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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18 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 ...
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27 views

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,...
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24 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 ...
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23 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 ...
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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 ...
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SCRAN encountered negative size factor estimates

I am running a public 10x dataset through SCONE in which one of the normalization techniques is from SCARN ...
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88 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 ...
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39 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 ...
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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. ...
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52 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 ...
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31 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 ...
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46 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 ...
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121 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 ...
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58 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 ...
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103 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 ...
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257 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 ...
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60 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 ...
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304 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 ...
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81 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 ...
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59 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 ...
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199 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 ...
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289 views

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: ...
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291 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 ...
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60 views

Normalization of data with rpkm

I'm very i difficult with normalization of 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 ...
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59 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 ...
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148 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 ...
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239 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 ...
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3answers
2k views

How to quantile normalization on RNA seq counts

I have a read count data (RNAseq) and want to perform quantile normalization. Could you please help me how to do it. I tried some scripts in R but it didn't work. I want the result output in matrix ...
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1answer
654 views

Calculating Z-score from logCPM values using edgeR

I have the raw counts for RNA-Seq data. I converted counts data to logCPM using edgeR package. Lets say I have a dataframe A with 15000 genes as rows and 100 ...
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549 views

How to normalise scRNASeq data for differential expression analysis

I wish to perform differential expression analysis for cluster-specific gene expression in single-cell data (with a tool such as MAST or SCDE). I have data for 3 biological replicates. I performed ...
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96 views

Why are TPMs per 10k or 100k in many scRNA-seq studies?

I noticed that many scRNA-seq papers normalize TPMs to 10k or 100k as opposed to 1M (as the abbreviation defines them). It doesn't really matter since you are just moving the decimal point, so why ...
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162 views

Convert TPM-normalized matrices back to UMI in python

I want to process a plenty of scRNAseq datasets in python, and I want to run TMM ...
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2answers
732 views

Input normalization in ChIP-seq

If I subtract input counts from ChIP counts (for every gene, since I have one peak per gene) I get negative values for most genes. This makes sense to me, because (as can be seen in the figure) input ...
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1answer
859 views

Normalize ATAC-seq/Dnase-seq sequencing reads coverage signals over estimated background

I'm trying to normalize the coverage signals of ATAC-seq reads against its own background using normal distribution, described in this paper It says: Finally, all open chromatin coverage ...
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509 views

Drawbacks of upper quartile normalization for scRNA-seq data

I would like to use Upper Quartile normalization for scRNA-seq data defined as: The upperquartile (UQ) was proposed by (Bullard et al. 2010). Here each column is divided by the 75% quantile of the ...
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685 views

Order of batch effects removal, data imputation and library size normalization in scRNA-seq data

I am preprocessing scRNA-seq data. What is the best practice in use to run both ComBat for batch effects removal, data imputation (to mitigate dropout) and library size normalization? I thought that ...
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274 views

Large dataset normalization for PCA

I need to normalize a large (400Mb) dataset for doing PCA analysis. I want to use scran for doing that: ...
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1k views

Normalizing RNAseq for PCA and CCA

Usually the expression data is transformed to log space using either RPKM, FPKM or CPM, this is required when looking for differential expression because the data is tested against the normal ...
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690 views

Normalizing microarray data for clustering heat map

I wanted to generate a clustering heat map for the microarray data. This is the first time I'm working on Microarray data. I read some tutorials but have few doubts. I'm using microarray (Affymetrix ...
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3k views

How to apply upperquartile normalization on RSEM expected counts?

I see that TCGA RNASeq V2 RSEM data is normalized with upper-quartile normalization. After doing Quantification with RSEM with the samples I have, I got "genes.results" as output which has gene id, ...
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1answer
439 views

Voom function from limma package and Normalization on counts data

I know that Voom function from limma package from Bioconductor converts raw counts into log-CPM values and then Normalization is applied on that, with normalize.method argument. I would like to know ...
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62 views

Analyzing Illumina Counts

I'm pretty new to all of this--forgive me if this is a simple question. When I download illumina counts from GEO (like the supplementary file in GSE89225). Can I do comparisons directly on that file?...
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5k views

RNAseq: Z score, Intensity, and Resources

I'm very new to bioinformatics in general, and I'm trying to understand some basic concepts. I have RNAseq data, and bioinformatics people tell me that intensities cannot be compared across patients. ...
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123 views

Minfi returning incorrect beta values

UPDATE: I found the solution. I was using normalized values and GEO was using raw beta values. I'm trying to link GEOquery and minfi. Specifically I want to obtain beta values from the idat files ...
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1answer
441 views

qPCR: Why is fold change and standard deviation calculated after transformation?

I am analyzing data from a quantitative polymerase chain reaction (qPCR) using R. After cleaning the raw data, it looks something like this: ...
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717 views

How to read and interpret a gene expression quantification file?

I have a gene expression quantification file from TCGA that contains the following lines: ...
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392 views

What are some good practices to follow during EPIC DNA methylation data analysis?

I recently got some EPIC DNA methylation data and I was wondering what are some good practices to follow? I am interested in knowing about normalization and differential analysis. Thank you.
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4k views

How can I extract normalized read count values from DESeq2 results?

The results obtained by running the results command from DESeq2 contain a "baseMean" column, which I assume is the mean across samples of the normalized counts for ...
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365 views

Confirm success or failure of RNA-Seq normalization

I am working with a set of (bulk) RNA-Seq data collected across multiple runs, run at different times of the year. I have normalized my data using library size / quantile / RUV normalization, and ...
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2k views

Normalization methods with RNA-Seq ERCC spike in?

ERCC spike-in is a set of synthetic controls developed for RNA-Seq. I'm interested in using it to normalize my RNA-Seq samples. In particular, I'd like to use the spike-ins to remove technical bias ...