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Questions tagged [normalization]

The tricky art of scaling quantitative data across libraries, typically to account for differences in sequencing depth. This can also be about scaling for read source length, like transcript or gene length, in order to enable comparisons across genes.

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Find most abundant transcript isoform of a gene across different tissues in long read RNA-Seq data

I want to understand which is the most abundant variant of a gene of interest across different tissues by looking at different publicly available databases (mouse/human). I have deduced from ...
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Inconsistent microarray expression levels after normalizing with log2

I know it is necessary to normalize the GEO microassay data before analysing. Howerver, after using log2 to normalize the data and checking with boxplot, I found the data is still not align in the ...
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When analysing microarray data, is it need to do normalization and standardization both?

When we do differential expression analysis, it is common to normalize the data using a log transform: exprs(gse) <- log2(exprs(gse)) This ensures that the ...
Bioinfotec's user avatar
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How to normalise Bulk RNA-seq data to account for transcript length, coverage depth, library size and batch effects?

I'm currently working on a project where I'm comparing aggregate measurements (mean, median, etc.) of expression data (RNA-seq) from different groups of genes across various samples with different ...
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DESeq2 throwing error while normalizing raw microarray expression data due to presence of negative values

This question was also asked on Biostars I am trying to download and analyze a miRNA expression dataset from NCBI GEO (GSE25631). I specifically want non-normalized ...
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Normalization and Merging of Gene Expression Panels with Overlapping Genes from Same Cohort

I have done some gene expression work using two Nanostring panels (glial profiling and neuroinflammation) on the same 18 tissue samples. There are some overlapping genes between the panels, about 150 ...
Carolyn's user avatar
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Using Multi-Dimensional Scaling (MDS) to produce a vector in order to account for patient bias when constructing DGE lists from RNA-seq datasets in R?

I am currently working on my PhD and as part of my thesis, I intend to analyse gene expression within multiple sclerosis (MS) lesions by looking at RNA-seq datasets on Gene Expression Omnibus (https://...
R_Cres_01's user avatar
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1 answer
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Error while using SummarizedExperiment() in R

I'm tryig to perform RKM normalization on raw counts for RNA-Seq Data: ...
Félix Agosto's user avatar
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PCA of methylation-values normalized by coverage

I have started analyzing methylation (EM-seq) data for the first time ~0.8M positions. In this data set I have 27 samples of 20 patients. I want to perform a PCA of the dataset to check for possible ...
llrs's user avatar
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257 views

plotting gene expression after EdgeR DE analysis using RUVg (RUVseq) covariates

I have used the empirical RUVg method (from RUVseq) to estimate the unwanted variation of my dataset (consisting of several public datasets analysed together, with controls and case samples in ...
FrAoJm's user avatar
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1 answer
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What is the difference between Normalized Expression in EdgeR vs DESeq2?

I am trying to access the normalized expression in both edgeR and DESeq2, yet the results are different. Does anyone know why? How to get normalized expression using edgeR: ...
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Normalization methods to combine scRNA-seq experiments with different sequencing depths

I am training a classifier to identify a cell type in a particular state of activity using scRNA-seq. There is a large variation in the sequencing depth (reads average per cell) of the testing data (...
Angus Campbell's user avatar
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A question on Homer normalization

When using annotatePeaks.pl script from Homer software to create histograms, the output is normalized per bp per peak (on top of normalization to 10 million tags). What does it mean to normalize per ...
Sam's user avatar
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What is the correct order of flooring-normalization-batch correction for microarrays?

This question was also asked on Biostars I am trying to learn and understand the correct order of data processing steps for microarrays. I have data which already was analyzed by a researcher using ...
Sam's user avatar
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Normalization methods for single cell RNA sequencing that take read count into account

I have two RNA-seq datasets. One was sequenced at an average read count of 1.5 million per cell the other at 43K average reads per cell. For the first I also have the meta data from reads alligned ...
Angus Campbell's user avatar
2 votes
1 answer
159 views

What statistics can I use on qPCR data composed of several runs if all normality tests fail on the dataset?

I have >100 qPCR experiments that I'd like to analyze together, each containing the same set of genes (10 genes of interest and 2 reference genes). I have four different samples (untreated & 3 ...
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How to perform a meta-analysis using data consisting of paired-end and single-end reads generated from Illumina and Ion Torrent?

So basically I have RNA-seq reads that were generated from Illumina and Ion Torrent platforms for yeast species. I have seen an article where they compared liver cells of a rat that were sequenced ...
Justin1609's user avatar
3 votes
2 answers
320 views

Calculating most abundant transcript from RNA-Seq data

vcf2maf uses VEP to annotate variants, and I believe selects the default Ensembl transcript to use for annotation. Sometimes the ...
Tomas Bencomo's user avatar
1 vote
2 answers
417 views

Problems about value log2(IP/Input) less than zero in ChIP-seq?

I have a question about the normalization for ChIP-seq. I used CPM to normalize my bam files of each IP and Input. Then I calculate the coverage of gene bodies for all genes on the genome. I have WT ...
Lingling Yang's user avatar
1 vote
5 answers
874 views

Normalize RNA seq data from multiple runs for expression analysis

I have RNA samples sequenced with TruSeq Stranded Total RNA kit protocol in Illumina HiSeq (2x125bp) and NovaSeq platforms (2x150bp) - almost 100 samples altogether. I have to use the samples data for ...
Praveen's user avatar
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1 answer
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Doubt about using TPM for statistics

We designed an experiment to explore the potential role of carbon dioxide on algae physiology using RNA-Seq. We analyse the differential gene expression using DESeq2 but now we are interested into ...
Manuel Sánchez Mendoza's user avatar
1 vote
2 answers
89 views

Bulk RNA-Seq Read Length Normalization across different samples

I have 20 samples out of which 14 are 100 bp in length and 6 are 150 bp. Is there a way to normalize the read length for cross-sample differential expression comparison? What would be the best way to ...
so_close_yet's user avatar
0 votes
1 answer
79 views

Differential expression analysis for a subset of transcripts

I want to check the differential expression of a specific class of transcripts (say, long non-coding RNAs) using DESeq2. Now, I know that the normalization step takes into account the total number of ...
Hart Radev's user avatar
1 vote
0 answers
1k views

Why is there a minus in the 2ˆ(–delta delta CT) method (qPCR)

Question: Why is there a minus in the 2ˆ(–delta delta CT) formula? My line of thoughs: Consider Ct values of some pPCR ...
Paul's user avatar
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213 views

GWAS phenotype data format and preprocessing

I have a set of different phenotypes which I want to use for a GWAS analysis (general linear model). I have a couple of questions and uncertainty about the phenotype data input. I have control and ...
snowflake's user avatar
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1 answer
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if I know the number of sequencing circles can I give this information to DESeq2?

I am trying to understand library normalization in DESeq2. I would like to ask the following: I know that some samples have been run 15 cycles and some others 20, can I give this information to DESeq2,...
marilu's user avatar
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2 votes
1 answer
945 views

median normalization for proteomics

I am using the data from a proteomics study were the data was log2 transformed and then a median normalization was applied. The data was normalized by groups of conditions (normal, mutant), not for ...
Mee's user avatar
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1 vote
1 answer
48 views

What is a good rule of thumb for the threshold of noise versus signal for RPK in RNA seq?

I have RPK values (RNA seq) and I'm wondering what is a good rule of thumb for what is considered to be noise versus what is considered to be signal? I.e what should I choose as a threshold value for ...
An Ignorant Wanderer's user avatar
2 votes
0 answers
146 views

scRNA-Seq: Account for sequencing depth and gene length?

Task: Normalize a single-cell RNA-Seq dataset to account for sequencing depth and gene-length. For UMI-count based protocols (like 10x) that don't suffer from gene-length biases, there are various ...
Bluescreen's user avatar
1 vote
2 answers
557 views

How to create a list of differentially expressed (DE) genes after normalization with RUVSeq?

I am using edgeR to perform differential expression (DE) analysis on a set of RNA-seq data samples (2 controls; 8 treatments). To correct for batch effects, I am using RUVSeq. I am able to get a list ...
Gawain's user avatar
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3 answers
162 views

What is a good RNA seq normalization method that allows for across sample comparisons and between transcripts

What is a good RNA seq normalization method that allows for across sample comparisons, and allows between transcripts comparisons as well? I read that TMM for example allows across sample comparisons ...
An Ignorant Wanderer's user avatar
1 vote
0 answers
62 views

Regarding RNA seq data analysis and building coexpression network

I have some questions regarding RNA seq analysis if you can suggest anything it will help me a lot. I am currently normalizing RNA seq data for comparing genes expression within and between samples. ...
Citu's user avatar
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1 vote
1 answer
763 views

What unit (TPM, FPKM/RPKM, or other) to use when working across samples

I have raw gene read counts and would like to perform an analysis across multiple samples. I've found conflicting info online on how this should be done. One commonality however is that FPKM/RPKM ...
An Ignorant Wanderer's user avatar
1 vote
0 answers
203 views

apply TMM on counts imported from salmon using Tximeta

I used Tximeta to import a summarisedExperiment from the salmon output (used with genocide transcriptome v34). I need to produce 4 matrix of counts: - tx counts in TPM - gene counts in TPM - tx counts ...
FrAoJm's user avatar
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-1 votes
1 answer
62 views

How are these definitions related to differential expression?

I have two groups of patients; for each patient I have an output file (RNA-seq) contains this information ...
Zizogolu's user avatar
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1 vote
3 answers
592 views

RNASeq: Normalization, stabilization, gene length and rlog

I was thinking about the best method for normalization, which takes gene length into account (in order to compare genes)... Do you think I can do that? : - taking raw counts and dividing each gene by ...
Nin00's user avatar
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1 vote
2 answers
175 views

Normal score transformation

This question is related to a normal score transformation which is performed in the paper by RUAN, Quansong, et al. Local similarity analysis reveals unique associations among marine bacterioplankton ...
FeRex's user avatar
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1 vote
0 answers
411 views

Should I use log2-CPM values (voom-limma) as input for my model?

We have created a model to integrate several OMICs data, but we realized that the maximum TPM values of RNA-Seq data were so big that had unexpected effects on our results. We hypothesized that this ...
Hart Radev's user avatar
1 vote
1 answer
590 views

Is re-normalization of RNAseq data recommended for analysis of gene subsets?

I downloaded an RNAseq dataset from TCGA database in 3 formats: 1) HTSeq counts; 2) FPKM; 3) FPQM-upper quartile normalized. The complete dataset contains ~60,000 genes. All of my analysis will focus ...
Marius's user avatar
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2 votes
1 answer
226 views

RPGC normalisation creates artefacts at centromere

I am studying ChIP-Seq data in HeLa cells and I've started using the RPGC normalisation of deepTool's bamCoverage. MACS2 also uses this normalisation for its peak calling. I am seeing a large number ...
Whitehot's user avatar
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1 vote
1 answer
1k views

Is it okay to use deeptools bamCompare (SES normalization) for comparisons across different ATAC-Seq datasets?

We are trying to use deeptools for analysis of ATAC Seq datasets. We have datasets with different sequencing depths and are wondering if bamCompare's SES based normalization is appropriate for ...
zerotimer's user avatar
0 votes
2 answers
144 views

In-sample and across samples normalized expression

I want to get the expression data that is in-sample normalized like FPKM and also across samples normalized as obtained using DESeq2 or else. What I am currently doing is that I first normalize the ...
Rajinder's user avatar
0 votes
1 answer
228 views

RNA-seq: How to get new expression count after normalization

I've RNA seq, Human, Paired-end data, Sample size is <40. These are aligned using STAR, RSEM processed. With RSEM I've TPM and expected counts, that is two files columns as individual IDs and row ...
Death Metal's user avatar
1 vote
0 answers
352 views

Normalization of single cell RNASeq data with ERCC spike-ins

I wish to normalize a scRNASeq dataset with respect to ERCC spike-ins, where "for some of the samples, ERCC spike-in RNA was added to the lysis buffer" and was wondering how to do so? I have seen ...
h3ab74's user avatar
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1 answer
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Cluster is split in 2-3 locations on tsne plot - Suerat

I am running a single cell dataset (count data - exon) through Seurat. After running tsne I see a cluster (13) split in 3 different locations on the plot. Here are the commands I am running: ...
user2998764's user avatar
1 vote
0 answers
434 views

What is the formula for Mg values in TMM normalization for RNA Seq data?

I am reading through the paper "A scaling normalization method for differential expression analysis of RNA-seq data" by Mark D Robinson, Alicia Oshlack, available here. In this paper they introduce a ...
Brady Gilg's user avatar
1 vote
1 answer
112 views

Normalization of data with RPkM

I'm having difficulty normalizing my data. I was searching for transposable elements in my genome, and after this step, I made counts of reads in some transcripts. I produced something like this: <...
jonny's user avatar
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-1 votes
1 answer
80 views

How I normalize these two sets of data

I have average log fold change for a cluster of cells versus another cluster of cells by Seurat like below ...
Zizogolu's user avatar
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1 vote
1 answer
225 views

Discordance in gene signature behavior between bulk and single-cell RNASeq

The objective of the following analysis is to identify an activation signature of a specific phenotype on bulk RNASeq and to apply it to single-cell RNA-Seq, in order to identify the population of ...
gc5's user avatar
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3 votes
3 answers
385 views

Normalization for two bulk RNA-Seq samples to enable reliable fold-change estimation between genes

I have two bulk RNA-Seq samples, already tpm-normalized. I would like to know what is a reasonable normalization procedure to enable downstream log fold-change estimation. The distribution of the ...
gc5's user avatar
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