Bioconductor Forum

I am working on expression classifiers for leukemic subtypes using Affymetrix Plus2 arrays. The training data consists of several batches. The developed classifier will be used to predict the subtype of new sets of samples...as well as single samples. So far, I co- normalized new arrays with the training set, but this is not ideal. I have read the frma paper by McCall et al, and it seems the per…

Normalization frma Normalization frma

updated 12.3 years ago • Guest User

analysis between different cell types, where I have four different cell types from two different experiments. Two of the cell types come from one experiment and four cell types come from the other. I have a total of 18 samples...I have also detected that there is a batch effect in the samples coming from the second experiment. How would I do a differential expression analysis when I have...cellTy…

edger batch effect differential expression

updated 9.2 years ago • sezimmer

Dear all, <span style="line-height:1.6">I've read some papers and vignettes related to calculate the sample size for RNAseq experiments. </span> Iv'e also tried to use some tools as...nbsp;   S4   N?    N?    N?    N?    N? (sample size for each group)   <span style="line-height…

rnaseqpower sspa sample size sequencing complex-design

updated 9.8 years ago • roser.navarro

design in which there are 3 conditions (control and 2 experimental). I need to estimate sample size based on desired power and FDR but I've been reading only SSPA allows for more than 2 groups. However, the example provided...in the original paper only contemplates a microarray experiment with 3 conditions and is pilot-data based, data which I don't have. Has anyone...used SSPA for 3-group sam…

SSPA ssizeRNA RnaSeqSampleSize

updated 2.3 years ago • Megan

Hi, I am currently working with MLInterfaces and have a question regarding the optimization of hyperparameters of the learning functions (that are given to MLearn by the .methods parameter). Is it somehow possible to do...optimization of hyperparameters, e.g the k in knn? Or in other words is it intended to have hyperparameter optimization included in MLInterfaces

MLInterfaces MLInterfaces

updated 12.7 years ago • Guest User

in response to temperature stress, where we replicated temperature treatments across two types of experiments (specifically, in culture and in symbiosis). These separate experiments were both sequenced using TagSeq, but...were sequenced at different facilities at different times. The result of both the distinct experiments and using different sequencing facilities is large size factor differences…

DESeq2

updated 4.4 years ago • Hannah

NOT be used before running association testing (lmFit); association testing should be run with batch as a covariate on original data.</span>" I have read the paper from Combat authors where they mentioned that Combat performs...better than SVD when sample size is small and comparably similar for large sample size. Now, my question is since my sample size is large and I am using limma...Ag…

combat sva limma

updated 9.7 years ago • AST

of magnitude difference in total counts between the two sample types, plus variation in library size within each sequencing type (table below). I know that DESeq2 can handle random variation in library sizes, but does this...default methods in order to better handle this situation? My experiment design is below, as is my info table (with an extra column showing the total counts in each sample)…

deseq2

updated 4.7 years ago • cja

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updated 17.1 years ago • sue@xlsolutions-corp.com

Hello, I am currently trying to do a sample size estimation for an RNAseq experiment I am planning, using ssizeRNA package in R. This package uses average read counts...and dispersion, proportion of DEGs and total genes mapped to estimate sample size based on power. Here is a link to the vignette (https://cran.r-project.org/web/packages/ssizeRNA/vignettes/ssizeRNA.pdf...per group (400+) . I am no…

ssize

updated 2.4 years ago • Nana

equipment; gas chromatography instruments). I have a degree in abstract mathematics, and some experience with probability theory on Wall Street. Bioinformatics is a very attractive field, but I need training. For example...I would work for free for a while in exchange for on-the-job experience. Or, I would take an intensive training course if one were available around the Boston area. Would you p…

updated 22.6 years ago • Ellis D. Cooper, Ph.D.

75 bp reads at ~32 million per sample.  We are following closely the EdgeR approach to finding batch effects given in the June 2016 version of the manual, including the TMM normalization.   The MDS plot shows a...batch effect pretty well.  We can get a generous number of DE genes at the end.  I am concerned that having only two reps...makes a test for,…

edger rnaseq batch effect read length

updated 9.1 years ago • Spollen, William G.

Hi, all I am a newbie to EdgeR. Now, I am following the introduction to EdgeR, and I need the training data set of Zhang, W et al. 1997. The link in the original paper doesn't work any more. was anyone able to locate this data

edgeR edgeR

updated 14.8 years ago • shaohua.fan

time Batch 1 GSM795538 irradiated_ctrl 4h 1 2 GSM795539 irradiated_ctrl 4h 2 3 GSM795540 irradiated_ctrl 4h 3 4 GSM795541 irradiated...targets$Sample.and.Data.Relationship.Format, ".", targets$time)),col= as.numeric(as.factor(targets$Batch)))</pre> </td> </tr> <tr>…

limma illumina microarray multifactorial design batch effect

updated 9.5 years ago • Konstantinos Yeles

3 MUT samples and 3 drug treated samples ``` WT1 - 12/14/2022 WT2 - 12/14/2022 WT3 - 11/03/2021 MUT1 - 11/03/2021 MUT2 - 11/03/2021 MUT3 - 11/03/2021 DRUG1 - 11/03/2021 DRUG2 - 12/03/2021 DRUG3 - 11/03/2021 ``` How can I make my sample info...so as to include the batch column. Right now this is how my sample info looks like: ``` Sample condition mut_rep1 mut mut_rep2 mut mut_rep3…

DESeq2

updated 3.3 years ago • gv

Hi, I am analyzing RNAseq data of two experiments with multiple time points and their own control condition. These two experiments were sequenced in two separate...runs. I wonder if I can merged the two raw counts tables and normalize them using the DESeq2 size factors method, or do I need to normalized each table separately with DESeq2

RNAseq normalization deseq2

updated 7.7 years ago • uguy

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Visualization PROcess Category Visualization PROcess Category

updated 22.9 years ago • rgentleman

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updated 20.6 years ago • christopher leeds JIC

This is more like a sanity check question for experienced users, because I don't have a lot of experience on differentially expressed genes analysis. I am trying to perform DEGA for a large experiment with 80 samples...in 10 conditions (not 8x10 unfortunately but each condition has a random sample size, min=2). I started with sample QCs, and just out of curiosity checked the library sizes before …

DESeq2

updated 21 months ago • HAK

of strains with mutation A and (B) containing a number of strain with mutation B. We performed two experiments of cell-model infection. The 1st experiment was done with half of strains from A and half of strains from B. The 2nd...experiment was performed later with the rest of strains from A and B. Both experiments used the same cell line and included...uninfected cells. RNA from both experiment…

BatchQC BatchEffect RNASeq

updated 18 months ago • ttt

concept clinical study (initially there were more patients involved, but at the end of the experiment only 10 valid pairs left). Things would be easy for simple paired design experiment, but 4 of the patients...all samples are mixed, except sample 3 pair stands far apart. And I also don't see any clear batch effect between 'baseline' and 'placebo' groups. Below…

deseq2 edger limma batch effect paired design

updated 7.7 years ago • Vladimir Krasikov

to my post completely. I am using scater/scran package for scRNA-Seq analysis and MNN for batch correction. As I know with multiple datasets; MNN integrates first and second datasets first and integrate the third...I want to do something like this if possible: Let's say I have 3 different scRNA-Seq (first experiment set) data and will have 2 more (second experiment set) in a month. I use…

scater singlecell scran BatchEffect

updated 5.1 years ago • hamza_karakurt

Hi, I am following a DESeq2 tutorial (see link below) and I have a question about setting up the experiment table and design formula.  https://www.bioconductor.org/help/course-materials/2015/LearnBioconductorFeb2015...3 groups (control, knockout\_a, knockout\_ab), and each group has 4 replicates.  Here is the experiment design table that I created: <table border="1" cellp…

rnaseq differential gene expression deseq2 r

updated 8.7 years ago • greenhawmatt

This appears to be a randomized complete block design. The way I would compute the sample size is: Use a routine that computes sample size for a randomized complete block design. If you are planning to use log2 expression...then enter "1" as the size of the difference you want to detect. That corresponds to 2-fold. Using someone else's data for the same experiment, compute...each gene (e.g. us…

Microarray Microarray

updated 16.6 years ago • Naomi Altman

Hi I want to perform a differential expression on RNA-Seq data. During the data exploration, the PCA plot indicated that a batch effect was present (plot not shown). I obtained additional information about the experiment and indeed, the samples were processed by two different persons. The metadata of the experiment is shown in this table: ``` sample treatment lab_tech 1 sample1 cont…

linear model batch effect DESeq2 design

updated 5.3 years ago • tim.meese

Also, we preforem the ATAC seq between the three groups. How we could calculate the sample size for ATAC seq and single cell RNA seq, respectively? Notably, we have found a paper titled "SCOPIT: sample size calculations...for single-cell sequencing experiments" . But it provide a procedure for prospectively planning single-cell experiments or retrospectively evaluating...of cells have been …

singlecellRNAseq ATACseq sample-size

updated 3.0 years ago • gogniu

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openPrimeR

updated 2.2 years ago • WebAsha

<div class="preformatted">Dear all, Currently£¬we are working with Affymetrix u133plus2 array and interested in classification disease/control using microarray data. We have identified a molecular signature and trained a svm-based classification model based on 200 samples (100 disease +100 control). We are satisfied with the model performance (sensibility/specificity) evaluated with cross…

Microarray Classification PROcess Microarray Classification PROcess

updated 16.0 years ago • qinghua.XU@as.biomerieux.com

Hello Everyone, I'm currently analyzing a series of RNA-Seq experiments that are splitted into 4 runs. In order to account for the batch effect of each experiments, I'm trying to correct...RNASeq workflow (http://www.bioconductor.org/help/workflows/rnaseqGene/\#removing-hidden-batch-effects) As I proceed in this phase, I got very confused regarding the methods and the procedures, so I wonder if…

sva deseq2 batch effect

updated 9.7 years ago • wariobrega

vs mutant. I am using a likelihood ratio test in DESeq2 using the following code. From a DESeq2 training page, the log2 fold change is printed in the results table for consistency with other results table outputs, but is...genes. Seeing as I cannot use the log2 fold change from the DESeq results to measure effect size, is there any other tool I can use in DESeq2 to quantify effect size of these…

DESeq2

updated 4.2 years ago • jd348

somebody would be able to clarify something for me regarding variance stabilising transformation and batch correction and subsequently extracting a matrix of batch corrected VST counts. I have run an experiment as follows...DESeq(dds, reduced = ~Organ+Extraction, test = "LRT", parallel = TRUE) Extraction being the batch (for this run, only two batches, 1 and 2). And i only want to see DE g…

deseq2 batchcorrection

updated 6.2 years ago • A

sva.html This version includes support for both surrogate variable analysis, as described in the papers: http://www.biostat.jhsph.edu/~jleek/papers/sva.pdf http://www.biostat.jhsph.edu/~jleek/papers/framework.pdf and...for Combat, an approach for removing batch effects when the source of batch is known as described in the paper: http://biostatistics.oxfordjournals.org/content...description of…

Regression limma sva Regression limma sva

updated 14.0 years ago • Jeff Leek

coverage between conditions etc.) will be applied to these windows, if ws =100. I've seen a paper (PMC4213696) apart from MEDIPS's original paper that researchers are using ws = 100 only.  My question is what is the...criteria for selecting window size? What if I increase the window size ? I guess the values such as 'counts', 'logFC' , 'rpkm' & eventually 'p-value' will cha…

medips medip-seq peak finding luckas

updated 8.6 years ago • anupriyaverma1408

div class="preformatted">Dear list, I have the following affymetrix microarray experiment: 2 fixed effects, each factor has two levels 1 random effect (patient) Can anybody tell me how to calculate the sample...size for it? Thanks, Shirley </div

Microarray Microarray

updated 16.6 years ago • shirley zhang

Due to experimental limitations, the samples have been treated in the same way but in 15 different batches. Each batch includes 8-40 different donors each, and 2 donors are present in every batch (donor A and B), while others are...present in a variety of batches (some in 2, some in 3 or 4). So the majority of donors are present only in one batch, and there is not a clear baseline. Each donor...h…

limma RNASeq DifferentialExpression

updated 21 months ago • mperalc

previously here: https://support.bioconductor.org/p/66067/ The way to access the normalized count table in DESeq2 can be done by the following script lines: dds <- estimateSizeFactors(dds) counts(dds, normalized=TRUE) I'm trying...to understand if the normalized count table that can extracted using the above is taking into consideration a batch effect that is modeled in the design …

deseq2 edger normalization

updated 6.0 years ago • Matan G.

We have an experiment where samples were collected and sequenced in several batch (matched ATACseq and RNAseq). We would like to include...of the batches have only one sample in them (we have several batches with many samples in and a few with only one). We have used DESeq2...and would also like to do more downstream analysis (such as clustering etc). We have included the batch in our design for…

batch effect deseq2 limma

updated 7.6 years ago • i.sudbery

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EDIRquery

updated 2.5 years ago • webasha23

div class="preformatted"> Dear All, at Texas A&M we are seeking candidates for our two-year training program in Bioinformatics, Biostatistics and the Biology of Nutrition and Cancer, starting July 2006. Please see...text attachment was scrubbed... Name: Bio-Ad_TrainingProgram2_2005-2006.pdf Type: application/pdf Size: 28245 bytes Desc: Url : https://stat.ethz.ch/pipermail/bioconducto…

updated 20.1 years ago • Marina Vannucci

the reason for this email, is the ability to upload a 5-10 minute video describing your scientific paper. For one of our first examples see http://www.scivee.tv/node/53. We cannot tell you what will be the long term impact of making...for the short term (see http://www.scivee.tv/worth_it) indicates that it renews interest in your paper. It also adds a human element to the work which can be parti…

updated 18.2 years ago • Phil Bourne

The raw counts data were in a dataframe `counts`. `coldata` looks like below: Samples Group Experiment Sample1 Control EXP1 Sample2 Control EXP1 Sample3 Control EXP2 Sample4 Control EXP2 Sample5 FOXCUT Overexpression...counts,group = group) ## Filtering keep <- ro…

edger r batcheffect clustering removeBatchEffect

updated 6.6 years ago • Biologist

<div class="preformatted"> Qunyuan Zhang writes.... > Hi, > > We just finished an initial inverstigation (50000-gene Affymetrix, 15 > cancered people and 10 normal people). 40 genes' RNA expressional levels > were found significantly different between the two groups (by two sample > t tests, p values corrected). We are now planning a second-stage &…

updated 19.9 years ago • Kort, Eric

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clusterSeq

updated 5 weeks ago • Meena

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splots CopyNumberVariationData

updated 2.3 years ago • zarahale

Re-attached in plots in PNG format. I am analysing Illumina micro-array data and seem to have batch effects (plots attached) in my data. For batch effect removal I am using Combat from 'sva' package. This is my sample info file...in any of the batch For ex. P4 is in batch 1 and never repeated again. I have few questions about implementation of ComBat. 1. Which column should...lt;- read.table('P…

updated 12.5 years ago • Bade

Hello, I have 5 experiments on bees, each experiments is done a new bees - never the same bee but always the same treatment at different doses...4 I am trying to figure out the best way to compare the effect of treatment vs control. All the experiments had same RNA extraction, libraries preparation, sequencing technology, cleaning, alignment and reads count...procedure. For exp 1 (which…

DGEs edgeR RNASeqData

updated 13 months ago • Philippine

generalizing. I have 36 iPSC, 9 primary CM, and 18 biopsy samples. My quality control plots showed batch effects in the data and I added 3 batches to the design matrix, using, e.g. this: https://support.bioconductor.org/p/69328...to be able to estimate more things than your experiment contains information about.  In this case, your batches are entirely confou…

limma batch effect

updated 9.7 years ago • smt8n

have 9 technical reps, so 54 total samples. I have a co-variate of number of mutations and a date of experiment of batch effect. The size of the gene expression matrix = 5000 genes by 54 samples. If I run Limma lmFit eBayes...and treatment in the design, not sure if this right? \# known batch = date of experiment \# known covariate is the number mutations \# want to preserve treatmen…

Limma removebatcheffect() duplicatecorrelation

updated 9.2 years ago • John

4 time points (1, 2, 3, and 4). For unavoidable reasons, samples were prepped in several different batches; however, all samples from each patient (all time points) are in the same batch (i.e. no patient has samples in more than...one batch). The following is the format of the sample metadata: <pre> PatientID SampleID Time Disease Batch p1 p1-1 1 A …

limma rnaseq batcheffect

updated 10.5 years ago • cfreije

close this forum. However, if not, here is my questions. So I have performed 2 different RNA seq experiments in the same cell lines. However, these 2 experiments were done in different time and also with different conditions...treatment, concentration, etc). In the 2nd experiment (which was done latter), I included several identical samples to the first experiment as the batch control. The probl…

deseq2 normalization probe

updated 5.7 years ago • l.s.wijaya

div class="preformatted">Dear all, I am now working on some time-course experiments and I have applied to them some classical statistic methods to identify genes that change their expression...between time points. However I have read few papers (such as Peddada et al. Gene selection and clustering for time-course and dose?response microarray experiments using...my background (I am biologist) …

Microarray Clustering Microarray Clustering

updated 22.2 years ago • edoardo missiaglia

Hi, I have raw read counts of 57 patients ranked with Mandard score as TRG12 and TRG45. Mandard score : **A histological assessment of the response of oesophageal cancers to neoadjuvant treatment (cisplatin chemotherapy and radiotherapy in the original paper). The tumour regression grades were defined as follows: TRG 1 Complete regression - absence of residual cancer and fibrosis extendin…

DESeq2 Edgeseq batch

updated 6.9 years ago • Fereshteh

I'm just asking here whether doing batch correction in a design with 3 batches where one of the batches only contains one sample is possible. From this post https...so I'm just asking for clarity whether the sample is removed or kept in the analysis. This is the experiment design ``` BATCH SEX RESPONSE 1 F A 1 F A 2 F A 2 F A 2 F A 2 M A 1 F B 1 M B 3 M B 2 F B 2 M B 2 M B 2 F …

DESeq2

updated 2.0 years ago • Tom

Hi! While re-analyzing GSE65106 from GEO, I have constructed a table containing a subset of samples, as follows: > a # see below for dput(a) accession cond donor batch pair A1.r1 GSM1587362...2 ASC2.r2 GSM1587377 Normal NN3 4a 2 Here, r1/r2 are the replications of experiment on the same subject, Rows named A1*\A2* are autistic (disea…

limma paired batch

updated 6.2 years ago • english.server

div class="preformatted">Hello, I'm a NIH scientists that is doing a HTS experiment and I'm using your Cell HTS2 software to analyze our results. I'm doing two different experiments with 2 repeats...for each of them. I want to combine the results and thought to try the batch function. Currently, I'm doing per plate normalization and no variance adjustment. I would like to do batch variance adj…

Normalization Normalization

updated 16.1 years ago • Baranes-Bachar, Keren NIH/NCI [V]

Hi I am trying to batch correct for samples from 4 different experiments that I ran in one fluidigm plate. I tried normalization with liima...using ``` norm_exprs <- limma::normalizeBetweenArrays(exprs, method="quantile") ``` followed by ``` batch <- factor(data$Experiment) # Perform ComBat batch correction modcombat <- model.matrix(~ Group, data=data) combat_data...lt;- sv…

sva limma

updated 2.4 years ago • Khushbu

Dear all, I'm analyzing a large number of microarray treatment experiments. I have modeled the expression level as a dependent variable of the continuous independent variables time...Dear all, I'm analyzing a large number of microarray treatment experiments. I have modeled the expression level as a dependent variable of the continuous independent variables time and...time + dose + time:dose<…

limma microarray effect size

updated 10.2 years ago • Moritz E. Beber

to do so. We generated RNA-seq data from a series of gene knockdowns in an insect cell line: <table border="0" cellpadding="0" cellspacing="0" style="width:260pt"> <tbody> <tr> <td>Sample</td> <td>Rep</td> <td>Library</td> <td>Batch</td> </tr> <tr> <td...td> <td>B</td> </tr> <tr> <td&g…

DESeq2

updated 11.1 years ago • mchamber0

Dear all, I am having a problem with removing/accounting for the batch effect in my RNAseq experiment using DESeq2. Initially we did 1 big time-course experiment in one batch of human cells...against 0 using contrasts. After getting some initial results, we decided to perform another experiment at 3h with a different fungus (lets call it 3D) to compare the difference between 0vs3 and 3vs…

deseq2 batch_effect

updated 6.7 years ago • gtechbio

In RNAseq experiments, batch effects are very strong. Here two situations I have observed often: 1. The same cell line is used to replicate...an experiment and results between two experiments are quite different (see case1 plot). 2. Two groups of patients (in this case three...their sequences exhibit a quite different profile and they cluster exactly according to sequencing batch. What to do w…

deseq2

updated 7.7 years ago • jovel_juan