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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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1answer
14 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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1answer
41 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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2answers
27 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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1answer
30 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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0answers
75 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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1answer
57 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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0answers
79 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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0answers
19 views

Normalization for microbiome 16s sequence analysis

The way I understand things, normalization (such as in DeSeq2, EdgeR, etc.) serves two purposes: 1) Model the "real" abundance in the original samples from the read counts, 2) Make the abundance ...
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0answers
51 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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1answer
53 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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1answer
107 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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3answers
144 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
683 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
199 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 ...
5
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1answer
225 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 ...
5
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0answers
46 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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1answer
79 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
409 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 ...
2
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1answer
705 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 ...
5
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1answer
302 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 ...
5
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1answer
357 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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1answer
222 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: ...
5
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1answer
715 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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2answers
408 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 ...
8
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1answer
2k 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, ...
5
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0answers
353 views

Voom function from limma package and Normalization on counts data

I know that Voom function from limma package converts raw counts into log-CPM values and then Normalization is applied on that, with normalize.method argument. But I would like to know clearly how ...
4
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1answer
56 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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2answers
3k 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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0answers
94 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
339 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: ...
11
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1answer
512 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: ...
5
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1answer
320 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.
13
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2answers
1k 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 ...
17
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2answers
275 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 ...
13
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2answers
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 ...