Bioconductor Forum

I am currently attempting to assemble and annotate various regions of unstranded RNA-seq data obtained from paired-end sequencing. I am currently using StringTie for assembly and have come across the option of incorporating the IsoformSwitchAnalyzeR package with the output from StringTie for 3' UTR annotation. I would like to know if I can analyze the...I am currently attempting to assemble and a…

IsoformSwitchAnalyzeR

updated 11 months ago • Henry

I am trying to figure out how to use IsoformSwitchAnalyzeR on Salmon quantification. I'm really stuck at the very beginning. I have not been able to import the...salmonQuant <- importIsoformExpression( + parentDir = system.file("salmonQuant", package="IsoformSwitchAnalyzeR") + ) Step 1 of 3: Identifying which algorithm was used... Error in dirList[sapply(dirList, FUN = func…

isoformswitchanalyzeR rna-seq splice-variants

updated 5.7 years ago • saddamhusain77

Hi I'm having issues getting IsoformSwitchAnalyseR working. When I put library(IsoformSwitchAnalyzeR) I get the message below which I dont understand. I've restarted, updated everything, removed and reinstalled...Hi I'm having issues getting IsoformSwitchAnalyseR working. When I put library(IsoformSwitchAnalyzeR) I get the message below which I dont understand. I've restarted, updated eve…

Isoform IsoformSwitchAnalyzeR

updated 21 months ago • Craig

Hello! I am interested to run the IsoformSwitchAnalyzeR package to assess differential transcript usage and related downstream analysis. However, I have

IsoformSwitchAnalyzeR

updated 6.0 years ago • maya.kappil

Hi all, I've just started to set up a workflow in IsoformSwitchAnalyzer, version 2.1.2. I'm having trouble with the analyzePFAM() function, as it doesn't seem to accept results

IsoformSwitchAnalyzeR

updated 20 months ago • Callum

if it possible to use directly, the output of a DEXSeq analysis and pass it in to analysis for isoformswitchanalyzeR? I have looked but cant find any information on this....   If so, which step can it be picked up from in

isoformswitchanalyzer isoform differential isoform usage

updated 6.2 years ago • A

aware of the option of wasabi --> sleuth, but I was wondering if anyone has tried directly to use IsoformSwitchAnalyzeR or TSIS with transcript counts/abundance? </span> &nbsp

isoforms diffrential expression noncoding rna TSIS isoformswitchanalyzeR

updated 6.8 years ago • rbenel

I am trying to use IsoformSwitchAnalyzeR package to assess Isoform switching on mouse RNA seq data. I am running into problems on the GTF file...that has haplotype info in it. Does anyone know where I can find this or how I would proceed using IsoformSwitchAnalyzeR without the "chr_patch_hapl_scaff.gtf" file. Per my code below on the 'isoformExonAnnoation', I can

rnaseq IsoformSwitchAnalyzeR

updated 2.6 years ago • Ryan

Hi, I am using R version 4.3.1, R studio version 2023.6.1.524, isoformSwitchAnalyzeR version 2.0.1. I imported Salmon dataset via Tximeta and saved as RData. I created aSwitchList using...the number of samples and the number of model coefficients are equal, i.e., there are no replicates to estimate the dispersion. use an alternate design formula &gt

salmon IsoformSwitchAnalyzeR tximport RNASeqData

updated 19 months ago • elva.gypsophila

Hi, i am trying to perform some analysis with isoformswitchanalyzer package. i used it before and it worked properly but now when i am trying to import Pfam result file

IsoformSwitchAnalyzeR PFAM.db

updated 3.1 years ago • mekili

Hi, I have a question regarding the test of differential isoform usage in IsoformSwitchAnalyzeR. I have a large dataset that includes 28 different pairwise comparisons (I actually only need a subset

IsoformSwitchAnalyzeR

updated 3.7 years ago • jbono

Dear All, I am using IsoformSwitchAnalyzeR package to identify recurrent isoform switches in a set of 24 rectal tumor samples. The isoform...Dear All, I am using IsoformSwitchAnalyzeR package to identify recurrent isoform switches in a set of 24 rectal tumor samples. The isoform and...CRC", and the program terminated with error. This is the code which I was using - ``` library(IsoformSw…

cancer Isoform limma IsoformSwitchAnalyzeR

updated 5.4 years ago • asmita

Hi, I am using IsoformSwitchAnalyzeR v1.21.0 in R 4.3.0 environment using RStudio (2023.06.0+421 "Mountain Hydrangea" Release). When I...Hi, I am using IsoformSwitchAnalyzeR v1.21.0 in R 4.3.0 environment using RStudio (2023.06.0+421 "Mountain Hydrangea" Release). When I try

IsoformSwitchAnalyzeR

updated 20 months ago • venura

Hi, I am analyzing dtu for a complex factorial experiment with three factors where I am particularly interested in interaction effects. I see that using satuRn I could pretty easily set up the contrasts I need. However, I would ideally like to use satuRn from within IsoformSwitchAnalyzeR so I can do some of the downstream analyses offered, but I don't see a way to construct contrasts in the..…

IsoformSwitchAnalyzeR satuRn

updated 17 months ago • jbono

Hi everyone, I am using RStudio Version 1.4.1106. I wanted to install the "IsoformSwitchAnalyzeR" packages. The code I am using is as follows. I tried changing the cran, reinstalling R and Rstudio, the...Hi everyone, I am using RStudio Version 1.4.1106. I wanted to install the "IsoformSwitchAnalyzeR" packages. The code I am using is as follows. I tried changing the cran, reinstalling R and Rstudi…

BiocManager

updated 3.8 years ago • Sasha

I am trying to run some alternative splicing analysis for two biological triplicates using IsoformSwitchAnalyzeR (with a pipeline that worked before for me). But in the ImportRdata step the program keep returning...error occurred. and then 'rlang tracing' gives me this: Backtrace: ▆ 1. └─IsoformSwitchAnalyzeR::importRdata(...) 2. ├─base::suppressWarnings(...) …

dplyr IsoformSwitchAnalyzeR

updated 22 months ago • ramon.dslps

Hi, I have a quick question on DEseq2 replicates. A good number of genes were marked as outliers (912, 5.7%) in my DEseq2 summary(res) output. I am wondering what that...flags genes which contain a Cook's distance above a cutoff for samples which have 3 or more replicates....__When there are 7 or more replicates for a given sample, the DESeq function will automatically replace counts...by the s…

deseq2

updated 6.9 years ago • Xianjun Dong

div class="preformatted">Dear friends, In my experiments there are 3 technical replications and 8 biology replications without dye swap. I have processed location and scale normalization for each print...tip-groups on 3 technical replications respectively. But next step what can I deal with them? Would I process scale normalization for 3 replications...and then take average or median of the …

Normalization Normalization

updated 21.0 years ago • zhao luo

treated conditions obtained from 4 different days. This SRA dataset obtained from NCBI doesn't have replicates but have mentioned in their article that they have merged the biological replicates drawn during sequencing...sample preparation section. When I try to run DESeq, I am getting error because of the absence of replicate sample read counts. How can obtain the differential expressed genes b…

deseq2 R bioconductor RNA_Seq replicates

updated 5.2 years ago • aishu.jp

Hello everyone, I would like to know if you could help me about someting in DESeq2. In the documentation, they talk about technical replicates ad biological replicates. From what I understand: - biological replicates are for example samples that do not come from the same people/animal/cellular culture. - technical replicates are for example samples that were sequenced twice on the same o…

DESeq2

updated 3.5 years ago • leo.dagata

if anyone knew of a function within bioconductor or method to provide some sort of metric for replicate quality analysis. I have typically been using multipanel graphs (having each replicate plotted vs each other) to...get a sense of the replicate qualities. However, I have not been able to find metric that would be good at determining how good a replicate is? I...using various outlier eliminati…

updated 21.8 years ago • Park, Richard

class="preformatted">Dear Users, I have Affymetrix Data. There are 5 animals and I have 2 technical replicates. I am trying to compare these technical replicates to ensure they are clean. I was wondering if anyone could please...or other factors caused a change in the values (readings) obtained between the technical replicates. So I would like to do some data analysis to compare these and ma…

updated 20.0 years ago • Shruti Subramaniam

Hi, I have just been passed a set of affy data that consists of 3 states, two technical replicates of each state (6 chips overall) 1. whats the best way (normalisations, algorithms) to leverage technical replicates...2. how do you tell algorithms such as rma which are the replicates? (I presume the phenoData in the AffyBatch specifies this, but the examples are in a binary format so you cant open

affy affy

updated 21.1 years ago • William Kenworthy

I did DE analysis on 80 libraries with DESeq2, the number of biological replicates is 2 (40 samples). For some genes, there is a big difference between read counts for 2 replicates, for example r1:2 and...r2:2436. If the number of replicates was more than 2, these cases were removed as an outlier. How to solve it with 2 biological replicates? Thanks

deseq2

updated 4.6 years ago • f_rahmdani

Hi, I have a set of data (cut and run) with 2 samples, three replicate of each. One set of replicate are separated from the other two replicates in sample heat map while the other two set...of replicates are segregated by sample (treatment replicate 1 next treatment replicate 2 and control 1 next to control 2). My question...is how do I go about to use mask to create a dba object just containi…

DiffBind

updated 4 months ago • Haiping

I have a question about how to analyze a mix of biological and semi-technical replicates. The experiment I am analyzing consists of 3 cell lines X 3 replicates of each cell line X 2 conditions. The 3 replicates...same cell line, but independently treated, processed and sequenced, so they aren't "hard" technical replicates, but they are not biological replicates as the 3 cell lines. They show …

limma technical replicates biological replicates paired analysis

updated 5.9 years ago • paul.alto

with 6 conditions (5 serial dilutions of a single drug and 1 untreated control) with 5 biological replicates for each condition. Our study uses low drug concentrations and so we expect to see only a moderate number of DEGs...due to condition. In fact most of the variation comes from replicate (generally global gene expression increases or decreases). Our coldata has Gene ID, Count, Condition and …

DEseq2 replicate DESeq2 normalization

updated 3.2 years ago • michael.morash

been using affy, affylmGUI, and limma for the analysis. Some of the samples in the latter group have replicates and I'd like to know how to handle these. My questions are: 1) In one case, the replicate is drawn from the same sample...but at a later time point. Is this a technical replicate (because it is the same sample but different chip), biological replicate (i.e., the different time point ma…

GO affy limma affylmGUI GO affy limma affylmGUI

updated 19.7 years ago • Jonathan Arthur

using the default 1-on-1 (pairwise) comparison for unpaired samples. My samples have both biological replicates and technical replicates. Shall I treat them the same way? Woud that be a little improper? Heyi </div

gage gage

updated 14.2 years ago • heyi xiao

have three samples with a simple design formula ~condition: sampleA - control sampleB - treatment (replicate 1) sampleC - treatment (replicate 2) DESeq2 returns one fold-change for control vs. treatment, but it is possible to consider...are three samples (A,B,C) for read counts Kij. Of course if geneX had extremely low counts in replicate 1 but not in replicate 2, we would expect strong var…

DESeq2

updated 3.6 years ago • P1000

class="preformatted">Dear List, I have a question regarding the use of biological versus technical replicates in a microarray experiment: I have two groups of samples, before treatment and after treatment. For some samples...I have biological replicates for before and after treatment (bucal swabs collected with a small brush) For some patients one of my biological...replicates usually afte…

Microarray Microarray

updated 14.2 years ago • Marcos Pinho

Looking at the oncogene2013 data set, they say they used 2 cell lines, three treatments, and 4 replicates. That's 24 samples, which is the number of columns in exprs(). What's not discussed is the number of technical replicates...which I assume they must have done. Can nondetects impute a dataset that includes technical replicates? Or do you just toss the replicates that don't work an…

qPCR nondetects

updated 15 months ago • Ed Siefker

I have an RNA-seq experimental design which includes replicates that are not exactly technical replicates and not exactly biological replicates. I compare tumor samples, but...I have an RNA-seq experimental design which includes replicates that are not exactly technical replicates and not exactly biological replicates. I compare tumor samples, but the same tumor is implanted sometimes in two or…

DESeq2 RNASeq limma

updated 3.0 years ago • Eyal

Hello, Here:[ https://imgur.com/z7Fkyjv](https://imgur.com/z7Fkyjv) you can find the PCA of the experiment that I am analyzing. Cond1 e cond2 were designed to have an over expression of two different genes. The sample cond1 near to controls has an expression of that gene much less than the others biological replicates. In this case I know that in that sample something …

deseq2

updated 6.7 years ago • ribioinfo

hi it is possible to use all dexseq script without replicates? ty efrat

replicates

updated 9.5 years ago • efratdahan21

analyzing some cDNA data; in the simplest case there are a total of 6 arrays, with three biological replicates; for each biological replicate, the arrays are duplicated and arrayed using dye-swap. Of course, for some genes there...might be missing values in some of the replicates. In addition, some genes are replicated within arrays 5 times, whereas other genes are replicated twice (or three...a…

Cancer limma Cancer limma

updated 21.5 years ago • Ramon Diaz

<div class="preformatted">Dear Dr. Smyth, I have "two groups Affymetrix" experiment with 5 biological replicates in one group and 2 technical replicates in other. In other words one group has all biological replicates and the other has all technical replicates. I would really be thankful to know how to use LIMMA (to identify differentially expressed genes) for such kind of design. After r…

limma limma

updated 16.2 years ago • Garge, Nikhil

How does HTqPCR handle technical replicates? Suppose I have 4 samples, 8 primer sets, and 3 replicates on a 96 well plate. "n.features indicates the number of features...read from each file. " 8*4=32 But I have 96 lines of data? Do I have to make each technical replicate it's own feature? How then would I indicate that they are replicates if they each have their own feature nam…

HTqPCR

updated 17 months ago • Ed Siefker

Hi I want to use the DESEQ package between a control (3 biological replicates) and treatment (1 biological replicate). IN DESeq I herefore used the following code, and got 266 genes with padj &lt

DESEQ deseq2 deseq replicates biologicalreplicates

updated 9.2 years ago • wd

Hi Michael, I am having hard time with one of the replicates in a three replicate experiment. This is a control sample. But the other replicates look good on PCA plot and also...Hi Michael, I am having hard time with one of the replicates in a three replicate experiment. This is a control sample. But the other replicates look good on PCA plot and also on pairwise scatter plot comparison. Can I …

deseq2

updated 10.2 years ago • Prasad Siddavatam

with the following issue that I have been struggling with. My experiment consists of 3 biological replicates per genotype (total of 3 genotypes; thus, 9 biological replicates) per time point. I have two technical replicates...per biological replicate. In my case, technical replicates were **not** made by running the library multiple times and/or on different lines; instead...replicates are mer…

edgeR

updated 3.9 years ago • mo17

I have 4 biological replicates of mRNA samples. For my dds, I am comparing type but have also added in the replicate factor to account for the biological...replicates. Is this correct? I have a column specifying the replicate number (1, 2, 3, or 4) per sample as they do not have the same names...dds <- DESeqDataSetFromMatrix( countData = counts_data, colData = metadata, design …

DESeq2

updated 3 months ago • shreya.kanade

Hi there, For some reason, one of the replicates of my htseq-count files is not normalizing correctly when run through DEseq2. I have attempted to change settings...re-map the original .fasta file, and all of my efforts still lead to the same replicate not normalizing. I believe that it has something to do with the htseq-count values of that replicate, but I haven't

deseq2 normalization

updated 6.7 years ago • mpw183

div class="preformatted">Dear limma experts, I have direct experiments with two biological replicates and two technical replicates. In each array sots are printted in 4 replicates. In duplicateCorrelation help it...this time it is not possible to estimate correlations between duplicate spots and between technical replicates simultaneously." The question is it possible to average on both techni…

limma limma

updated 19.8 years ago • Ron Ophir

ovarian primary tumor. After reading information about the package, I am still confused by the word , "replicates". In the context of my data, each RNA sequencing sample represents a unique patient's primary tumor, and DESeq2 would...treat these as individual replicates of the primary tumor condition, correct? Therefore, I can conduct analysis on this data using this tool? I want to

DESeq2 RNASeq

updated 11 months ago • kcarey

The thing is that i need to compare 5 conditions (stages) versus "control", each condition with two replicates. My question is... can i compare all in one run or do i need to compare by pares? And... how can i manage the replicates? I started...a count matrix (with data from the htseq count) comparing one condition versus control (with the 2 replicates of each one), and this command line:&nb…

deseq2 bioconductor R

updated 6.5 years ago • anaQ

upgraded to R version 2.9.1 and CellHTS2 version 2.8.1. Using my previous version I could miss replicates from the plate list file without generating an error - for example : Filename Plate Replicate plate_1_1.txt 1 1...3 plate_3_2.txt 3 2 plate_3_3.txt 3 3 Where plate 3, replicate 1 is missing. However, with my current ver…

Cancer cellHTS2 Cancer cellHTS2

updated 15.6 years ago • Becky Saunders

Dear Marcelo, Only one of your biological replicates has technical replicates, which isn't enough for `duplicateCorrelation()` to give a reliable estimate of the technical...0300 > From: Marcelo Laia <marcelolaia at="" gmail.com=""> > Subject: [BioC] limma technical replicates and biological replicates > in the same design > To: Bioconductor <…

limma SANTA

updated 5.7 years ago • Gordon Smyth

<div class="preformatted">Hello all, I am looking for a quantitative measure of replicate quality to know if one should be tossed or not. Right now, its a gestalt from pairs and mva plots after running rma on...div class="preformatted">Hello all, I am looking for a quantitative measure of replicate quality to know if one should be tossed or not. Right now, its a gestalt from pairs and …

GO GO

updated 21.9 years ago • Luckey, John

f1_A2 f1_B1 f1_B2 f2_C1 f2_C2 f2_D1 f2_D2 Here f1 and f2 are two families A1 and A2 are technical replicate. same for B1 and B2,C1 and C2, D1 and D2. A,B,C,D are biological replicate, say A,C are affected while B,D are unaffected. Noted...have more than 1 affected or unaffected samples. How does LIMMA take the two-level (technical replicate and familial relationship) dependence into account? C…

limma limma

updated 14.8 years ago • kuangqin

Hi, I am finding the differential peaks using DiffBind, but my sample has no replicates. And my `SampleSheet` is ``` > dbObj <- dba(sampleSheet="SampleSheet.csv") trisomy_21 fibroblasts trisomy_21 trisomy_21...33153 sites in matrix (47495 total): ID Tissue Factor Condition Treatment Replicate Caller Intervals 1 trisomy_21 fibroblasts trisomy_21 trisomy_2…

DiffBind

updated 5.3 years ago • zhangdengwei

<div class="preformatted">Hi, Does anybody know how to merge replicate spots in R Bioconductor? I use home-made spotted arrays and every probe is printed in triplicate. I normalize my data...div class="preformatted">Hi, Does anybody know how to merge replicate spots in R Bioconductor? I use home-made spotted arrays and every probe is printed in triplicate. I normalize my...using limma pa…

probe limma probe limma

updated 15.8 years ago • Barbara Cegielska

by family. I also tried method like GEE to > take familial relationship by averaging technical replicates. At this point, > looks the methods like LIMMA/GEE can not? two-level/type of dependence (like > technical replicate...deleted' vs 'undeleted'. > > In your point 2, what do you mean "combine families as biological replicates > and do cases vs. control…

Cancer limma Cancer limma

updated 14.8 years ago • Steve Lianoglou

a lot about testing on datasets with batch effects, but in all cases the effect is on biological replicates and not technical replicates. Just some quick terms for a better understanding: technical replicates: same sample...and loading onto the machine I noticed two different types of variation between technical replicates: A) systematic/plane shift in PCA --> batch effect due to…

rnaseq deseq2 differential expression batch effect replicates

updated 5.4 years ago • Nik dAK

wrote: > From: Leutz Buon <lb089 at="" research.dfci.harvard.edu=""> > Subject: Biological replicates with Gage > To: "Weijun Luo" <luo_weijun at="" yahoo.com=""> > Date: Sunday, July 24, 2011, 8:01 PM > > Hi, > I'm doing a gene set...samples at different time points : > 1hr,8hr,16hr,24hr. > For example: > c1r1 (co…

gage gage

updated 13.5 years ago • Luo Weijun

<div class="preformatted">Hello Bioconductorers, I have a question regarding biological v technical replication in a continuous culture model. These are very expensive experiments to carry out so are not normally repeated to get the usual biological replication required. So the following experimental design has been suggested. Cells are grown in a continuous culture fermenter...div cla…

updated 17.7 years ago • PeadarÓGaora

Hi, For input data for WGCNA, can I input all my data including the replications for each samples, or use average of the replications for each sample? Also to have a better chance to find modules

WGCNA

updated 4.8 years ago • bahmanik@msu.edu

Hi Rory, I very well realize the importance of replicates as well as your advice in this forum multiple times to other questions earlier that no meaningful statistics...can be done without replicates. However, the data I have received to analyze has bad replicates. There are three matched pairs for two conditions...and the biologist says that they can be loosely considered as replicates, b…

DiffBind ATACseq replicate

updated 4.3 years ago • CodeAway

vs. mut, with one gene knocked out in mutant. For one of the transcription factors, I only have one replicate as the second replicate has failed. As a result, I am getting errors from diffBind. Any advice on how to proceed? Thank

diffbind chipseq

updated 5.5 years ago • danielaperry2015

Hi, I have 6 samples each with 6 replications. One replication is missing from one sample.  T1\_LD\_FW1-6 T1\_LD\_SW1-6 T2\_LD\_FW1-6 T2\_LD\_SW1-6 __T3\_LD\_FW1

deseq2

updated 8.9 years ago • John