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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blood contamination rna seq

I have tumor rna seq samples. I want to find out percentage of blood contamination within these samples. What is the best possible way to find % of blood contamination within the rna seq samples?
1 vote
1 answer
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Error while using SummarizedExperiment() in R

I'm tryig to perform RKM normalization on raw counts for RNA-Seq Data: ...
2 votes
0 answers
24 views

Does the contrast matrix I have made address the question I am trying to answer?

I have the following design matrix: mm_noreps.interactions <- model.matrix(~condition*TRAPed) Both variables are factors condition has 4 levels and TRAPed has 2 ...
2 votes
1 answer
53 views

Is it possible to do homology inference across species using different kinds of NGS data?

Background: I have a list of species that I want to put through homology inference. The goal of homology inference is to investigate the evolution of a trait on a species tree. I want to use the ...
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2 votes
1 answer
44 views

Examples of machine learning approaches to validate results where ground truth is lacking?

I am currently looking into some of the published methods for deconvolution of spatial transcriptomics data where each spot does not have single-cell resolution. These methods all rely on cell type ...
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1 vote
2 answers
45 views

Display row and column names in R

I have plotted a heat-map with 235 rows and 570 columns which is below: how do we make clear names of row and columns on this graph using R?
4 votes
1 answer
96 views

Kallisto error: index input file could not be opened!

I am utilizing Kallisto in Anaconda/miniconda for RNA sequencing. I have successfully made ...
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3 votes
1 answer
82 views

How to remove double '/' from file path, bash script

I'm using the following script to detect strandedness of my paired end RNA-seq data. ...
1 vote
1 answer
56 views

How to run how_are_we_stranded_here?

I'm trying to run the python library called how_are_we_stranded_here. I have installed with pip install how_are_we_stranded_here. I have also installed its ...
0 votes
2 answers
57 views

How do I determine read length from a user-inputted fastq file and then input that information into a tool's command line?

Can you guide me on how could take an input fastq file, read the length from read and then feed it into the findtail's command? I have suggested concerns about documentation and program defaults to my ...
2 votes
1 answer
49 views

How to make unrooted tree for Likelihood mapping result by using IQ2-tree?

I am a biologist, and I do not fully understand the tree topology of the experimental species. I used four-taxon set (4 sequences) to identify the Four-cluster Likelihood-Mapping by using ...
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2 votes
1 answer
36 views

What are the conditions to perform Gene Expression Analysis on data you've performed RNA Sequence Analysis on?

I want to perform Differential Gene Expression Analysis. I recently completed RNA Sequence Analysis using the Seraut Tutorial up to this point: Finding Differentially Expressed Features (Cluster ...
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1 vote
1 answer
58 views

Can I perform Expression Analysis on this 10x dataset?

10x Genomics: 20k Human PBMCs is the dataset. Description of the dataset: Inputs/Libraries Human peripheral blood mononuclear cells (PBMCs) of a healthy male donor aged 30-35 were obtained by 10x ...
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2 votes
2 answers
39 views

Calling isoforms from long read data generated from partially degraded RNA

What will be the best tool to call isoforms from long read data generated from partially degraded RNA. By mistake we processed some samples with poor quality RNA to generate long read. Now we are ...
0 votes
1 answer
70 views

How to find closely related genes for a specific gene from WGCNA modules?

I have used WGCNA by integrating several datasets which are processed in a similar way. Identified 17 modules, followed by performed pathway analysis with the genes present in those modules. Among all ...
1 vote
0 answers
37 views

Benchmarking for variant identification using RNA-seq data

I am in need to benchmark the variant identification pipeline which uses RNA seq data alone without any matched-normal. I would like to know the reference dataset (and the pipeline on which the ...
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5 votes
3 answers
73 views

Ubiquitous regulation of highly specific marker genes

I am fairly new to scRNAseq analysis and keep running into the same problem in the two datasets I am currently working with. We work with kidney inflammation and have two new mouse models, which we ...
1 vote
1 answer
31 views

Snakemake MissingRuleException

I'm trying to run a snakefile for the first time with limited coding experience using salmon to index a reference genome. I'm not too sure what I'm doing wrong based on this error message. ...
1 vote
0 answers
16 views

Number of Spots in a Spatial Seurat object

I have got an integrated seurat object of 21 Spatial samples. I want to know how many spots are in all the samples together, Is there a way I can do that? Also, Please suggest any R Packages that do ...
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1 vote
0 answers
45 views

Stringtie/DEprep.py gene/transcript IDs are wrongly formatted

Hi my RNAseq workflow is ending up with wrongly formatted gene IDs (and separately transcript _IDs) after a Hisat2 ->samtools sort -> stringtie -> DEprep.py workflow. DEprep.py outputs a ...
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0 votes
2 answers
55 views

Is it possible to integrate multiple gene expression datasets and use it for WGCNA?

I have 8 RNA-seq datasets and am interested in looking at genes co-expressed with a specific gene. Among 8 RNA-seq datasets, 6 have less than 20 samples. Rather than working on each dataset ...
0 votes
1 answer
65 views

select highly variable genes out of dataframe

lets say I have the following dataframe: ...
2 votes
2 answers
49 views

WCGNA - Relate modules with Y features when the % of variance explained of each eigengen is low

I'm doing a WCGNA analysis (signed network) on microbiome 16S data. I have transformed counts to centeres log-ratio transformed data (CLR) to address the compositional characteristics of the data and ...
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2 votes
0 answers
23 views

Avoiding false anticorrelations between individual genes per cell in single-cell RNAseq

When analysing a single-cell RNAseq dataset, one question I like to ask is: At a single-cell level, are these two genes correlated (expressed together) or anticorrelated (only one or the other is ...
2 votes
0 answers
41 views

What codes represent what genes?

I want to run experiments on the data used in PatternMarkers & GWCoGAPS for novel data-driven biomarkers via whole transcriptome NMF link So far, the paper has reduced the dimension to ammon's ...
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0 votes
1 answer
33 views

Is loss/gain of function reflected in RNA-seq transcript counts?

Do LoF/GoF transcripts count toward the RNA-seq TPM count? Or would these LoF/GoF transcripts only be detected by isoform quantification?
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3 votes
2 answers
75 views

How should I interpret DGE results if only one HLA-A gene shows up as significant but not the others?

I have done a DGE recently and have been looking at the DGE list. One of the genes is HLA-A. However, when I dug deeper I realised there are hundreds of HLA-A genes with unique ENSEMBL number (of ...
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1 vote
1 answer
52 views

Expression analysis of miRNAs with normal RNA-seq data without small RNA-seq data

I am looking to perform expression analysis of miRNAs with normal RNA-seq data lacking small RNA-seq data? Which path should I choose for known miRNAs and unknown new miRNAs? Data set: rna-seq data ...
3 votes
0 answers
19 views

Genome-guided transcriptome reconstruction: should I filter my reference annotation by transcript_support_level or other tags?

Is there really any advantage to filtering the GTF annotation for transcriptome reconstruction (or, for example, for pseudo-alignment quantification)? Are there any downsides (i.e. reasons why I ...
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1 vote
0 answers
29 views

GSEA. Subsetting Gene Ontology, Biological Processes, by gene set families of interest

I have run GSEA - on Gene Ontology , Biological Processes (GO and BP hereafter) - on some Mouse bulk RNA-seq data, and am currently in the process of going through the results. To do this, I have used ...
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3 votes
0 answers
31 views

Can multiplexing in Sequel II SMRTcells reduce the coverage?

I would like to have high depth of coverage of the transcriptome, but I need to analyze several tissues. I was suggested to pool all 5 tissue samples in same SMRT 8M cell. Will this result in a ...
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1 vote
1 answer
35 views

How can Iso-Seq reverse transcriptase artifacts be avoided?

My end goal is annotate a de-novo assembled genome. When trying to select the best method for transcriptome assembly I read Iso-seq was the preferred method. However other people suggested Nanopore as ...
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1 vote
0 answers
30 views

Transcriptome analysis

I am trying to assemble reads belonging to two different readlength. Is it a valid way since I am looking for common genes among the species I am assembling.
1 vote
0 answers
40 views

RNASeq NMF clustering using what kind of expression unit?

I would need some help regarding certain points in a RNASeq gene expression analysis (sorry if there are stupid questions, its my first project and i have no capable supervision). My workflow is the ...
1 vote
2 answers
60 views

Tool for cross referencing a newly found MOTIF to database of known MOTIFS

I need a tool or a function I can use in my code (R, or Python) that I can cross reference a MOTIF against known MOTIFS, a function that will take as input a MOTIF (a probability weight matrix, PWM ...
2 votes
1 answer
52 views

How to use the contrast function in ebayes for making different comparisons

The design matrix i have is this I would like to know how to use they way its done in deseq2 where we can use the contrast function to make particular comparison The code im running to make ...
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1 vote
0 answers
48 views

How to assess quality of WGCNA module identification in blockwisemodules()

I'm exploring WGCNA for bulkRNA sequencing analysis with human subjects. I have healthy, myocarditis, and heart failure patients (which can be further broken into ischemic or nonischemic) I have been ...
1 vote
1 answer
31 views

Gene/protein expression specific to a group in omics

I am wondering what is the significance of finding a particular protein specific to a disease or control group? when we detect 1000s of proteins in a proteomics experiment, how can one be sure that ...
2 votes
4 answers
111 views

nested samples in design DEseq2

I'm running a DEG analysis with Deseq2 by specifing the following design that includes 12 samples from 6 pts in 4 different conditions: ...
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2 votes
0 answers
104 views

How to adjust by multiple variables using ComBat-Seq?

I am trying to adjust my RNA data using ComBat-Seq (from sva R package) since I realised that there are 3 batches that I need ...
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2 votes
2 answers
170 views

PWM (4xM matrices) to Sequence Logo Visualizations in MATLAB or Python

Is there a MATLAB, or else Python (not R) tool for visualizing sequence Logos from probability weight matrices (PWMs)? This seems like a basic and simple tool to implement, yet just above the ...
2 votes
1 answer
76 views

How to identify genomic regions / peaks associated with enhancers (TF binding sites)? Is there a tool or a formal recipe?

I am attempting to identify DNA sequences/regions associated with enhancers in my NGS sequencing data. How can I identify (from peak files/counts for chip-seq, atac etc.) genomics regions/peaks ...
2 votes
2 answers
82 views

How to identify which isoforms of a gene are actually expressed in my data?

I am new to bioinformatics and have been assigned the task of discovering which isoforms of a certain protein-coding gene are present in the RNA-seq data that I have. There are several isoforms of ...
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1 vote
1 answer
64 views

plotting gene expression after EdgeR DE analysis using RUVg (RUVseq) covariates

I have used the empirical RUVg method (from RUVseq) to estimate the unwanted variation of my dataset (consisting of several public datasets analysed together, with controls and case samples in ...
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2 votes
1 answer
72 views

Extracting clusters, hierarchical clustering - bulk RNASeq

I have been attempting to extract particular clusters, given by hierarchical clustering outputs from the pheatmap function (in R). Please find the ...
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1 vote
1 answer
132 views

Tutorial for the WGCNA: changes in heatmap colours

I am trying to reproduce the results of the R tutorial of the WGCNA package. In section I number 5, when generating the heatmap it is quite similar to the one provided by the pdf but the colours of ...
2 votes
1 answer
47 views

Find enriched processes in modules obtained by WGCNA

I am doing a gene enrichment of different clusters obtained by WGCNA, but there are some of them in which I don't get enriched processes, is there any way to find processes to these modules, for ...
1 vote
1 answer
68 views

Can I Incorporate svaseq() into GSEA/GSVA analysis?

I understand GSEA/GSVA can take microarray-like expression matrix such as the output of voom() or vst(). However, I have a question on how we can also use svaseq() variables, correct the output from ...
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3 votes
1 answer
125 views

Different results of spearman correlation between TPM and FPKM

TPM and FPKM of RNA-Seq data form GDC TCGA calculated based STAR were retrieved, respectively. The correlation between a specific gene, e.g. HIF1A, and other genes were calculated based on TPM and ...
2 votes
0 answers
22 views

DeconRNASeq: Extract gene names from returned mixing proportions

This question was also asked on Biostars I am using the Bioconductor package "DeconRNASeq" to perform tissue deconvolution. Let's say I run the following code (this is from the manual): <...
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