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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4
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1answer
62 views

Analyzing Illumina Counts

I'm pretty new to all of this--forgive me if this is a simple question. When I download illumina counts from GEO (like the supplementary file in GSE89225). Can I do comparisons directly on that file?...
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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 ...
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2answers
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Can gene co-expression networks be used to help identify differentially expressed genes?

In my RNAseq dataset of differentiated stem-cell lines, some samples have far fewer significantly differentially expressed genes than others. QC shows that this is because there are way fewer reads ...
8
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1answer
466 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 ...
10
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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. ...
6
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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 ...
2
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0answers
66 views

ngs time course experiment [duplicate]

I have a question regarding the RNA-Seq analysis. We have a time course data regarding a mouse model wt and mutant treated with a drug (10uM) and the taken down at different time points: 8 time ...
11
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5answers
210 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 ...
4
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2answers
5k views

RNAseq: Z score, Intensity, and Resources

I'm very new to bioinformatics in general, and I'm trying to understand some basic concepts. I have RNAseq data, and bioinformatics people tell me that intensities cannot be compared across patients. ...
8
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1answer
351 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: ...
7
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2answers
434 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 ...
4
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1answer
346 views

insert size pre and post trimming

I have a problem here with my rna seq data: Sequencing details: rRNA was removed, followed by cDNA preparation and generation of stranded libraries using the TruSeq Stranded Total RNA Sample Prep Kit. ...
3
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1answer
443 views

ribo interval list for picard ensembl mm9 gtf

Could some one please help me with understanding how to generate the ribosomal interval list that is required to when using picard metrics this step ...
6
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3answers
203 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
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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 ...
4
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5answers
3k views

using python to write bioinformatics pipelines tutorial

I was wondering if there is a tutorial or a small code snippet to understand how to write bioinformatics pipeline using python, for example use a aligner (say hisat) get the output and process it ...
5
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1answer
246 views

PASA pipeline: compare experimental transcripts to the reference annotation

I would like to ask if anyone has experience in running a subset of the PASA pipeline, in particular for the reconciliation of some experimental 'transcripts' with the reference annotation. In more ...
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 ...
4
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1answer
92 views

Correlating gene expression with qualitative variables

I have a gene expression dataset that I want to investigate. Particularly, I would like to understand whether there is any correlation between each gene's expression and some quantitative or ...
8
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2answers
794 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 ...
3
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2answers
2k views

Handling sample identity in aggregated 10x libraries?

cellranger aggr can combine multiple libraries (samples), and appends each barcode with an integer (e.g. AGACCATTGAGACTTA-1). The sample identity is not recorded in ...
4
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3answers
552 views

Analysis of differential transcript usage (DTU)

Recent breakthroughs in bioinformatics tools for quantification (e.g. Cufflinks/Kallisto/Salmon etc.) and tools which can identify differential transcript usage (DTU) (e.g. DRIMSeq, Cufflinks etc.) ...
4
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1answer
2k views

10x Genomics Chromium single-cell RNA-seq data analysis options?

Provide an overview of 10x data analysis packages. 10x provides Cell Ranger which prepares a count matrix from the bcl sequencer output files and other files (see bottom of page https://support....
9
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3answers
440 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 ...
5
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1answer
65 views

Should the cell sorting marker genes be excluded during clustering?

We sort different populations of blood cells using a number of fluorescent flow cytometry markers and then sequence RNA. We want to see what the transcriptome tells us about the similarity and ...
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 ...
12
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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 ...
7
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2answers
728 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 ...
5
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1answer
605 views

What are the ways to process a list of differentially expressed genes?

We are studying six different human macrophage/dendritic cell types isolated from healthy skin. They all differ from each other in a few cell surface markers. We are interested in the characteristics ...
7
votes
1answer
139 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 ...
8
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2answers
440 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 ...
10
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4answers
679 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 ...
6
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2answers
453 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, ...
18
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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 ...
7
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2answers
309 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?
10
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1answer
640 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 ...
8
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4answers
222 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 ...
11
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1answer
192 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
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2answers
579 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 ...
17
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2answers
365 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 ...
16
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4answers
6k views

How to compute RPKM in R?

I have the following data of fragment counts for each gene in 16 samples: ...
13
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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 ...
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 ...

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