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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2answers
495 views

tx2gene file for tximport issue

I am trying to use tximport to get raw count matrices of a bulk RNA-seq sequencing experiment. I followed the tutorial: https://bioconductor.org/packages/3.7/bioc/...
2
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1answer
304 views

Salmon tximport

I ran bulk RNA-seq experiment and got quant.sf file. Now, I am struggling with understanding what ...
8
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3answers
964 views

How to identify gene expression signatures from gene expression data?

I have TCGA gene expression data. I'm interested in identifying gene expression signatures using the data. I would like to know whether there are any tools or R packages for identifying gene ...
2
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0answers
574 views

Which R package to use for differential analysis with TPM values?

I'm using hisat2, stringtie tools for the RNA-Seq analysis. After stringtie using ballgown I get FPKM and TPM values for every gene. I have seen that edgeR, Deseq2 can be used for Counts data. I ...
3
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1answer
277 views

Cufflinks Error: sort order of reads in BAMs must be the same

I am running Cufflinks for transcriptome assembly using the .bam file generated by Hisat2. I tried both bam and sorted bam files ...
4
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1answer
139 views

GOseq analysis with evidence code filter

When using GOseq analysis on RNA-seq data, I often find many 'false positives'. What I mean with that is that some genes in certain categories are not really involved in the process, but are only ...
3
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1answer
137 views

How to download gene expression data from NCBI gene database

In the NCBI gene database, I can add the expression tracks (circled in picture blow) through 'Tracks' button, but How I can download the expression data directly, not just look the picture?
7
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2answers
656 views

Convert R RNA-seq data object to a Python object

I have done some work in R and would like to try a Python tool. What is a good way to import the data (and its annotations etc) as a Python object? I am particularly interested in converting a ...
4
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1answer
90 views

LRT or LRT-like test on cyclical (Sleep) data

I have RNA-Seq data from 4 time points (3 hours awake, 9 hours awake, 3 hours asleep, 9 hours asleep). I'm interested in doing something similar to a LRT where genes are found to be significant if ...
5
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1answer
186 views

Which tools for differential expression analysis in scRNA-Seq?

I am starting to run analysis for differential expression in scRNA-Seq. Which tools are available for this kind of analysis? Can tools for bulk RNA-Seq like DESeq be used for scRNA-Seq?
6
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1answer
156 views

Run gffread in multi-thread mode

Is there any possibility to run gffread in multi-thread mode? The answer seems to be 'no' from the manual (or gffread -h), as no multi-thread option is mentioned. ...
6
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1answer
154 views

Salmon output: transcripts quantified with zero reads support

I quantified some samples using Salmon. According to the documentation of output format, the last column of the 'Quantification File' represents the number of reads that supported the given transcript,...
7
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2answers
777 views

Stranded vs. unstranded library preparation protocols in RNAseq

I've been reading this paper lately: Sailfish Enables Alignment-Free Isoform Quantification from RNA-Seq Reads Using Lightweight Algorithms I don't really understand the second paragraph under ...
1
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1answer
237 views

gffread: GFaSeqGet errors on coordinate overhang

Disclaimer: I had this issue posted on Tuxedo Tools users group and shared it on Twitter, but could not get an answer to this from the developers, nor find documentation of this issue online. So, I'll ...
1
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1answer
78 views

Negative binomial modeling of RNA-Seq data

A common way to model RNA-seq data is using a negative binomial distribution, where each sample-gene pair is modeled by a different negative binomial distribution with mean $\mu_{ij}$ where $i$ and $j$...
1
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1answer
168 views

How to fix 5'UTR annotation with RNA-seq data?

I am studying the transcriptome of Arabidopsis. Interestingly, the 5' UTR of the latest annotation is usually too long. Here is an example (this gene is right to left). You can see both EST (orange ...
4
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3answers
811 views

Is it possible to merge scRNAseq data from experiments with different design?

I have 4 different single-cell RNAseq experiments, each one representing a different sample of cell type population. I'd like to merge them to a single dataset. However, different cell types are ...
3
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1answer
771 views

Strange per sequence GC content results

I would be grateful if someone could take a quick look at these FASTQC results. This is rna-seq paired-end data. From the FASTQC manual, an unusual distribution seems to be suggestive of contamination ...
5
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1answer
1k views

5' and 3' bias in Rna-seq data

I'm working with rna-seq samples. I see 5' bias and also 3' bias in the per-base sequence content plot. From this link I see that the bias at the start of the sequences appears to be the result of ...
4
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1answer
84 views

Why is ribosomal RNA difficult to remove even with Poly(A) selection?

In this answer (actually in a comment), it is stated that: As you've noticed from your own analysis, the ribosomal genes have quite variable expression across cells. They're expressed everywhere, ...
1
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1answer
110 views

Optimal design for an RNA-seq experiment with ERCC RNA Spike-In Control Mixes

I am planning an experiment on gene expression. I have 6 conditions and 3 replicates in each condition. I would like to use ERCC spike-in mixes for quality check: lower limit of detection, dynamic ...
1
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1answer
288 views

Alignment QC differences between HISAT2 and Qualimap

I used hisat2 for aligning reads to to the genome. I have an alignment summary for sample1 as follows: ...
4
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2answers
141 views

Exon-exon junctions: compare experimental transcripts to reference annotation

My aim is to parse an experimental transcript set (obtained by RNAseq) to check which splice junctions are already reported in a reference annotation and which ones are new. I tried ...
2
votes
1answer
565 views

Error in checkFullRank(modelMatrix) deseq2

Trying to use deseq2 for differential expression analysis (rna-seq) between three groups and also account for batch effect as the control were sequenced at a different time point. control: con ...
1
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2answers
942 views

ComBat for batch effects removal on scRNA-seq data

Is it possible to use sva's ComBat for batch effects removal on scRNA-seq data? Is there any difference between RNA-seq and scRNA-seq data which doesn't allow to use ComBat on single-cell data? (I am ...
3
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2answers
100 views

Detect transcript isoform abundance for a specific gene in scRNA-seq

I want to detect the count of isoform transcripts for a specific gene in scRNA-seq data. Data is coming from cells of Mus Musculus. For transcript isoforms I mean the different alternatives provided ...
5
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1answer
250 views

What are the count units in DEseq2?

This is a pretty silly/simple question, I suppose. What units are DESeq2 counts? Or are the units arbitrary but internally consistent estimate of actual reads?
1
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1answer
167 views

TCGA data sets joining multiple files

I have downloaded harmonised the TCGA-HNSC dataset and extracted it. It has 546 samples in total, so what I see in it are FPKM normalised values. The first column remains the same with 60000 rows and ...
7
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2answers
3k views

Are mitochondrial genes to exclude in scRNA-seq such as ribosomal genes?

In this answer, it is stated that ribosomal genes should be excluded prior to normalization in scRNA-seq as contaminants. Do mitochondrial genes have to be excluded as well? I plotted the top 50 ...
4
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2answers
690 views

Counting reads within the intron

I have paired-end RNA-seq samples and a list of intron coordinates in bed format. I want to count the intronic reads such that: ...
1
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3answers
147 views

Intron retention events for a condition sample with 3 replicates

I have 3 biological replicates of RNA seq data for a particular condition. I want to find out intron retention events from those biological replicates for a given condition. There is no comparison I ...
5
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1answer
335 views

What is the ICGC normalized_read_count?

I downloaded gene expression data (exp_seq) from the ICGC file browser. For each sample and gene, the file contains a ...
11
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2answers
2k views

determining doublets in single-cell RNA-seq

Doublets are a known problem with scRNA-seq experiments, where 2 or more cells are sometimes captured instead. To determine their presence, there are studies that mix multiple species (such as human ...
5
votes
2answers
475 views

Difference between de novo transcriptome assembly methods

I have been looking around (including read the original papers) to understand what is essentially the difference between StringTie in non-reference based mode (de novo) and Trinity de novo assembly. I ...
1
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2answers
1k views

How to filter ribosomal RNA from scRNA-seq data

I want to filter out ribosomal RNA from scRNA-seq data (downloaded from here). Is there a list of known ribosomal RNA? The only solution I found is SortMeRNA, however it works with raw sequencing ...
5
votes
1answer
335 views

Single-cell RNA sequencing (scRNA-seq): filtering cells by transcript counts, how to choose cutoffs?

I am running a notebook with example for the MAGIC algorithm. In the data preprocessing step, a filtering operation is required to filter out cells with a small count of transcripts. My question is ...
3
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1answer
315 views

R: 'Matrix' can not be unloaded, but 'writeMM' method not found

I want to save a sparse and very large dgcMatrix onto disc. I read that it could be done with writeMM method. So, when I am ...
1
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1answer
1k views

Error in colSums(assay(sce)) : 'x' must be an array of at least two dimensions

I am having a SingleCellExperiment (sce) object: sce <- SingleCellExperiment(list(counts=UMI_count)) which has two dimensions: ...
5
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1answer
70 views

How to compute the chance of failing to detect a gene given the detection limit of a protocol

In Shapiro et al., when discussing about loss of molecules as source of error in single-cell sequencing, it is written that: Another source of error is losses, which can be severe. The detection ...
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0answers
225 views

Error in .computeSumFactors: cells should have non-zero library sizes

While trying to run the following code in R: ...
3
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1answer
235 views

Large dataset normalization for PCA

I need to normalize a large (400Mb) dataset for doing PCA analysis. I want to use scran for doing that: ...
5
votes
3answers
121 views

Show presence of known mutation in RNA-seq data

We have RNA-seq fastq data from control (WT) patients and a patient with a point mutation at a known location in one gene. I'd like to retrieve the reads aligning to that gene and show the presence of ...
5
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1answer
795 views

Normalizing RNAseq for PCA and CCA

Usually the expression data is transformed to log space using either RPKM, FPKM or CPM, this is required when looking for differential expression because the data is tested against the normal ...
5
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1answer
201 views

Filter out outliers of the scRNA-seq (heterogenous cells)

I am new to data science. I have a dataset of single-cell gene expression from multiple cell types in C. Elegans. The dataset is from the paper Comprehensive single-cell transcriptional profiling of a ...
2
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0answers
389 views

Filter out PCA outliers automatically

I am new to bioinformatics and PCA. What I am trying to do is to remove bad cells from a dataset that was obtained with scRNA-seq for ...
2
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1answer
62 views

Pipeline (Pypiper) returns error, but individual command works fine

I am running a script file on a cluster with slurm through sbatch. Locally it runs perfectly well on 2 different linux versions (...
3
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1answer
835 views

sorting BAM file error using samtools

I have few bam files and would like to get read counts using samtools idxstats [Data is aligned to hg19 transcriptome]. To use that command I need a sorted bam ...
4
votes
1answer
839 views

Read counts from BAM file

I have few BAM files which are generated from Ion Torrent Server (ampliseq) aligned to hg19 genome. I want to extract read counts from the bam files and I know that "featureCounts" can be used for ...
4
votes
1answer
87 views

Determine if a gene is mitochondrial or not

I need to determine whether a gene is mitochondrial or not for C. Elegans automatically from its name by running a regular expression on a dozen of thousands of gene names. Currently I am having gene ...
2
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1answer
45 views

Get gene annotation type from gene name

I am having RNA-seq data set for C.Elegans. I do not know which annotation scientists used for naming the genes of it, but I ...