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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18
votes
3answers
4k views

How exactly is “effective length” used in FPKM calculated?

According to this famous blog post, the effective transcript length is: $\tilde{l}_i = l_i - \mu$ where $l_i$ is the length of transcript and $\mu$ is the average fragment length. However, typically ...
17
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2answers
360 views

Confirm success or failure of RNA-Seq normalization

I am working with a set of (bulk) RNA-Seq data collected across multiple runs, run at different times of the year. I have normalized my data using library size / quantile / RUV normalization, and ...
17
votes
3answers
1k views

What is the actual cause of excessive zeroes in single cell RNA-seq data? Is it PCR?

First, sorry if I am missing something basic - I am a programmer recently turned bioinformatician so I still don't know a lot of stuff. This is a cross post with a Biostars question hope that's not ...
17
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2answers
2k views

How can we distinguish between true zero and dropout-zero counts in single-cell RNA-seq?

In single-cell RNA-seq data we have an inflated number of 0 (or near-zero) counts due to low mRNA capture rate and other inefficiencies. How can we decide which genes are 0 due to gene dropout (lack ...
16
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4answers
5k views

How to compute RPKM in R?

I have the following data of fragment counts for each gene in 16 samples: ...
14
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2answers
1k views

Alignment based vs reference-free (transcriptome analysis)?

I want to focus on transcriptome analysis. We know it's possible to analyze RNA-Seq experiment based on alignment or k-mers. Possible alignment workflow: Align sequence reads with TopHat2 Quantify ...
13
votes
2answers
2k views

Normalization methods with RNA-Seq ERCC spike in?

ERCC spike-in is a set of synthetic controls developed for RNA-Seq. I'm interested in using it to normalize my RNA-Seq samples. In particular, I'd like to use the spike-ins to remove technical bias ...
12
votes
4answers
2k views

What methods are available to find a cutoff value for non-expressed genes in RNA-seq?

I have a gene expression count matrix produced from bulk RNA-seq data. I'd like to find genes that were not expressed in a group of samples and were expressed in another group. The problem of course ...
11
votes
5answers
209 views

How do I efficiently perform a metagenome screen of “all” species?

I’ve got an RNA-seq dataset with a large proportion of environmental RNA “contamination”. BLASTing random reads reveals that much of the data comes from bacterial, plant and viral RNA. My target ...
11
votes
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 ...
11
votes
1answer
186 views

Classifying samples based on marker gene expression

I have a few sets of marker genes that I can classify RNA-seq samples using semi-supervised clustering. I would like to automate the process, however, I am struggling to find the ideal algorithm that ...
10
votes
4answers
671 views

What methods exist to calculate RNA expression profile similarity

Some of the work in our lab requires a comparison of a strain across several experimental conditions. We are looking to identify most similar experimental conditions based on the gene transcription ...
10
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2answers
2k views

Run kallisto iteratively across many samples

I am on a Mac using UNIX. I am trying to use the kallisto quant command on all files in a directory (instead of manually entering them). Because I'm running the analysis against the same index file, I ...
10
votes
2answers
12k views

Difference between CPM and TPM and which one for downstream analysis?

What the difference between TPM and CPM when dealing with RNA seq data? What metrics would you use if you have to perform some down stream analysis other than Differential expression for eg. ...
10
votes
2answers
571 views

Can I model technical replicates in DESeq2?

I’d normally use collapseReplicates (or do the collapsing upstream) to handle technical replicates. However, in my current RNA-seq experimental design, samples ...
10
votes
1answer
631 views

Quantifying reads mapping to multiple loci

I have been using STAR for our RNA-Seq samples. The final.out log file reports percentage of uniquely mapped reads along with percentage of reads that map to ...
10
votes
2answers
634 views

*very* unbalanced group sizes for DE

I downloaded some publicly available RNA-seq data and want to compare those samples carrying a mutation (~4) against the rest (~800!). I ran both EdgeR and DESeq2, and the first results in an ...
9
votes
3answers
1k 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 ...
9
votes
3answers
431 views

Visualisation of long read RNA-Seq splicing

I have a dataset of Oxford Nanopore cDNA reads. Many of my reads are full-length or close to full-length transcripts, and I and am interested in examining alternative splicing. For this, I would like ...
9
votes
2answers
819 views

Duplicate genes with RSEM counts: Which one to choose?

I have Ensembl ids in the first column and samples with RSEM counts data in other columns. I converted Ensembl ids to gene symbols. Now I see there are three genes repeated twice. ...
8
votes
3answers
1k 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 ...
8
votes
2answers
433 views

Building STAR Genome Index for nanopore RNA sequencing

I am aligning a dataset of 1,000,000 reads oh human mRNA sequenced on Oxford Nanopore Technologies' MinION, and would like to use the STAR aligner, using the parameters recommended by Pacific ...
8
votes
4answers
220 views

How can the cell line contribution be estimated from RNASeq data?

Using a laser-capture microdissection of cells a group of cells stained with the marker of interest was sequenced. In another cohort of patients (this is all human liver tissue) the whole tissue was ...
8
votes
4answers
95 views

Introduce errors in reference transcripts according to external dataset error model

I would like to modify some reference transcripts from Ensembl (D. melanogaster) to introduce a controlled rate of random errors in the sequences. The idea would be to introduce random base ...
8
votes
2answers
781 views

Getting a “system is computationally singular” error in sleuth

I am analysing 142 samples belonging to 6 batches. Additionally, those samples belong to 72 strains, which means that for most of the strains there are two samples. I could fit simple models (for ...
8
votes
1answer
350 views

When performing differential expression analysis, should genes with low read counts be removed before or after normalization?

I have RNA seq data which I've quantified using Kallisto. I'd like to use tximport to transform the read count data into input for EdgeR, following the R code supplied in the tximport documentation: ...
8
votes
1answer
3k views

How to apply upperquartile normalization on RSEM expected counts?

I see that TCGA RNASeq V2 RSEM data is normalized with upper-quartile normalization. After doing Quantification with RSEM with the samples I have, I got "genes.results" as output which has gene id, ...
8
votes
1answer
459 views

Sleuth: transcripts with beta close to 0 are considered differentially expressed in a likelihood-ratio test

I'm comparing the results that I obtain when doing a DE analysis with the Wald test and the likelihood-ratio test. One the thing that I've noticed is that there are many genes with 'beta' close to ...
7
votes
2answers
431 views

differential gene expression complex design no replicates

We have an experimental design as seen below Where we administered drug at 0 min for each mouse genotype and took them down at given below intervals. wt and ko mouse models were administered only ...
7
votes
2answers
965 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 ...
7
votes
2answers
2k views

Filtering step for read counts data

I have around 1200 samples as columns and 60,000 genes with Htseq-Counts data. Before normalization with voom function I want to do filtering step. I want to remove genes whose expression is == 0 in ...
7
votes
2answers
305 views

Which tools can detect chimeric RNA (fusion genes) from WGS or RNA-Seq data?

Given WGS data or RNA-seq data, which tools can I use to detect gene fusions?
7
votes
2answers
2k views

Which measure should be used in a PCA or RNA-seq data? TPM or counts?

I'm trying to understand the magnitude of batch effects in my RNA-seq samples, and I was wondering which expression units are more suitable to draw a PCA. I'm thinking of either ...
7
votes
2answers
4k 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 ...
7
votes
1answer
764 views

Coverage calculation: long reads (RNA-seq)

Say your aim is to calculate the coverage of an RNA-seq experiment generated with long-read sequencing (so, uneven read length). Up to now, I relied on the Lander/Waterman equation: $$C = L*N / G$$...
7
votes
1answer
453 views

Interpreting Intergrative Genomic Viewer (IGV)

I was following a tutorial on "Tuxedo Genome Guided Transcriptome Assembly Workshop" and was wondering how to interpret the following: From what I understand from 'Color Legends', the color blue ...
7
votes
1answer
138 views

How can HISAT2/StringTie report decimal coverage values

I have performed RNA-seq analysis using HISAT2 & StringTie workflow suggested in: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Some of the ...
7
votes
2answers
690 views

Spliced vs. unspliced ratios for transcripts in RNA-seq data

Is there a computational tool for measuring what percentage of RNA is spliced in an RNAseq experiment? I'm not particularly interested in complicated analyses that give ratios for all possible ...
7
votes
1answer
83 views

Network comparison of single cells (from sequencing data)

What methods can we use to compare networks (PPI, gene regulatory etc) created from single-cell gene expression (or proteomics) data? There are methods that construct networks by comparing two ...
7
votes
2answers
334 views

Correct for gene length or read counts in GO enrichment analysis

It is a well reported fact that GO analysis of RNAseq results is affected by a number of biases, including length bias and expression level bias. The bioconductor ...
7
votes
1answer
477 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 ...
7
votes
0answers
83 views

Run cuffcompare in strand-agnostic mode

Is there a way to run Cufflinks' cuffcompare in a strand-agnostic mode? I would like to do this because I have some RNA-seq datasets derived from an unstranded run, that should be compared to a ...
6
votes
3answers
7k views

Volcano plot in R

This question has also been asked on biostars How can I reproduce this volcano plot? I'm only able to do the traditional one, I'm kind knew too these field.
6
votes
2answers
162 views

Clarification on Gene Enrichment

When I run a GSEA analysis on two conditions from the same RNaseq (negative control PBS injection VS positive control CpG injection) from the same dataset/same gene list, I get results that look ...
6
votes
3answers
117 views

How can I compute gene expression for a set of RNA reads?

I'm trying to compute a gene expression profile for an organism. I have gene nucleotide sequences of the mentioned organism stored in a fasta file and a set of paired reads stored in two separate ...
6
votes
3answers
3k views

Removing PCR duplicates in RNA-seq Analysis

After reading some of the forum posts in Biostar and SeqAnswers I find it very confusing whether to filter out the duplicate reads from aligned files or not. As far I understand it's very difficult to ...
6
votes
3answers
1k views

RIP-seq analysis?

Given an experiment consisting of an input (baseline RNA) and IP (pulldown to find RNAs bound to certain protein of interest)... Is a DE analysis performed over the RNA-seq data from the samples (lets ...
6
votes
3answers
200 views

Filter Trinity transcriptome based on RNASeq reads

I have recently generated a genome-guided transcriptome with Trinity, and would like to apply an additional filter to exclude transcripts that don't have good support from the RNASeq reads. This is ...
6
votes
2answers
452 views

Missing genes and normalisation of RSEM output using EBSeq

Without going into too much background, I just joined up with a lab as a bioinformatics intern while I'm completing my masters degree in the field. The lab has data from an RNA-seq they outsourced, ...
6
votes
2answers
2k 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 ...

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