Bioconductor Forum

region1_condition1) res <- lfcShrink(dds, contrast = contrast, res =res, type="ashr") ``` As a result, I will have four sets of Differentially Expressed Genes (DEGs). Subsequently, I aim to compare each set of DEGs

DEGcomparison DESeq2 InteractionSet pvalue

updated 9 weeks ago • paria.alipour

in advance for your help and insights, Theo Edit 1: The MA plot looks similar with `lfcShrink(type='ashr')`: ![MA plot after LFC shrink, type=ashr][6] I suspect something is off with the data but I have not been able to track it down... ``` > sessionInfo

DESeq2

updated 12 weeks ago • theophile

method for the comparison of two conditions. However if i prefer the lfcshrink FC (apeglm or ashr methods) then what would be the value to be used for GSEA? Indeed the "stat" column is generated using the normal method and

lfcshrink GSEABenchmarkeR DESeq2

updated 4 months ago • delphine.rossille

Hi everyone, I am trying to carry out some analysis for an experiment which has three dimensions (treatment, genotype and time) and while I have managed to extract the pairwise comparisons I am interested in, I would like to go further and look at the genes which behave differently upon my drug treatment depending on either the genotype or the time factor (for a given genotype) - I believe that …

DESeq2

updated 8 months ago • james.zhang20

res = res, type = "ashr") } ``` Any help or advice, would be really appreciated! Thanks

shrinkage DifferentialExpression DESeq2

updated 9 months ago • Abir.khazaal

Hello, We have RNA-seq as well as functional data of various kinds. For some of the most important genes in our study, we quantified protein and transcript levels using wet lab assays to confirm the size and direction of effect of the results. Our original data were from microarray studies, these data showed massive downregulation of interferon responsive genes in the microarray data; su…

DESeq2 apeglm DESeq ApeGLM Ashr

updated 9 months ago • Ndimensional

Hello, @rory I have a question regarding the 3 scenarios in which lfcShrink is applied in DiffBind package. The source code looks like this ``` if(shrink) { if(contrast$contrastType=="byname") { res$de <- suppressMessages(DESeq2::lfcShrink(pv$DESeq2$DEdata, coef=contrast$contrast, …

FoldChange DiffBind lfcShrink

updated 9 months ago • bhandary.8590

the default algorithm that is used is "aplegm", while the lfcShrink function in DiffBind uses ashr. Is this right? and if so, what is the reason for this? It seems that aplegm is more suited for RNA-seq data while ashr is for other...sequencing data. Could you offer more insight as to why ashr is used by DiffBind? 3. For some of the pairwise DiffBind results, we found some very interesting resul…

FoldChange DiffBind lfcShrink Cut&TagData

updated 10 months ago • bhandary.8590

Hi everyone, I am trying to analyse some RNA-Seq data in DESeq2, but am struggling a little conceptually in understanding how exactly to investigate what genotype specific changes I have. My data consists of 2 genotypes, 3 timepoints and 2 treatments (treated/untreated) - 48 samples in total (4 replicates per). Therefore, in the sample information file, I have formatted it like so: Sample Geno…

RNASeqData DifferentialExpression DESeq2

updated 10 months ago • james.zhang20

list(c("status_als_vs_con"))) res1 <- lfcShrink(dds, contrast = contrast, res =res1, type="ashr") '' Thanks, Paria

DESeq2 SingleCell log2FC

updated 12 months ago • paria.alipour

changes as well as the visulal representation. I tested this mostly using apeglm, but when I tried ashr, the absolute values didn't match up either. There are not too many DGEs in this data set, otherwise I might have missed it

DESeq2 apeglm

updated 13 months ago • Daphnia

Since I'm analyzing my SLAM-seq data for the first time, I thought I'd follow this guide: https://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#quick-start In the guide, it says to use contrast (or name) because contrast sets the log fold change to 0. I have two conditions treatment and control. And for my dds$condition, I releveled it so that the ref = "control". …

DESeq2 MAplot

updated 14 months ago • La

not redone? I hope that the question is clear. Also, I already have implemented a for loop with ashr that produces a list (res as in the example above) with all given comparisons. [1]: https://support.bioconductor.org/p/9147589

DESeq2

updated 15 months ago • theodore.georgomanolis

0 expression, the results have much lower number of DEGs. Also, after updating some packages (as ashr and mixsqp), I am getting lower number of DEGs when running exactly the same code. Any explanation please? Many thanks

DESeq2 filter

updated 15 months ago • ayabalbaa1990

this sound reasonable? 2. Here is the MA plot from the same dataset used for volcano plots. with ashr shrinkage (I need to use contrast, so I select this method for shrinkage) I wonder if this MA plots looks okay. They are slightly

DESeq2 RNASeq

updated 16 months ago • BT

I got this message every time: **Error: package or namespace load failed for ‘DESeq2’ in loadNamespace(j <- i[[1L]], c(lib.loc, .libPaths()), versionCheck = vI[[j]]): there is no package called ‘Biostrings’ In addition: Warning message: package ‘DESeq2’ was built under R version 4.1.1** ```r > sessionInfo() R version 4.1.0 (2021-05-18) Platform: x86_64-w64-mingw32/x64 (64-bit) Ru…

DESeq2 Install

updated 18 months ago • Hannah000111

listValues = c(1/3,-1))) resLFC <- lfcShrink(dds, contrast=contrast, res=res, type="ashr

DESeq2 log2FC

updated 19 months ago • User000

I have been working with DESeq2 for the past couple of months analyzing my data, I have read over the vignette many times, found other workshops, read message boards, but I still second guess my decisions and the options I have chosen. Basically my design is that I have multiple clam lines, lets say 3 (A,B,C), and two salinities I am comparing (35 ppt vs 15 ppt). Salinity 35 ppt is my control…

DESeq2

updated 19 months ago • lgspeight

We have RNAseq data with 4 cell lines with different knockout genotypes (WT, A, G, GA), and for each cell lines, we have two treatment types: treated (H) and untreated (C) with 5 replicates for each condition. we are interested in the treatment effect on each of these cell lines: WT.H_C, A.H_C, G.H_C, or GA.H_C (H_C means treatment vs untreat comparison). These are easily to be done in DESeq2 bas…

DESeq2 Complicated.contrast DEGs

updated 19 months ago • Mike

Enter the body of text here Hi, there: I read the previous posts about deriving confidence interval for fold change using DESeq result at URLs: https://support.bioconductor.org/p/9142141/ and https://support.bioconductor.org/p/80725/#80729 one comment from Micheal Love mentioned: Estimated standard errors for the estimated coefficients on the log2 scale are given by the lfcSE column. Ye…

DESeq2 Confidence.Interval

updated 19 months ago • Mike

c("ConditionB","ConditionC")), listValues = c(1,-1/2), type="ashr") using 'ashr' for LFC shrinkage. If used in published research, please cite: Stephens, M. (2016) False discovery rates: a new deal...the `contrast` or `listValues` or is there likely something wrong with my input data? I reinstalled `ashr` but it doesn't help

DESeq2

updated 19 months ago • Kent

My dataset has a total of 53 samples (with outliers removed) of breast cancer patients with two conditions. I have read that DESeq2 doesn’t provide automatic shrinkage, and I have to use lfcShrink to shrink the log2FoldChanges. The manual states that apeGLM and ashr perform better than the normal option and that apeGLM is quite strict compared to the others and performs well with small...and I h…

DESeq2 RNASeq RnaSeqSampleSize apeglm

updated 21 months ago • Riley

control"),listValues = c(1/2,-1))) resLFC <- lfcShrink(dds, contrast=contrast, res=res, type="ashr") up <- res[which(resLFC$log2FoldChange > 0 & resLFC$padj < 0.01),] down <- res[which(resLFC$log2FoldChange < 0 & resLFC

log2 DESeq2

updated 21 months ago • User000

Hi I have done a DESeq2 DGE analysis and it showed an error when I was using ashr shrinkage ``` using 'ashr' for LFC shrinkage. If used in published research, please cite: Stephens, M. (2016) False discovery rates...contrast = c(-1/12,-1/12,-1/12,-1/12,1,-1/12,-1/12,-1/12,-1/12,-1/12,-1/12,-1/12,-1/12,0), type = "ashr") ``` Edit: Thank you Michael for pointing towards the solution here: htt…

sva DESeq2

updated 22 months ago • Kent

Hi everyone, I'm currently getting some warnings when using lfcshrinkage with "ashr", regarding solve(). Example Code should be placed in three backticks as shown below ``` library(zebrafishRNASeq) data(zfGenes...c("group", "Trt","Ctl")) ashr_res<-lfcShrink(dds, contrast = c("group", "Trt","Ctl"),type="ashr") ``` This leads to the production of the corresponding output:…

RNASeq DESeq2

updated 23 months ago • andrebolerbarros

based on Zhu *et al.*, 2018 but am curious as to what criteria others use to justify `apeglm` vs `ashr` for this sort of analysis. My current dataset is 160 samples, but I'm narrowing focus to 4M + 4F per experimental condition...I have an FDR < 0.05 for ~ 200 genes, but `apeglm` shrinks all my fold changes towards zero, and `ashr` gives LFCs between 1 and 28 for all 200 genes. Base mean…

lfcShrink DESeq2

updated 23 months ago • Allison

which is why I don't want to run the model for each coefficient separately. I also had a look at ashr, but testing against a threshold is not implemented there. Best, Frederik

shrinkage apeglm

updated 2.0 years ago • Frederik Ziebell

Edit": ```r ddsShrink_t <- lfcShrink(ddsObj2, contras=c("cell_group","DC3_TLR","DC2_TLR"), type="ashr") ``` It now worked with ashr. But is this correct

lfcshrink R DESeq2

updated 2.0 years ago • LHA_trash

equivalent to: ``` res1 <- lfcShrink(dds, contrast=c("condition","treated1","control1"), type="ashr") res2 <- lfcShrink(dds, contrast=c("condition","treated2","control2"), type="ashr") ``` Given the statistical benefit on the DESeq2

tximeta DESeq2 swish fishpond

updated 2.2 years ago • jeremymsimon

design = ~ sex+month+method+cat) I've used the default wald test and am applying ashr for LFCshrink which I think is the correct method, however I have previously used IHW for multiple testing correction...of using both please? Or what is used for multiple testing correction otherwise when running with ashr? Many thanks, B

Ashr LFCShrinkage IHW DESeq2

updated 2.3 years ago • Bex

3 UP 843 > res_tableOE_shrunk <- lfcShrink(dds, contrast = contrast_oe, type = "ashr") using 'ashr' for LFC shrinkage. If used in published research, please cite: Stephens, M. (2016) False discovery rates: a new deal

DESeq2 lfcShrink

updated 2.5 years ago • Deevanshu

RcppArmadillo Suggests: testthat, knitr, rmarkdown, vsn, pheatmap, RColorBrewer, apeglm, ashr, tximport, tximeta, tximportData, readr, pbapply, airway, pasilla (>= 0.2.10), glmGamPoi, BiocManager License: LGPL (>= 3) MD5sum

Install Bioconductor DESeq2

updated 2.5 years ago • ladypurrsia

Hi all, I am using DESeq2 v1.32 for RNAseq analysis. I am using the following code to apply a threshold/ FC cutoff of 1.25 in the wald stat. However, I am seeing some significant genes in res_sig have |LFC| < 1.25. From an old discussion thread in this forum, it seems to be normal if lfcShrink type = "normal". Is it okay in ashr also? Or am I supposed to add a post hoc threshold also? Wil…

ashr lfcShrink lfcThreshold DESeq2 diffGeneAnalysis

updated 2.7 years ago • lamia11me

Hi, Dr love. I post a question about [weird MAplot or volcano plot of DESeq2 diff result](https://support.bioconductor.org/p/9139060/) and also in [biostar](https://www.biostars.org/p/9484986/#9485059). @atpoint give a useful answer about too many 0 count genes and prefiltering. It seems that too many 0 count genes makes lfc shrink have a probelm. And I find the `apeglm` and `ashr` result i…

ashr DESeq2 apeglm

updated 2.7 years ago • Guandong Shang

type") dds <- DESeq(dds) res_lfc <- lfcShrink(dds = dds, type = "ashr", coef = "type_Fx600_vs_Fx593") plotMA(res_lfc) as_tibble(res_lfc) %>% mutate(padj = case_when( is.na(padj) ~ 1, TRUE ~ padj )) %>% ggplot

DESeq2

updated 2.7 years ago • Guandong Shang

instead of p-adjusted values in results. Is there a way I can get the nominal P-values by using the ashr shrinkage estimator? I only see the s-values. If not, which P-values is it base on? How can I examine my test by P-value histogram

DESeq2

updated 2.8 years ago • haasroni

Hi, I'm using Deseq2 (v1.26.0) and ashr (v2.2-47) to shrink the log2FoldChange of the genes resulting from interaction term. DEseq2 returns the different shrunken...among Mut.T/UT vs WT.T/UT using the following **design = ~cell+trt+cell:trt** followed by ashr fold-change shrinkage. I get different results with ashr with reference levels compared to the ones without defining...for example**).…

DESeq2 RNASeq

updated 3.0 years ago • sofiagreen72211

I obtained negative svalues from the lfcShrink function of DESeq2 using the ashr shrinkage estimator. The values are very small and close to 0: the smallest is `-1.110223e-16`, the largest negative is `-1.199592e...I obtained negative svalues from the lfcShrink function of DESeq2 using the ashr shrinkage estimator. The values are very small and close to 0: the smallest is `-1.110223e-16`, t…

ashr DESeq2 lfcShrink svalue

updated 3.1 years ago • Enrico

Risso et al. 2014) to estimate > variables that capture the unwanted variation. In addition, the ashr > developers have a specific method for accounting for unwanted > variation in combination with ashr (Gerard and Stephens

BatchEffect DESeq2 RNASeq

updated 3.5 years ago • igor

for LFC shrinkage, the Normal prior from Love et al (2014). Note that type='apeglm' and type='ashr' have shown to have less bias than type='normal'. See ?lfcShrink for more details on shrinkage type, and the DESeq2 vignette

deseq2

updated 3.5 years ago • helenhuang.math

me 1618. This only happens if I use lfcShrink with "apeglm". It doesn't happen with "normal" or "ashr" or if I just use "results". Attached is an example of my code and also my ColData for dds1. Due to sequencing issues, I have some

DESeq2

updated 3.5 years ago • serdarT

did comparisons within each culture condition by grouping condition and genotype and using lfcshrink/ashr. For instance for genotype A: dds <- DESeqDataSetFromMatrix(countData = countdata, colData = coldata, design = ~ Experiment...2D contrast A_2D_v_WT_2D_lfcShrink <- lfcShrink(dds, contrast=c("group"…

deseq2

updated 3.5 years ago • Nick F

Dear all, I analyzed RNA-seq data using DESeq2 extracting the results with or without shrinkage without shrinkage: res21 <- results(dds2, contrast=c("combined", "XX.DMSO", "XX.dTAG"), cooksCutoff=FALSE, alpha=0.05) with shrinkage: resLFC1 <- lfcShrink(dds2, contrast=c("combined", "XX.DMSO", "XX.dTAG"), alp…

deseq2 ashr

updated 3.6 years ago • da.de

ashr" generally performs better than "normal" and that it preserves large LFC, but these LFC are unrealistically large with...ashr. I have 2 questions: 1. Is this expected behavior or does log2FC on this scale imply there is something fundamentally wrong...with my design or samples? 2. If this is expected behavior, should I go with "ashr" or "normal"? I am not using apeglm because I need the …

deseq2 ashr rna-seq

updated 4.0 years ago • Alex

I have created 12 pairs of DGE for my 3x2 design matrix with deseq2 and 10/12 shrinking by ashr works fine however,for 2 of the pairs, this message appears > Optimization failed to converge. Results may be unreliable...increasing maxiter and rerunning. I saw error in a thread here https://github.com/stephens999/ashr/issues/76 Does installing mixsqp to resolve this issue? Am I m…

ashr

updated 4.0 years ago • kavator

Hi, I have pretty simple but probably weird question. I have complex design with interaction terms in my model (see below), I run DESeq2_1.20.0 on rna-seq data. It seems that I can't change my contrasts to end up using only apeglm shrinkage and lfcThreshold, instead I'm stuck with using ashr shrinkage method without being able to incorporate lfcThreshold into calculation of null. What bothe…

ashr Deseq2 lfcThreshold

updated 4.0 years ago • Iryna

for LFC shrinkage, the Normal prior from Love et al (2014). Note that type='apeglm' and type='ashr' have shown to have less bias than type='normal'. See ?lfcShrink for more details on shrinkage type, and the DESeq2 vignette

deseq2 software error

updated 4.0 years ago • junli1988

the column “stat” , which seems to be lost after the lfcshrink calculation using either apeglm or ashr. My question therefore is: Q1: How can I still apply the fdrtool in a correct way? Or am I doing something wrong? I do not think

fdrtool

updated 4.1 years ago • Paul

I have RNA seq data an experiment consisting of an untreated and treated samples in triplicates; a total of 6 samples. The culturing of the used tissue can be challenging so a batch effect is expected. I would like to apply apeglm shrinkage and use adjusted p-values as a cutoff to explain effects between conditions, but am encountering some problems. My questions are mainly related to the lfc sh…

deseq2 fdrtool apeglm

updated 4.1 years ago • Paul

for LFC shrinkage, the Normal prior from Love et al (2014).` Note that type='apeglm' and type='ashr' have shown to have less bias than type='normal'. See ?lfcShrink for more details on shrinkage type, and the DESeq2 vignette

deseq2

updated 4.2 years ago • nabiyogesh

1] ``` > resLFC_sh2 <- lfcShrink(dds, contrast=c("Treatment", "Sh2", "Control"), type="ashr") using 'ashr' for LFC shrinkage. If used in published research, please cite: Stephens, M. (2016) False discovery rates: a new deal

deseq2

updated 4.6 years ago • MatthewP

the `lfcShrink` function, but no matter what type of shrinkage estimator I give (*normal/apeglm/ashr*) I get the same results. Also - I get NA values in the **padj** column. Could this have to do with the number of samples expressing

deseq2 normalization

updated 4.8 years ago • ronif10

lt;- if(l2FCShrink){ as.data.frame(lfcShrink(dds, contrast=rn, type = "ashr")); } else { as.data.frame(results(dds, contrast=rn)); } results.df$log2FoldChange <- round(results.df$log2FoldChange, 2); results.df

deseq2 nanopore

updated 5.0 years ago • David Eccles (gringer)

logical, should p-values and adjusted p-values be replaced with s-values when using apeglm or ashr. s-values provide the probability of false signs among the tests with equal or smaller s-value than a given given's s-value

deseq2 apeglm

updated 5.0 years ago • ATpoint

lfcShrink` does not say much on the topic, except that `contrast` and `coef` are ignored for `type="ashr"` when `res` is provided (and for `ashr` neither `contrast` nor `coef` are required). There is also the error message `one of coef or...reason why `contrast` or `coef` should be required if `res` is given (even for for types other than `ashr`), or am I missing something? In fact, if `res` is…

deseq2

updated 5.1 years ago • tdanhorn

above for my own dataset. However, I am struggling to perform lfcShrink() using types "apeglm" and "ashr". Does any one have any suggestions on how to perform log fold change shrinkage on this design? I look forward to your replies

deseq2 lfcShrink interactions

updated 5.2 years ago • hannepainter

Hello DESeqers, I have two groups of patients whose gene expression is expected to change differently over time. In one group, the change in gene expression is expected to be greater or at least different, but individual patients are also expected to have different baseline levels of gene expression as well. Therefore, I'm interested the interaction of time, and patient, and not in indi…

deseq2 lfcshrink contrast

updated 5.4 years ago • jurbach

Hello everyone, I am currently working on RNA-Seq data using DESeq2. As it is in the manual, you can perform pre-filtering (e.g.: keep <- rowSums(counts(dds)) >= 10 dds <- dds[keep,] However, it's also said that: _"While it is not necessary to pre-filter low count genes before running the DESeq2 functions..."_. So, from what I gather, using this threshold (1…

deseq2 rnaseq

updated 5.4 years ago • andrebolerbarros

Hello, I have a small dataset with 3 replicates per condition. One condition is a gene over-expression, the other is a control. I'm used to run DESeq2 with betaPrior = True to have a comparability with the 'old' DESeq2 behavior. I can't apply this old workflow because the expression differences between my conditions it quite small and one gene, namely the over-expressed ![](https://ibb.…

deseq2 lfcshrink betaprior

updated 5.7 years ago • mat.lesche

better just use non-shrunken log2foldchange for all? (I'm looking forward to `` apeglm ```` ``and `` ashr ``) Thank you

deseq2 rnaseq

updated 5.9 years ago • Yuya Liang