Questions tagged [rna-seq]

Questions should include this tag if they pertain to issues related to bioinformatics analysis of RNA-seq data, e.g. normalization, differential expression analysis, sequencing and experimental design or transcriptome assembly...

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1answer
320 views

WGCNA: Problem with selecting soft threshold

I have 25 tumor samples with counts data. Initially, I filtered out low expressed genes and then converted counts to varianceStabilizingTransformation using ...
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2answers
138 views

Differential gene expression bias due to effect of an individual sample

I am analysing a human single cell RNA seq experiment, where we have 4 groups, four samples each. Data has been analysed using Seurat, with the canonical workflow. I have tried DE using various ...
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2answers
66 views

Is the quality score of fastq used somewhere besides trimming/fastqc?

In the fastq format every 4k+4-th line contains the positionwise qualityscore (ascii encoded): ...
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0answers
89 views

Error in my RNAseq analysis

I was following the RNAseq analysis tutorial online(https://combine-australia.github.io/RNAseq-R/06-rnaseq-day1.html) but am not obtaining the same variance values for each row in the logcounts matrix....
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0answers
43 views

include a glimma interface in a shiny app

I am trying to code a shiny app for RNA-Seq data analysis. I would like to include glimma interactive plots in it. However, in my current interface, clicking the action button ...
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1answer
29 views

mapping and de novo assembly of plant without reference genome

I'm studying on a plant that has no reference genome and only has one scaffold assembly and one gff3 annotation file. Can I create an index with the same assembly and gff3 in STAR and do the mapping? ...
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3answers
1k views

How to convert gff to gtf?

My annotation file is in .gff format. I would like to convert it to .gtf format or to know if there is a way to directly download the annotation file in .gtf format? I am working on sequences from ...
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0answers
28 views

What should I do when I have “#NAME?” in my Log Fold Change data to calculate Z-score? replace with zero or what?

I am trying to calculate the Z-score for several columns with Log fold change (LFC) data from RNA-seq expression values. But the mean for all columns, except one of them, is -inf and the standard ...
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1answer
255 views

how to interpret of “pvclust” dendrogram and finding height for cutting dendrogram?

I study on RNA-seq expression dataset about one cancer in TCGA. I downloaded FPKM dataset and removed batch effect by ComBat() function. Now, I used pvclust for ...
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0answers
23 views

How to detail the specific GO terms

How can I get more specific GO terms when using clusterProfiler? I got my Dotplot by: ...
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0answers
93 views

Model matrix design for limma-voom and batch effect correction

I've posted it on Bioconductor but didn't get a response, so I thought maybe I could get some help here (most likely the same audience but I'll try) I have multiple clusters in a 270 leukaemia sample ...
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2answers
55 views

Pre-filtering genes for Principal Component Analysis

I have a raw counts data-set of 20,502 genes and 137 samples. I want to find out Principal Components which best explain variation between samples in different stages of tumor. I am new to Machine ...
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2answers
39 views

about GO terms's name

Did anyone know whether the GO terms can include more detail information? Like I can get the DotPlot of the GO terms as below: The problem is that some of the genes, in the GO terms, is more about ...
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1answer
50 views

Where can I get ensembl Medaka genome for RNAseq

I am trying to map a RNA-seq dataset using ensembl genome for medaka fish. From here http://uswest.ensembl.org/Oryzias_latipes/Info/Index when I click on a ...
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1answer
113 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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0answers
98 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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0answers
25 views

How to analyze expression of certain group of genes in certain types of cell?

I need to analyze expression of ~ 400 genes with a certain function in the embryonic stem cells. All these genes are combined into one database with RefSeq IDs. What is a recommended workflow to ...
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1answer
110 views

CIBERSORT runtime error eval failed

I was running CIBERSORT but caught this error right after it started permutation: ...
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1answer
244 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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1answer
70 views

RNAseq data analysis

I am currently doing a Differential Gene Expression of RNAseq data in Lung Adenocarcinoma using TCGAbiolinks. In the data Preprocessing step TCGAanalyze_Normalisation, I am confused as to which method ...
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1answer
50 views

How I deal with this expression set?

I have a gene expression raw counts like below for 16 patients ...
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0answers
31 views

How to segment genome into homozygote/heterozygote blocks based on RNA-Seq data

Given a RNA-Seq data of a progeny between two inbred lines (for example an F4 plant which was derived from two inbred lines (parents) and the F1 selfed 3 times; however this is probably not crucial ...
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1answer
88 views

Tree cut issue in WGCNA

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3answers
485 views

Which correlation method to compute the correlation score between different clusters of Sc-RNAseq data?

I'm analyzing single cell rna-seq data and trying to compute the correlation score between different clusters. Wondering how to choose the correlation method("pearson" (default), "kendall", or "...
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2answers
143 views

too long header for fasta file

I have a fasta file, like this: ...
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0answers
61 views

Selecting genes with more contribution from PCA

I have RNA-seq data in response to treatment vs non response; By machine learning I selected three principle components likely can predict the response based on the gene expression. Now I have ...
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1answer
161 views

Convert FPKM to RPKM

I have my 24 samples analyzed by Hisat2>Stringtie, from paired end sequenced data. I have got FPKM data and am planing to convert them to RPKM. to be able to compare my samples with another already ...
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1answer
20 views

How to map selected genes to Metabolic pathway Maps

I have a selected Arabidopsis Genome Initiative (AGI) list for RNA seq and proteomics data, how can I map them to metabolic pathway maps to vilualize(e.g. TCA cycle / FA / Photosynthesis) in KEGG or ...
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1answer
505 views

Changing the axis limits of ggplot objects

By EnhancedVolcano R package I have this plot with this code ...
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1answer
85 views

How I deal with this kind of gene expression comparision

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example tumor 1 in batch 1 and tumor 1 in batch 2 , ...
2
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2answers
595 views

TPM or rlog(CPM) for comparing expression?

I want to see the expression of a gene in a group of patient amongst the entire cohort using my RNA-Seq data. While I can do a differential expression analysis with limma or DESeq2, I want to see how ...
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2answers
75 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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0answers
136 views

Module preservation analysis WGCNA

I have done a WGCNA (weighted gene co-expression network analysis) analysis for various brain disorders. So along with control, I have 3 different disorders such as BPD, MDD and SCZ. Now I have to ...
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2answers
204 views

Interpreting this PCA plot for RNA-seq

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example ...
2
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2answers
73 views

Find most similar genes to a set of genes of interest in time series RNA-seq data

I have gene expression data (RNA-seq) for 30 different time points (from 0 to 60 min each 2 min). I have a set of 8 genes that behave similarly (although not identically) and I want to find the top X ...
2
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2answers
72 views

How to best detect the “peaks” in RNA-seq data that are not assigned to any gene?

I encountered that many reads from single-cell RNA seq data were lost in the analysis because not assigned to any gene (genome: galgal6). I am trying to find an approach than could give me all the "...
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1answer
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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0answers
74 views

length of 'dimnames' [1686] must match that of 'dims' [3]

Please if anyone has experience with the use of the BSEQ-SC package for the deconvolution of bulk RNA sequencing data with single cell RNA sequencing data I will be very grateful for your suggestion. ...
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1answer
48 views

How do I create a filtered gene list using expression medians

Forgive the simple noob question I have TPM data of ~50k genes (rows) across ~1k cell lines (columns). In R, I would like to output an "intermediate expression" gene list for each cell line, like: <...
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0answers
46 views

Patient-sample mapping in GSE72056 dataset

I want to use the single-cell data from the following expression profiling: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE72056 This is also the paper published and analyzed the data https://...
0
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1answer
71 views

After running a DRIMSeq pipeline, how do I know which genes are upregulated in the different conditions?

After running a DRIMSeq pipeline and obtaining the genes that are differently used between the null and full models, how do I select the genes that are differently used in the different conditions? ...
1
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1answer
96 views

Comparing RNA-Seq and Ribo-Seq

I'm attempting to compare total RNA-seq with Ribo-Seq, to determine if changes in Ribo-Seq are due to changes in transcriptional expression (akin to the analysis performed by Anota2Seq). I am, however,...
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0answers
46 views

Identification of differential genes across 8 groups [closed]

I need to identify differential genes across 8 groups. I know this can be done using DESeq2 or EdgeR as these are better suited and equipped. However, I was thinking if something like this piece of ...
0
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1answer
132 views

Correlate DEGs from DESeq2, EdgeR and Limma results

I have a lists of DEGs identified by DESeq2, EdgeR and Limma. I would like to correlate the the gene rankings in the lists to decide on a package to use in downstream analysis. I am havig a few ...
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0answers
100 views

Error using bseqsc

I will be very grateful for any hint on how to overcome the error. I wish to deconvolve my bulk RNA seq data obtained from the lungs of mice using single cell RNA seq data. For practice, I am ...
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2answers
130 views

How to transform and sort the matrix to make a heatmap showing signatures?

I have a matrix data with cells as rows and samples as columns. Here I am giving the data with dput ...
5
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1answer
72 views

design formula question

I am not sure I am building the proper design formula for the question I want to answer I have the following samples with three factors; clone, the structure and the condition. ...
2
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1answer
66 views

Single Cell RNA seq Analysis for low input Cells?

I am having a single cell RNA seq data from ~500 Cells. I do understand this number is very low for present day analysis tools like Seurat. So, I want to know what if one have a such a small number of ...
1
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1answer
83 views

How do I select a proper design matrix to use in DRIMSeq?

I am trying to use DRIMseq for DTU with 2 treatments on two different strains of an animal model. How do I select a proper design matrix to use in DRIMSeq ? How I know that one is the correct one to ...
3
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1answer
979 views

How to find novel transcripts using GFFcompare?

I am trying to find novel transcripts from an RNA-seq database. Based on the advice I got, it seemed that using Stringtie for transcript assembly is a good way to go, and it supports novel transcript ...

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