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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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How much should the 5' adapter and i7 sequence differ?

In a multiome (RNA and ATAC) project I'm working on we chanced upon a strange situation. We're not sure if it poses a problem or not and would like to know if and where I can look it up if necessary. ...
Assa Yeroslaviz's user avatar
2 votes
2 answers
49 views

Reasons for extremely low number of DESeq identified differentially expressed genes after RNAseq?

I am fairly new at RNA-sequencing analysis, but I have attempted to analyse the data I obtained from RNA sequencing on my own by following an online EdX class and by basing the majority of the ...
Adrianna V's user avatar
3 votes
1 answer
64 views

RNA-seq QC and alignment error in script

I am analyzing bulk RNA-seq data for Paired-End. I have separate scripts for fastqc, STAR & qualimap but want to run them in a single script which looks like this USAGE: sh rna-qc.sh <path/to/...
S_Malik's user avatar
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1 vote
1 answer
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time*treatment series with repeated (i.e: NOT independent) sampling of replicates

I am performing the analysis of cell cultures in suspension, untreated(U) and treatment A and B, at t0, t1 and t2, 4 replicates per treatment. The experiment started with 12 cultures, 4U, 4A and 4B, ...
Alfredo Pagliuca's user avatar
1 vote
0 answers
26 views

Assessing the quality of an assembly

I am trying to run a script that assess the quality of a transcriptomic assembly, a de novo assembly using a tool called Transrate. To install the tool I followed the prompts in https://bioconda....
thole's user avatar
  • 153
1 vote
0 answers
16 views

Find most abundant transcript isoform of a gene across different tissues in long read RNA-Seq data

I want to understand which is the most abundant variant of a gene of interest across different tissues by looking at different publicly available databases (mouse/human). I have deduced from ...
user2998764's user avatar
0 votes
0 answers
22 views

Predicting gene expression, RNA-seq, from regulatory signals, ChIP-seq

For a given cell line, I have ChIP-seq and RNA-seq data. I am trying to construct a model for predicting gene expression from ChIP-seq. Basic models I have seen [a very simple model is described in ...
Zebra Fish's user avatar
0 votes
1 answer
17 views

Is it possible to integrate a bulk and a pseudobulk (previously scRNA or snRNA seq) dataset

I have recently sequenced a bulk dataset. However one of my conditions has a lot of contamination from other cell types. I was thus thinking of using a publicly available single-cell dataset of my ...
John's user avatar
  • 1
0 votes
1 answer
22 views

Plenty of unmapped reads in human RNA-seq data, why does the largest contig from SPADES alignes to human 18sRNA?

Pretty much the title. We have performed RNA-seq using SmartSeq3 and processed it with zUMIs using human genome (GRCh38) as reference and gencode 44 for annotation. We noticed plenty of unmapped reads,...
Karthik Nair's user avatar
1 vote
0 answers
20 views

I want to know what are the main differences between two R Objects constructed for analysis of microbiome data: phyloseq and TreeSummarizedExperiment

I am looking for main differences between two R Objects constructed for analysis of microbiome data: phyloseq object and TreeSummarizedExperiment object First, I will provide a little information ...
akspat's user avatar
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Surrogate variable analysis for paired RNA seq experiment

I have an RNAseq experiment with 1000s of samples. Each sample comes from a patient and was treated either with a drug or DMSO (as control). I am currently deciding between SVA and RUVseq to account ...
nhaus's user avatar
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1 vote
2 answers
60 views

Abnormal representation of differentially expressed genes in volcano plots located at the extremities

This question was also asked on reddit Does anyone know how to correct the abnormal genes that seemed to be located on the curves near the extremities? The analysis was done using R package edgeR on ...
Timi's user avatar
  • 51
1 vote
0 answers
26 views

Is it normal to have low mapping to bacterial genome in a total RNA sample of plant root-colonized bacterial cells?

I had isolated the total RNA from a sample of plant roots with colonized bacteria by excising the roots (harboring the bacterial cells) and crushing them into a powder using liquid nitrogen. The RNA ...
K_081's user avatar
  • 149
1 vote
1 answer
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missing additional visits in GSE data

When downloading GSE data, often I encounter that the authors refer to additional visits in the data description, yet, they are not found in the data. For example, the following study. They mention ...
Kozolovska's user avatar
1 vote
1 answer
34 views

rna seq matrix design hybrids

I am struggling to get my head around the correct design to use for my experiment. I have RNAseq of 2 pure species (s1, s2) and of the hybrid (h). I aligned all three genotypes to the s1 genomes. I am ...
RomainL.'s user avatar
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2 votes
0 answers
292 views

GSEA vs GSVA: Pros and cons of each method

Trying to understand when GSEA is more appropriate than GSVA and vice versa. I have seen cases when running GSEA and GSVA on the same task - compare enrichment of a geneset between two groups - gives ...
Tomas Bencomo's user avatar
5 votes
2 answers
92 views

Multiple genome assemblies of the same bacterial species

I have some RNA-seq data where there are reads from the "host" as well as from several bacteria species. In this experimental context, I am interested in the host associated reads and the ...
haci's user avatar
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1 vote
1 answer
61 views

Bedtools genomecov gives me coverage files showing zeros at all nucleotide positions

Also posted on biostars I have bam files that I've obtained through STAR alignment. Here is the first 10 lines of one of my bam files: ...
gcabebe's user avatar
  • 21
2 votes
1 answer
30 views

Salmon Multiple File SCRIPT giving error

Hi I am using Salmon quantitation for multiple fastq paired files using following code ...
S_Malik's user avatar
  • 61
1 vote
1 answer
63 views

After running nf-core, is there a way to map a gene_id to a specific gene's DNA sequence?

I have been running nf-core in Python and it works great! But I have a seemingly simple question that I'm struggling to find an answer for online. After running the nf-core pipeline on my RNA ...
user18959's user avatar
2 votes
1 answer
117 views

WGCNA: Extract genes within the different modules to create a dataframe with two columns

My inquiry pertains to WGCNA. After identifying the modules, I would like to extract genes within the different modules to create a dataframe with two columns: ...
nann's user avatar
  • 23
1 vote
2 answers
37 views

How to normalise Bulk RNA-seq data to account for transcript length, coverage depth, library size and batch effects?

I'm currently working on a project where I'm comparing aggregate measurements (mean, median, etc.) of expression data (RNA-seq) from different groups of genes across various samples with different ...
Duhlky's user avatar
  • 11
1 vote
0 answers
53 views

Continuous predictor variable to identify responder genes using DESeq2

I have samples from 45 individuals at 2 time points and these individuals were given a specific diet. The BMI of these individuals were recorded at both time points. The responders to the diet are the ...
Angelo's user avatar
  • 237
0 votes
1 answer
135 views

Featurecount output .txt file from bam file

After running this command: featureCounts -p -s 2 -a $genome -o $dir"/"$specie"/count_table/"$value".txt" $output_loc -T 8 on the BAM output of ...
ToTheMoon's user avatar
0 votes
0 answers
17 views

RNAseq and TTseq results

I'm working with two sets of data. One of two sets of data, one contains the results of a RNAseq analysis and the other contains the result of TTseq analysis. I want to check which genes overlap in ...
Adrián Coto's user avatar
0 votes
0 answers
25 views

Suggestion for way foward with upregulated genes

I am working with single cell RNA data- control and treated. I analyzed and got a list of upregulated genes. Upon doing pathway analysis all the genes were of apoptotic pathway. But these genes can ...
subhiksha sundaram's user avatar
0 votes
1 answer
41 views

problem with feature counts of RNA bulk seq paired data- in Rsubread

I did manage to bild index file (from NCBI transcriptome) and perform alingment in Rsubread in R with my fq files. I did get BAM files as a result and no error. However i am having trouble with next ...
MKE1508's user avatar
2 votes
1 answer
95 views

Classical alignment with HISAT2

This question was also asked on Biostars I have this script ...
Emmanuel Aroma's user avatar
1 vote
0 answers
44 views

What method uses "diverge probability" in RNA-Seq differential expression analysis?

I am replicating differential expression analysis from a paper (here) on RNA-Seq data. I extracted the regulated genes using DESeq2::results with a threshold of 1 ...
Hadeer Ibrahiem's user avatar
1 vote
1 answer
184 views

Kallisto RNA-seq errors because of incompatible indices when running <kallisto quant> using downloaded mouse index

I'm trying to run RNA-seq on single-end mouse RNA data (50bp reads) but have very limited RAM (<4 gb) - I don't think I really have a way to work around this memory restriction, and wanted to try ...
mz.'s user avatar
  • 11
1 vote
1 answer
73 views

issue with rna seq analysis

I am working on RNA seq analysis and I would like to know the following things: Current Methods: downloaded genome fasta file for non-coding rna from ensembl and got the gtf file for hg38 performed ...
subhiksha sundaram's user avatar
2 votes
1 answer
114 views

Mitochondrial genes - TPM calculation bulk RNA-Seq

This question was also asked on Biostars I was wondering if any of you have encountered a situation for bulk RNA-Seq where, possibly due to low sample quality or presence of dead cells, mitochondrial ...
nick_b55's user avatar
2 votes
0 answers
54 views

How to incorporate negative controls in DESeq2

We are doing a comparison between two outcomes (positive and negative). We could not have any positive controls as we do not have any "control" data to set as baseline, either from ...
Karthik Nair's user avatar
0 votes
0 answers
109 views

how to use CIBERSORT

I have seen in several articles, the use of CIBERSORT to calculate the proportion of immune system cells using gene expression data, I would like to try it but I have not found how to install it for R,...
María José's user avatar
0 votes
1 answer
197 views

HISAT2: RNA strandedness

My library is unstranded and the code I'm trying to use is this: ...
Neophytos Kouphou's user avatar
0 votes
0 answers
29 views

Calculating p-value and adjusted p-value for pre-normalized microarray data with fold change precalculated

I have a microarray dataset with two mutants dataset that has already been normalised, and the fold change values for each gene in each mutant versus the wild type have been calculated. I'm interested ...
shaimaa Hassan's user avatar
3 votes
0 answers
120 views

how to install cibersort in R, to do deconvolution using my RNA-seq Data with public single cell data

I have RNA-seq Data from lung cancer immunotherapy patients but i don't have any single cell data from my sample, so i wanted to do a deconvolution with a public single cell data. I thought of doing ...
Rita Soares's user avatar
3 votes
1 answer
48 views

Discrepancy with featurecounts analysis using a forward stranded and reverse stranded protocol

My RNAseq analysis pipeline is as follows: fastqc (read quality is good, some overrepresentation of adaptor sequence) → trimmomatic (trimmed adaptor sequence, qc report after trimming suggests the ...
xtian's user avatar
  • 31
3 votes
2 answers
112 views

Cannot obtain alignment summary after running Bowtie2

I am aligning my Small RNA Seq data with Bowtie 2. Although the alignment performs well, the only information I obtain after finishing running the alignment is the following: ...
ALEJANDRA PANDO CACIANO's user avatar
1 vote
0 answers
111 views

Working with Smart-seq3 data, have some questions regarding UMI and bar coding

Most of my experience has been working with genomic data, this time I am working on data from Smart-seq3. RNA excreted by cells in culture medium were processed with Smart-seq3 (From the looks of it, ...
Karthik Nair's user avatar
4 votes
2 answers
263 views

Question about umap using different numbers of pca components as initialization

I am new to the scRNA-seq field and I have been doing some experiments of visualization of UMAP using different numbers of PCA components for initialization. The process involves projecting scRNA-seq ...
Zack's user avatar
  • 43
0 votes
1 answer
99 views

scRNA: What are good dimensionality reduction/clustering parameters to get biologically plausible groupings?

I've got a moderately large set of PBMCs, over 1M cells. That means I can't easily do a grid search of dimensionality reduction/clustering parameters/methods. Some examples results I'm getting with ...
Henry Gong's user avatar
0 votes
1 answer
70 views

Paired end reads with different read lengths

I downloaded some raw RNA-seq reads from NCBI using the sra-toolkit (Accession numbers: SRR2053159-64). The reads were generated using ABI SOLiD, and in colour space. I downloaded them in base space ...
Arkajyoti Banerjee's user avatar
-1 votes
1 answer
227 views

Using CSV files continaing scRNA-seq count data from GEO [closed]

I'm completely new to scRNA-seq analysis (and to most of Bioinformatics as well) so I apologize if this was asked a million times before. I am trying to get the single cell expression data of lymph ...
Newbie Bioinformatician's user avatar
1 vote
1 answer
74 views

What are the output files of RNA-Seq from facility?

This question was also asked on Reddit I am new in our lab and I am going to do bulk RNA-Seq. What type of files will we get from the company (Genewiz)? Will it be a bunch of Fastq files? or they give ...
Sina Asadi's user avatar
-1 votes
1 answer
55 views

Is there a data analysis software (free) or code that I can use to view normalized CDR3 amino acid length distributions?

Is there a data analysis software (free) or code that I can use to view normalized CDR3 amino acid length distributions? Regarding code, preferably using Python or R. EDIT: Added a picture of the non-...
SData11's user avatar
3 votes
1 answer
152 views

Applying glmnet to identify predictors for subtypes

I am new to glmnet and other ML techniques, so apologise in advance if this sounds a trivial question. However, your guidance is very much appreciated. So, I have nearly 700 genes with their ...
Angelo's user avatar
  • 237
3 votes
1 answer
148 views

Issue with Differential Gene expression analysis with Deseq2

I am using the Deseq2 package to perform a DE analysis on multiple factors. So, basically to perform the analysis I consider patients characterized by the Brugada Syndrome type 1 and other patients ...
Alessia Avon's user avatar
2 votes
1 answer
61 views

DE analysis tool and method for non-coding features

I am currently working with the non-coding features of A. thaliana, and trying to get the DE features. Among the three DE test methods in edgeR such as ...
Arkajyoti Banerjee's user avatar
2 votes
0 answers
24 views

Feasible to find genetic variations of two samples using RNAseq data?

I have bulk RNAseq data from two strains of mice from Jackson Lab: C57BL/6 and B6.SJL. The former expresses a Ptprc-b allele and the latter expresses a ...
geom_na's user avatar
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