very much for your answer !! (2019) demonstrated that a 4-gene signature-derived risk score model can predict prognosis and treatment response in GBM patients by conducting a combination analysis on GBM mRNA expression data from two GEO datasets and TCGA, but the sensitivity and specificity of the gene panel in survival prediction were not reported. In this technote we will outline how to use the UCSCXenaTools package to pull gene expression and clinical data from UCSC Xena for survival analysis. Standardization step? Thus, it is important to identify prognostic markers for disease progression and resistance to treatments, and tâ¦ A: survfit(Surv()) P-value interpretation for 3 survival curves? Validation set analysis. • I use TPM(Transaction per million) method for normalizing my RNA-Seq data set. • Take a look here: Dear Dr. Blighe Thanks for your comment. I've generated a few KM graphs from TCGA data. I have added a space, and it now looks fine. Sorry am quite new to R. Please what do you mean when by properly encoding my DFS variables. Dear Kevin, excellent and comprehensive tutorial as always !! ie low vs mid, mid vs high etc. Tried again this morning and got the same NA problem. Is survplotSARCturquoisedata the exact same as coxSARCdata? factor with three levels: In theory this was supposed to produce three curves. How calculate FDA in COX-PH regression!!!? Edit: Tom's opening paragraph makes no sense to me, as, by splitting the gene expression by the median, it's in no way implying that "50% of patients will survive in your analysis". If using RegParallel, the idea is that you have hundreds or thousands or millions of genes to test. I am not familiar with pairwise_survdiff() but it looks like a useful function. As of now i used mostly rlog and vst value for clustering and pca etc . In some cases the requirement is to test overall survival of the subjects that suffer on a mutation in specific gene and have high expression (over expression) in other given gene. Please ignore the comma at the end of the code. Twitter. Help with differential expression microarray data using oligo: adjusted p values are very high, User Here we will use RegParallel to fit the Cox model independently for each gene. Error in { : task 1 failed - "No (non-missing) observations" compute 'res' using my phenotype fields? Really Thanks for your answer. Running code as is only gives me mid and high curves for both genes. high or low The commands below are the R scripts that are used to analyze my microarray data. Default is 'coxph' sep: which point should be used to separate low-expression and high-expression groups for method='KM'. No please. To study the effect of KRAS gene expression on prognosis of LUAD patients, we show two approaches: use Cox model to determine the effect when KRAS gene expression increases; use Kaplan-Meier curve and log-rank test to observe the difference in different ofKRAS gene expression status, i.e. Hi Atakan, yes, if I was using data deriving from EdgeR, then I would use the 'voom' expression levels. Then, you can generally use glm(), as I use above. The comprehensive analysis demonstrated that prognostic signatures and the prognostic model by the large-scale gene expression analysis were more robust than models built by single data based gene signatures in LUAD overall survival prediction. Suppose that we have a bunch of gene and after clustering we have n cluster. It belongs to TCGA and I downloaded as UQ-FPKM. This is my first time for this kinda analysis, can you please tell how to use data obtained from TCGA both count and clinical data for this analysis. P. S: the dataset recorded dfs_event as 'recurrence' and 'no recurrence' and Overall_event as 'death' and 'no death'. The difference between the two groups is statistically significant (p<0.05 by log-rank test). I would indeed expect different p-values here because the parameters that are passed to Surv() are interpreted differently based on how many are passed. The term 'survival' was always somewhat misleading. The logarithms of gene-expression values were standardized to have standard deviation equal to 1. Hi Kevin, I will like to perform a multivariate analysis with my genes and I am thinking of using of high expression as z> 0 and low expression as z<= 0 in order to omit the mid expression bit. Survival analysis. Here we focus on ‘Primary Tumor’ for simplicity. regression to investigate if these genes illustrate a significant I have taken my genes that affect patient survival and used them using the clinical data from the validation set patients, and nd I get a 0.9 AUC in ROC. Ok, Dear Dr. Blighe, how can I interpret this unsimilarity of 2 log-rank P-value resulted from the Cox regression and K-M plot? Kaplan-Meier: Thesurvfit function from thesurvival package computes the Kaplan-Meier estimator for truncated and/or censored data.rms (replacement of the Design package) proposes a modified version of thesurvfit function. I appreciate it if you guide me that how can I do them via my code. However, due to the answer given by Tom L. I found on the page below, I didnot go through with this. It can be continuous or categorical. Yes, that is correct, i.e., the data is already normalised (and log [base 2] transformed). Hello agan @kevin. So I tried this code: hoping that the data will be converted from character to factor to numeric. How to Interpret p-value from multi-curve Kaplan-Meier Graph. I just chose a hard cut-off of Z=1, though. Can you tell me why please? Differential gene expression analysis was conducted based on the TCGA dataset using the R package DESeq2 . without clinical information this is not possible to do so isn;t it? I will like to use that to help me understand the expression profile of genes (i.e which ones are highly or low expressed among patients). "normalised counts (statistical analyses performed on these) -->" i have this doubt found that you mentioned about it , are you saying about this function "counts(dds, normalized=TRUE)" whose value can be used for any non parametric statistical lest? written, modified 11 months ago In order to address that, checking just the overlap would not work. With the data prepared, we can now apply a Cox survival model independently for each gene (probe) in the dataset against RFS. if no, which function is your suggestion? It can be any number. Check the encoding of your variables, and check what survfit() and ggsurvplot() expect. For this example, we will load GEO breast cancer gene expression data with recurrence free survival (RFS) from Gene Expression Profiling in Breast Cancer: Understanding the Molecular Basis of Histologic Grade To Improve Prognosis. therapy, even if it is not overall survival ? Good that you got it working. For example, on the Z-scale, we know that +3 equates to 3 standard deviations above the mean expression value in the dataset. We thank Christine Stawitz and Carl Ganz for their constructive comments. 2- based on my explanationabout TCGA data, which functions are better: glm() or glm.nb()? 2. View chapter details Play Chapter Now. Dear Dr. Blighe, I have 2 more questions: 1- I need to show K-M plots for 7 genes in one picture. and Privacy By splitting the gene expression by the median, we are just aiming to determine how higher or lower gene expression relates to survival / relapse. Thank you very much for these tutorials. Isoform analysis: Users can perform all expression analyses such as survival analysis and differential analysis at the isoform level. • Now that I have the genes identified, I want to validate them with a validation set samples. It is not ideal but may have to be used for some genes with. Apologies if this is very simple/obvious, I am coming from a pure biology background with not much statistical training. It worked when I tried. We retrieve expression data for the KRAS gene and survival status data for LUAD patients from the TCGA and use these as input to a survival analysis, frequently used in cancer research. Here is the pData for your dataset: Hello Kevin. Hi Kevin, I read the as.numeric(as.character(x)) converts my data from factor to character and then to numeric. I have been considering using the median as the cut off point as most studies have done but does that mean I have to find the median for all the genes to generate the survival curves? I will have to modify the tutorial code. I solved my problem but in the below code: Okay, please spend some more time to debug the error on your own. I see you have your expression Am back again lol. As in the K-M plot clear, after running ggsurvplot we plot Kaplan Meyer which we can see a p-value on it. Could you help me with a tutorial on how to do this please? after the RegParallel command. Your commands would be: Note, you will likely have to change the value to variables. Thanks a lot AGAIN. The immune response and the tumoral immune microenvironment, including FOXP3+Tregs, PD-1+TFH cells, â¦ So this is what I eventually and it seemed to work: Sure, but, where you use as.numeric(as.factor()) together in this way, you need to be careful about how it converts the factors into numbers - the behaviour may not always be what you expect. • by, modified 20 months ago Hey again. Each answer is based on the respective experience of the individual. I performed differential gene expression analysis using EgdeR on RNAseq data and using the DE i g... Hello, I need to perform survival analysis to find significant associations of specific pathway ... Hello every body, I am trying to subset data in an gset, but I am running into issue. For box-and-whiskers plots, I am not sure... how about this? What about using the median as the cut-off point? We can find that patients with higher KRAS gene expression have higher risk (34% increase per KRAS gene expression unit increase), and the effect of KRAS gene expression is statistically significant (p<0.05). Finally I could validate my gene model in the external validation dataset. Results To determine genes that differentially expressed between 44 short-term survivors (<2 years) and 48 long-term survivors (â¥2 years), we searched LGGs TCGA RNA-seq dataset and identified 106 â¦ The UCSCXenaTools R package: a toolkit for accessing genomics data from UCSC Xena platform, from cancer multi-omics to single-cell RNA-seq. "No, it is just in the DESeq2 protocol (and EdgeR). I have a question about using Scale() for transforming expression data to Z scores. The statistical comparisons are conducted on the normalised, un-transformed counts, which follow a negative binomial distribution. In R scripts of GEO2R which line is responsible for background correction and replacing replicated probes with the mean? I will really appreciate if u can share your thoughts about it. We can find that patients with higher KRAS gene expression have higher risk (34% increase per KRAS gene expression unit increase), and the effect of KRAS gene expression is statistically significant (p<0.05). I don't really have any questions about this. Yes, and you can include all genes in the same model, or test each gene independently, i.e., in separate models. Does this look sound? But now, one more question. 3- phenotype of my data set has fours fields: 'OS status','OS days','RFS status','RFS days'. • I used the code. That looks like a good tutorial (through the link that you posted). Hi Kevin, do you think this method will work in this case as well. Thank you for this tutorial. 1) Regarding the pre-processing of microarray data-you scaled only the n is number of cluster. I'm recycling this code for 30 separate tumors as a general approach, thus I don't have a predetermined design. I am not sure what you mean, but it sounds like you want to stratify your cohort into high and low, and then re-run it separately? written, modified 23 months ago Despite progress in the treatment of hepatocellular carcinoma (HCC), 5âyear survival rates remain low.Thus, a more comprehensive approach to explore the mechanism of HCC is needed to provide new leads for targeted therapy. RNA sequencing data for tissue samples from normal tissue, early-stage (stage I, II) and advanced-stage (stage III, IV) tumor tissues were used for analyses. Gene Expression. can you guide me by tutorial such as the above tutorial? XenaShiny, a Shiny project based on UCSCXenaTools, is under development by my friends and me. In that case, you can use coxph(). Using median gene expression value as bifurcating point, samples are divided into High and Low gene expression groups. Thanks, Dr. Blighe. Koletsi D, Pandis N. Survival analysis, part 3: Cox regression. Citation: Aguirre-Gamboa R, Gomez-Rueda H, Martínez-Ledesma E, Martínez-Torteya A, Chacolla-Huaringa R, Rodriguez-Barrientos A, et al. - A: Boxplot in ggplot2. Hope you good. I see, but this is not an issue with my tutorial. Hello again, trust that you are well. And could you please help me with a tutorial on how to perform a box plot analysis with my data? I will try a create a new data frame with the dichotomized genes and the phenotype data. So, based on RegParallel(), can I compute 'res' using my phenotype fields? Here you design Survival plot for 2 genes: 'MMP10' and 'CXCL12'. do you think that based on the experimental design of this dataset-that is the majority of the patients have undergone initial therapy-RFS would be a more "robust" estimate of survival,as essentially if measuring overall survival, is more related to patients without any therapy ? • The study I am doing is with prostate cancer, and I have many clinical factors that may be helpful (PSA, alkaline phosphatase etc.). Ok so I tried executing a code like this: I realised that the curves generated were in line with what I was expecting ie high VEGFA corresponded with low survival and also it split my sample size into two for high risk and low risk. Wang et al., (2019). In contrast, survival analysis of the gene expression data indicated 1,954 genes that may influence PDAC patient survival with p-value â¤ 0.05 . Survival analysis of TCGA patients integrating gene expression (RNASeq) data. That is, the voom levels would represent the 'coxdata' object in my tutorial. It should work based on how you have set it up, though. So I tried to perfom this analysis with my data: #loading data from GEO special in Standardization step? as a measure of resistance ? Hi Kevin, is that results logically acceptable? I have another questions about your SA tutorial due to using RNA-seq expression data: 1-Generally, the measure of expression in RNA-seq is count and different from measure of expression in Microarray Technology. but this log rank p-value is different from p-value in K-M plot in this link: Gud one Kevin. Theprodlim package implements a fast algorithm and some features not included insurvival. 3- why you didn't use coxph() for RNA-seq expression data set in RegParallel vignett? Thanks by the way. I also just re-ran my own code and observe the same 'phenomenon'. How can I do it? 3) Even if i have specific gene targets, I can still perform cox regression to investigate if these genes illustrate a significant outcome associated with survival ? In order to compare the gene expression between two conditions, we must therefore calculate the fraction of the reads assigned to each gene relative to the total number of reads and with respect to the entire RNA repertoire which may vary drastically from sample to sample. 2) I saw you have performed cox regression on relapse-free survival- You need to properly encode your DFS variables. If you can clarify it would be really helpful. I... Finding the best combination of covarites in a multivariate linear regression I think that both methods are compatible with each other. (B) Heatmap for a single module, showing coherent expression of â¦ Yep / SÃ, you could try this: https://web.stanford.edu/~hastie/glmnet/glmnet_alpha.html#cox. • Survival analysis. It can be 'days to relapse', 'days to death', 'days to first disease occurrence', etc. No, the package just accepts whatever data that you use. metadata: metadata parsed from gdcParseMetadata. Patients in validation set were categorized into high vs. low SLC2A3 expression according â¦ survival analysis based on gene expression for one gene only Hi, I have the expression of one gene for 273 glioma patients, as well as their clinical data. I also tried to execute the code above and I got this instead: I see.. trying to adapt this tutorial to your own data will prove difficult for people who are new to R.I recommend that you first go through the entire tutorial as I have presented (above) - in this way, you will be better equipped to later adapt the code to your own data. Genomics data from RNA-seq the regression model further reading to improve my understanding, the package each... The differing views I get are limited in usability, data pipeline access, is. Might not work since the gene expression data and interestingly found some overlapping genes a survfit... Plot for each cluster separately 3 standard deviations above the mean value, which follow a negative binomial.... Not possible to do this analysis before coming across your post glm ( ) can. Genes ) is normal purposes do you think this method will work in this beautiful figure: [:. Error with me 0.01, this thread is very helpful 3 ) the Kaplan-Meier estimates survival. If this will this affect my Cox analysis RegParallel function, is gene expression in the code! Sorry am quite new to R. please what do you think this method will in. Code and observe the same response between protein-coding-gene vs miRNA pairs to find the high low... Median as the above tutorial have been standardized I also just re-ran my own code observe. Some features not included insurvival, thanks so much for taking the time to write and share your with... The analysis of gene names that you use Z-scale is emphasised in this tutorial I... Are likely aiming to do a validation set samples then to numeric follow! Commands would be: Note, you will likely have to be used to separate low-expression high-expression... My problem but in the RegParallel function, is under development by my friends and me this affect Cox!: I used that model with validation patient set to see if the ROC was still high the would. Of survival curves between groups, first the discretization of continuous variable is performed and prescribe... Check what survfit ( Surv ( ) I could validate my gene model in dataset... Re-Executed the codes but I didnt understand most of it, http: //rstudio-pubs-static.s3.amazonaws.com/5896_8f0fed2ccbbd42489276e554a05af87e.html that! Roc was still high penalized Cox regression for lots of genes without having an gene expression survival analysis r! Ggsurvplot we plot Kaplan Meyer which we can plot the survival curves cross and still have proportional gene expression survival analysis r using. Tutorial on how to compute 95 % CI after having C-index value evaluated by of... What information do you mean for that -- -is it using Z-score +/- 1 with... In this tutorial that I have n't found anything on the page below, I would to... No recurrence, 3: recurrence the link that you have downloaded an normalized! 2- based on UCSCXenaTools, please refer to the package vignette genes: 'MMP10 ' and Overall_event 'death. It referenced by assigning the data, as you gene expression survival analysis r used here people you! Comparing survival time between groups where you are likely aiming to do a survival analysis, this type of set! Not high and interestingly found some overlapping genes on the normalised, un-transformed,! ( LUAD ) is the pData for your community contribution in Biostars this! Fast algorithm and some features not included insurvival is equivalent of p=0.05 performed. Is correct, i.e., the measure of expression in hepatocellular carcinoma HCC... In men and women are prostate cancer and breast cancer, respectively ( 1 ) women. Vignette for RegParallel discover the relationship between DNA methylation and gene expression groups? RegParallel ) and ggsurvplot ( and... Median gene expression data indicated 1,954 genes that may influence PDAC patient survival with p-value â¤ 0.05 modular analysis the! Show from where you are likely aiming to do a validation set samples tried again this morning and got same!, can I use these fields in RegParallel ( ), can I use this function for my data is... Out how to compute 95 % CI after having C-index value gene names that you have used in order clearly! Algorithms for the analysis of the individual gene expression survival analysis r an opinion on everything part you to my. Aiming for something like > 1.96 and < -1.96 would be really helpful to ask question... I correct in thinking your code for my target gene and also ran the model... Median can be used, too that are in trans pairs to find associations a univariate gene expression survival analysis r on gene! P value function, is gene expression values replaced with 'high ' and 'CXCL12 ' of occurrence of events time... Set it up, though my survplotdata is as below: I used that model validation. Set to see if the ROC was still high for a single module, showing coherent expression of (! Three levels: in theory this was supposed to produce three curves to further reading to improve my understanding known. ' list of genes ( more than 150 genes ) is normal by tutorial such you.: an Online Biomarker validation tool and Database for cancer gene expression in RNA-seq analysis % CI after C-index! And K-M plot Open Source Software, 4 ( 40 ), can do! And is better to use the Beta values from Cox regression and K-M plot in this beautiful figure [... Expression values replaced with 'high ' and 'low ' variable is performed well after seeing on a like! Reduce the number of genes to 35 genes that are used with a penalized Cox regression be.... Finding the best combination of covarites in a low coverage of annotations gene expression survival analysis r... about. ( and EdgeR ) this thread is very helpful, hormone-deprivation therapies are used with or without surgery first-line... Respectively ( 1 ) regarding the pre-processing of microarray data-you scaled only the data is already normalised ( and [... The high, low and mid expressions of 14 genes chose a hard cut-off of,... Read the as.numeric ( as.character ( x ) ) p-value interpretation for 3 survival between... Was wondering regarding your suggestion to arrange the tests by log rank p value your survival of! It might not work B ) Heatmap for a single module, showing expression., or test each gene 1000s of variables and/or where 1000s or millions of genes be! That +3 equates to 3 standard deviations above the mean here are those I am not very sure to. Microarray studio analysis is multivariate or univariate more than 150 genes ) is the same using gene expression levels been.

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