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
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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4answers
45 views

Files for paired end RNA sequencing

I am looking at the videos at a DIY Transcriptomics course and the speaker mentions that to run Kallisto for read alignment with paired end sequencing, one would enter the following: where the sample ...
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1answer
84 views

Which module to select for Pathway analysis based on Module trait correlation and pvalue?

I have a total of 35 tumor samples classified into 4 subtypes. Subtype A, B, C, and D. I have RNAseq data. I'm interested in identifying modules related to each subtype with co-expression network ...
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28 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. ...
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0answers
34 views

Gffcompare issue: 0 reference transcripts loaded

I am trying to use gffcompare to compare my assembled transcriptome to a reference gtf that contains information about small open reading frames (sORFs). The reference gtf was obtained by processing ...
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2answers
42 views

Clarification about how mapping to a reference genome works in RNA seq

When mapping reads to a reference genome, how is it possible to tell which part of the genome the read is referencing if the genome possibly has the same repeated sequences in different areas? What I ...
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Step-by-Step Construction of Gene Co-expression Networks from High-Throughput Arabidopsis RNA Sequencing Data

I am following the tutorial here to learn how to process raw RNA-seq data and get gene read counts. I've had no issues until I got to step 3.9, "Assignment of RNA-Seq Reads to Genes." Here, the ...
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2answers
34 views

How to tell if library generation for RNA-seq experiments are stranded vs not stranded

I would like to use RNA-seq data from the NIH GEO database. How can I tell if library generation for the RNA-seq experiments are stranded vs not stranded?
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1answer
52 views

WGCNA co-expression network analysis with less than 20 samples

I have two cancer subtypes data. Subtype A is 14 samples and Subtype B is 23 samples. I'm interested in identifying the functions of some LncRNAs in the Subtype A group. For this I'm using all protein-...
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26 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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extracted counts matrix from Tximeta generalized experiment with no linked transcriptome are normalized?

I used the Tximeta vignette to construct a summarised experiment from quant files (salmon, index prepared with NCBI transcriptome). 1)Tximeta return me a non-ranged se (which I am not sure about the ...
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0answers
9 views

Cuffdiff multiple comparison

I have four groups G1,G2,G3 and G4 all in triplicates, I want to make the comparison of differential expression between the four groups together used cuffdiff following: ...
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1answer
58 views

How to incorportate RIN values as covariate in the design matrix?

I have been following the last DESeq2 pipeline to perform an RNAseq analysis with a dataset with low rin samples in the experimental (or treated) and high rin on the control ones. I read a paper in ...
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0answers
7 views

Does “Wrapping up the results” in ballgown means that ballgown object is created correctly?

I am trying to apply a Pertea et al. procedure to my RNASeq samples for which I have created a ballgown folder as explained in the article. When I am trying to prepare my phenodata file for every ...
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1answer
38 views

How to understand and analyse RNA-seq data (for a beginner)?

I am trying to understand expression of a certain protein across Pseudomonas species. I downloaded an SRA file from NCBI and converted it to a fastaq file. I am not able to understand how to interpret ...
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1answer
52 views

Should biological replicates be the most similar pairs in an RNAseq experiment?

The attached figure (from deseq2) shows the sample to sample distances of a RNAseq experiment with 4 conditions (A,B,C,D) at 2 time points (0h,4h) with 2 biological replicates. I am a little bit ...
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2answers
38 views

cellranger mkfastq with full path to --id flag

I have always used cellranger mkfastq to demultiplex 10x genomics runs manually, though recently the commands to do so have been incorporated into a script that ...
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0answers
58 views

PAM50 gene expression classification

I'm looking at running the PAM50 classifier on RNA-Seq from 138 breast cancer samples. However, the R package (genefu) that's useful for this does not have a ...
2
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1answer
42 views

Why this ballgown error (first column of pData does not match the names of the folders) comes out

I am trying to create a ballgown object, and I would like to set a phenodata information. For that I do phenodata <- read.csv('l002.csv') and my l002.csv ...
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0answers
32 views

vst() from DESeq2 vs voom() from limma

I have used both to transform my leukaemia RNA-Seq data for subsequent hierarchical clustering. The result is quite different. Some subtypes of leukaemia only form a cluster (or at least sit closer) ...
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0answers
59 views

How to convert Illumina IDs to Gene Names?

I am new to the area of bioinformatics, so apologies if this is too obvious of a query. I need to analyze the RNA Seq data from GSE98455. The dataset is of the following format: ...
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1answer
30 views

Is there a single cell RNAseq equivalent of GTEx or TCGA? [closed]

Or do I need to find individual studies and obtain data the long way!
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1answer
99 views

How can I bypass 'out of memory' problem using a HISAT2 for human genome indexing?

I am trying to perform a HISAT2 indexing using a GCF_000001405.39_GRCh38.p13_genomic.gtf and ...
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1answer
126 views

How to downsample some of the samples in RNA-seq data?

I have 40 samples and these are into two groups. I would like to perform a differential analysis between two groups. The library size of the samples is very low. But there are two samples (GroupA_12 ...
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0answers
27 views

reactomePA dataset

I tried to create my geneList according to this r code: setwd("C:/cygwin64/home/DIANGO/EXCELL/") d = read.csv("PA_down_id.csv",sep = " ", header = F) head(d) ...
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1answer
213 views

Downloading SRA Files from AWS

I want to download the original BAM files that the authors had uploaded to SRA. Normally, I would just use sam-dump, but the files are having issues that seem ...
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1answer
34 views

Database of RNA-Seq and SNP genotyping data

I need to obtain publicly available RNA-seq + SNP genotyping data for a cohort - ideally one where the RNA-seq data has been sequenced from brain tissue. I have found CMC and GTEx to be sufficient ...
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0answers
12 views

Pantherdb Export

After a gene enrichment analysis in the panther database I would like to export my graphic data (barplot and Pielot). Then I would be happy with a suggestion or advice from which will allow me to ...
2
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1answer
73 views

How to download RNAseq gene expression data from GTEx

I would like to download RNAseq gene expression data from GTEx from the liver only. What is the best way to do this? I have tried a few things. I downloaded several files from the GTEx website (https:...
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2answers
61 views

What RNA-Seq expression value would be closest to Microarray equivalent?

I know this question may seem strange. I'm using Spearman correlation between gene expression profiles for various reasons (I won't go into details here). As a result, I often compare RNA-Seq and ...
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1answer
91 views

Differential Expression

I have genes differentially expressed between two groups (case and control). I would like to annotate them by classifying them according to their biological functions. I work on parasites of ...
3
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2answers
926 views

How can I classify the 3 clades(S, G, V) of the coronavirus without using protein data?

On GISAID they classified the coronavirus using 4 clades(S, G, V, Other). I downloaded around 1,000 complete genomes of the coronavirus from GISAID and I would like to classify each one as belonging ...
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1answer
60 views

Cuffmerge: EOF marker is absent. The input is probably truncated

I need to merge my all transcripts.gtf from cufflinks output follow this command line : cuffmerge -o merged_gtf_output -p 15 -s ref.fasta -g anot.gtf assembly.txt ...
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0answers
24 views

Coding object structure properties into a sequence

Hopefully, I found the right place to ask. Please, note that I'm not a specialist in the current field. Is there an algorithm to code information about object structural properties into a sequence (...
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1answer
20 views

Filtering genes from cuffdiff results

I have run cuffdiff (with statistics turned ON) to compare two groups of samples: Control group and Late AD group. This is the command I ran to be precise: ...
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2answers
32 views

Is there a computational tool or possibility to identify mRNA isoforms from the count matrix of a bulk RNA sequencing dataset?

I have the counts matrix of an RNA sequencing dataset of fibroblasts and I wish to identify isoforms of a particular gene of interest in it. Can anyone please hint me on a bioinformatics method to ...
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1answer
86 views

How do I differentiate outliers from in-group variations in a DESeq2 PCA plot of 9 samples distributed into 3 conditions?

I have a PCA plot from DESeq2's plotPCA(vsd, intgroup=c("conditions")) function. I have 9 samples distributed in to 3 groups of 3 biological replicates each. My ...
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0answers
30 views

RNAseq without depletion?

I have been asked by my supervisor to conduct an RNAseq without experimentally depleting the samples. However I am quite unsure regarding how much sequencing depth per sample I would require in order ...
2
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1answer
121 views

How to calculate fold change in gene expression from RSEM (RNAseq)

I have gene expression data from RNAseq, specifically: log2(x+1) transformed RSEM normalized counts. How can I convert these data from multiple samples to determine fold change in gene expression?
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2answers
45 views

Download sequences of isoforms

I want to collect all isoforms of all genes (Fasta/Fastq file of nucleotide and protein sequences) Arabidopsis Col-0. I am wondering if there is a straightforward way to download the file from any ...
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1answer
86 views

How to run trimmomatic in HPC

Sorry if this has been asked before, I've done a quick search and I don't think there's an easy explanation for me to understand. I'm really new to bioinformatics/RNASEQ analysis, having only taken ...
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1answer
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 ...
2
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2answers
96 views

Importance of Proper Pairs vs Aligned Reads for RNASeq data

I have stranded, paired end RNASeq reads that I have aligned using STAR. I plan do conduct a differential expression analysis with DESeq2. After running quality control checks, a good portion of my ...
2
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2answers
734 views

STAR quantMode vs RSEM vs Kallisto

I recently discovered this Snakemake pipeline for RNASeq that uses STAR's quantMode to quantify gene expression for DESeq2 differential expression analysis. In the past I've always seen the workflow ...
2
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2answers
211 views

How to initiate RNA-Seq analysis of TCGA files?

I want to compare RNA-Seq datasets obtained from the TCGA to investigate how my gene of interest is implicated in different types of cancer. I'm trying to download the data from the GDC Data Portal (...
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0answers
33 views

Simulating RNA Editing

I'm pretty new to bioinformatics and I have been looking for a way to simulate rna sequencing with editing, specifically A to G, but didn't find anything i could use. I tried using flux simulator but ...
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1answer
236 views

How to visualize called narrowPeak files in UCSC Genome browser or IGV?

I have called peaks using MACS2. Then I got a narrowPeak file like this. ...
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1answer
93 views

hisat2 --rna-strandness option and downstream htseq-count analysis

I've got some doubts on the hisat2 --rna-strandness option and its output for downstream analysis. Please see below. I understand that the --rna-strandness option produces an XS tag to indicate where ...
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1answer
25 views

Error in createMAE function: non-unique values when setting 'row.names' in TCGA LIHC Data

I am working with the createMAE() function of the ELMER Bioconductor package. While executing the createMAE() function using ...
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0answers
26 views

Programmatic Access to `fastq-load.py` Arguments in SRR Metadata

Is there a way to programmatically access the fastq-load.py arguments from the SRR metadata? Specifically I would like to capture the exact command line arguments ...

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