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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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 ...
5
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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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0answers
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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31 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 ...
1
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0answers
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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0answers
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 ...
1
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0answers
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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0answers
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 ...
0
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0answers
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 ...
0
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0answers
24 views

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 ...
0
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1answer
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 ...
0
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0answers
30 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. ...
0
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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 ...
0
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1answer
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 ...
0
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0answers
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 ...