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
30 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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1answer
35 views

What are the right parameters to trim a small RNA transcriptome with trimmomatic?

I'm having some problems with finding the right parameters to trim my small RNA Illumina reads (51 nt long) with Trimmomatic. Before trimming, one of the samples (21M reads) looks like this: So for ...
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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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78 views

Strange p-value histogram for differential gene expression analysis

I'm trying to perform pretty standard differential expression analysis using RNA-seq data. I've used Kallisto to perform RNA quantification and am using Sleuth to perform the differential expression ...
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0answers
16 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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26 views

“perl: warning: Setting locale failed.” in RepeatMasker

I'm trying to run Repeatmasker in Linux on the command line with: ...
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16 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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1answer
26 views

Why does DESeq2 convert numeric columns to factor during differential expression analysis?

I'm attempting to perform differential expression analysis using DESeq2 on a dataset of ~90 samples and ~20000 transcripts. I have a number of variables I'd like to test against, some of which are ...
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2answers
181 views

How drastic would the result be if I use fasta or reference assembly from ucsc and gtf from gencode?

There are difference annotation file for UCSC and gencode. But if I use the reference assembly from UCSC and the GTF from Genocode or vice versa would my downstream results would be wrong?
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44 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
43 views

how can i download rna-seq data sample for starting rna-seq analysis by R? [closed]

I am looking for an induction into RNA-seq analysis using R. I have yet to decide a data set, or a subject area. Any help in this regard would be welcome.
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1answer
23 views

bedtools feature out of bounds

I'm trying to count the GC in regions leading up to polyA clusters. I found the bedtools command nuc, one of the outputs of which is the GC content. I took the mouse polyA cluster database from here,...
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2answers
54 views

Can we use GTEx data as control data for TCGA data?

I am using Recount2 TCGA data and was wondering is it right to use GTEx data as control data for this. I would really appreciate your views on this?
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1answer
41 views

bowtie2 options when mapping stranded single end reads

I'm trying to map RNA-Seq reads generated using the NEB Ultra Directional kit is first strand reversed protocal. I'm using bowtie-2.3.5_1 to align against the mus ...
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1answer
80 views

Align legends to each heatmap in a ComplexHeatmap with 2 heatmaps

I have created a ComplexHeatmap containing 2 Heatmaps, each with their own legend. The legend on the first Heatmap is continuous, whereas that of the second is ...
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2answers
84 views

GSEA - Gene Set Enrichment Analysis - in R studio

I have been trying to conduct a classic GSEA using the broad institute software and can not figure out how to compile it in R for the life of me. I am aware that there exists other packages in R that ...
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2answers
58 views

Calculating p-value for introns which are retained (or not) between 2 conditions

I am trying to calculate p-values and ultimately FDR values for RI introns for 2 conditions of experiment I have(control and knockdown) For every condition I have 3 biological replicates each: 3 ...
7
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1answer
256 views

Why are my kallisto and salmon results differing so much just for lncRNA transcripts?

I am running some analysis on an RNA-seq dataset. I have a list of transcripts that are potential lncRNA for which I ran both Kallisto and Salmon aligners. The input data for index building and ...
2
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0answers
53 views

What could cause differing counts of R1 and R2 in Paired End Sequencing (RNASEQ)

I recently finished mapping an RNAseq run using STAR2.3.0 and noticed the read1 and read2 counts are different, according to samtools flagstat. The map% is ~80% but the R1 R2 counts are: R1=...
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1answer
46 views

A GTF annotation for GRCH375d version

I have been given a bunch of .bam files, that come from the alignment on GRCh37d5 of human genome by Star tool. I want to extract raw read counts from these bam files by FEATURECOUNTS tool so I need a ...
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0answers
45 views

How to write the subclusters in file?

I have constructed a gene co-expression network from RNA-seq data. The network file is in edge list format of memory around 1gb which was created by calculating Pearson correlation of each gene pairs ...
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0answers
54 views

Is it advisable to remove X and Y chromosome genes in a bulk RNA-seq dataset at the level of the count matrix?

From this link remove X and Y chromosome genes in RNA-seq data using DESeq2 pipeline, I have learned that depending on context, it is perfectly valid to remove X and Y chromosomal genes in an RNA-seq ...
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1answer
59 views

log2fold change as heatmap

From a paper: (D) Expression analysis of multiple lineage-specific differentiation markers in WT and PUS7-KO EBs (14 days). Heatmap shows log2 fold change (FC) PUS7-KO to WT for each individual ...
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2answers
97 views

How I debug this code?

I am trying to run CMSclassifier::classifyCMSfunction on my data but I am getting this error ...
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1answer
112 views

Clustering of gene co-expression network by igraph R package

I have constructed a gene co-expression network from RNA-seq data. The network has more than 10000 nodes and more than 1 million edges. The network file as an edge list format of memory around 1gb ...
2
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1answer
38 views

How to get log2 fold change of RNA-Seq data for time series experiment?

I know if there is one control and one treatment group it is pretty straight forward to interpret log 2 fold change. But, I have time course experiment. I have infected cells with viruses and I ...
2
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1answer
77 views

Significant gene set testing - limma

While I have used my title, it is more of a general statistics question regarding how these significance tests are carried out, these include decideTests and ...
2
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0answers
33 views

load the phenotype data for ballgown

I am trying to reproduce the work of this paper [1], and I have run StringTie successfully, but after that I have to run Ballgown but could not understand this command: ...
1
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1answer
43 views

RNA-seq analysis of mixed viral/host reads with salmon

I did RNA-seq of mammalian cells infected with pox virus. Now, I have read files which contain both host and virus reads. I want to align the reads both to host and viral genome. I was thinking I ...
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1answer
36 views

Differential expression analysis when nested effects

I have 3 tumour samples from 3 patients from an experiment, I also downloaded 10 normal samples from TCGA. My design is like this ...
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1answer
31 views

Matching these matrices in R

I have two matrices; I want to convert the row names of first matrix to gene symbol from matched ensemble=gene symbol from second matrix ...
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1answer
36 views

Finding genes especific to microenvironment

I have RNA-seq .bam files for 3 patients, tumour and its matched derived model, namely organoid, but I don't have any matched normal sample. Differentially expressed genes between a tumour and its ...
3
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1answer
166 views

Running htseq-count over BAM files

I am trying to derive an expression matrix from BAM files using htseq-count on the server. These are bulk RNASeq BAM's by the way. I have read the ...
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1answer
64 views

Assembly by stringtie

I run this cmd ./stringtie G1_sorted.bam -B -o G1.gtf -G Triticum_aestivum.IWGSC.42.gtf -p 4 -C G1.refs.gtf -A G1.abund.tab Error is: ...
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1answer
100 views

Error in formation of correlation matrix-(corrupt matrix — dims not not match length)

I am working on a script to create a correlation matrix and mutual_rank matrix from RNAseq gene counts for 57992 genes in 7027 samples. I have already tested this script for a smaller file (for 10,000 ...
2
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0answers
124 views

lower mapping rates in salmon v0.13 compared to previous versions

Hi there :) Thanks for the tool! I recently updated to the new salmon (from 0.8... its been a couple years) and I noticed that my mapping percentages change dramatically between the two versions. ...
0
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1answer
52 views

I need some tips and suggestions for further analysis of NGS expression data (log2cpm)

I am a PhD student who inherited some log2cpm data of expression data from bulk kidney tissue from a UUO(unilateral urethral obstruction) experiment that tests a new drug. The sample material consists ...
1
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1answer
79 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 ...
0
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1answer
10 views

Empty .result in MirDeep Star

I am using MirDeep star tool for RNA sequence alignment. I have tried both GUI version and command line version of this tool. But every time, I am getting empty . result file and .known_mir file. This ...
3
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0answers
66 views

Getting genes specially up or down regulated

I have 6 RNA-seq samples like this 4 patients (005, 036, 121, 013) I have 3 tumour samples and 3 cancer models (organoid) This is PCA of log transformed data by ...
2
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3answers
119 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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0answers
32 views

What Ensembl genome version of wheat should I use for alignments? [duplicate]

There are many genome files available from Ensembl. Which one is the best to use/download for wheat RNA seq differential gene expression analysis? Also, tell me which tool or aligner or mapper to use? ...
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0answers
31 views

P21 mutant Colon Cancer cell lines (RNA SEQ)

I need to obtain RNA sequence datasets for my project. The datasets should include P21 mutation, knockout and wild type in any Colon Cancer cell lines, Preferably in the same study. I'm not sure if ...
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0answers
74 views

Making a bed file for RSeQC

I making a bed file for RSeQC, so it can do things like compute the number of reads from exons, introns, 5"UTRs, etc. I want to use a bed file that corresponds to my GTF file, so I use gtf2bed to ...
0
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1answer
62 views

Where to Download Cancer Raw Reads (fastq)?

Does anyone know where I can download cancer raw reads (fastq files), tumor and Germline for non-humans? I wanted to make a study with human data but I don't have the control access to download raw ...
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0answers
28 views

Common Mouse and human DEG analysis

I have DEGs from human and mouse (equivalent of a human disease model), and would like to generate a logCPM correlation plot between the overlapping mouse and human gene (275 genes). Prior to ...
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2answers
56 views

Using preprocessing/alignment functions on the server

I am new to bash and the processes behind cluster computing in general and need some help with understanding some basics. After looking all over the internet and this forum (+ askUbuntu) I found ...
2
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1answer
353 views

Error in as.vector(x) : no method for coercing this S4 class to a vector

I tried to run the following code in R studio. Everything worked fine, except at the last step [write.table(mdat, "recount_mdat.csv")] when I tried to export the 'mdat', I got the following error: <...
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2answers
35 views

Get & Annotation of isomiR (isoform of miRNA) profile from RNA-seq

I should get and analyse isomiR (isoform of miRNA) profile from RNA-seq with FASTA format. The best tool for this is "DeAnnIso". But here is a problem with this tool, it only allows you to upload ...
2
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1answer
22 views

RSeQC provided bedfile does not work with read_distribution.py

I frequently make use of RSeQC, I used to use it with my own bed file, generated using gtf2bed, but now I thought I'd use the one provided by RSeQC themselves from here, I got the file ...