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Questions tagged [rna-sequencing]

Determining the sequence of RNA. This technique is used for discovery and quantification of mRNAs and ncRNAs.

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10k views

Difference between strand-specific and not strand-specific RNA-seq data

I would like to ask the difference between strand-specific and not strand-specific dataset. As far as I know, strand-specific data means that we know which strand the transcript is from. I do not ...
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1k views

RNA-Seq library construction challenges: the biases of RNA fragmentation vs cDNA fragmentation

I recently watched a presentation on RNA-seq that covered some of the choices one can make along the way, and I didn't fully understand one of the choices in particular. Near the beginning of the ...
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1answer
148 views

Iron metabolism in the brain

I did an expression profiling from publicly available RNAseq data for mouse tissues. While for liver and testes I am getting expected proteins in the top highly expressed mRNAs, for brain I am getting ...
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859 views

Biological meaning of read length

I have some FASTQ files in two datasets which are sequences from 16Srna region. The first dataset is amplicons form V4 region and the second is V3-V4 region. However all the reads are 250 nucleotides ...
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1answer
953 views

RNA seq and using of Poly(A) or non-Poly(A) based amplification of RNA

I'm studying "Deep sequencing the circadian and diurnal transcriptome of Drosophila brain" Hughes et al., 2012. I've got some problems with the materials and methods. Before RNAseq, the authors ...
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1answer
139 views

How can I normalize mRNA samples for sequencing?

Is there an easy, inexpensive, not too labor intensive way to normalise mRNA samples so that even though one loses information of gene expression levels, each of the transcripts in the transcriptome ...
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1answer
969 views

How much total RNA can be extracted from Drosophila brain

I am wondering how much total RNA could be extracted from a single D. melanogaster brain. I could not find this information from the literature. The closest hit was this paper, that claims that 16-21....
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2answers
120 views

Sequence of ribosomal RNA

Is it possible to sequence rRNA directly, that is, using the ribosome rather than the DNA from the nucleus? For example, this paper, Complete nucleotide sequence of a 16s rRNA gene from E. coli, ...
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4answers
352 views

Tools to analyze RNA-seq data

I hope this is a good place to ask such question. I have to do some data analysis on RNA-seq data from human cells. I am currently searching for tools to help me with that. Specifically, I would need ...
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1answer
1k views

Where to find E.coli gene expression data?

I am searching E.coli whole genome expression data with different conditions, any suggestion is appreciated. Condition could be for example different growth temperature, different medias, etc. I have ...
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2answers
4k views

Transcript(omics) terminology: cDNAs, ESTs, RNA-seq, etc

I've worked pretty frequently with genome and transcriptome data for several years now, but I'm still not 100% sure I understand the proper usage for certain terminology related to transcripts and ...
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2answers
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How were the first primers made?

I keep reading about how primers are useful in pcr -- they allow you to select a specific dna region. Similarly, in NGS or Sanger sequencing they give you a starting point. The primers I see are about ...
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2answers
1k views

Why is assembling paired end illumina without any input parameters an important problem?

In one of the comments in this question about multiple sequence alignment, it was stated @5heikki: btw if you want a good bioinformatics problem, come up with an assembler that assembles any paired ...
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2answers
471 views

The meaning of RNA-seq data

many papers I read mentioned "RNA-seq data". While searching for the meaning of this word, I could not find any layman's definition. As far as I understand, RNA-seq data is the complete RNA ...
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2answers
939 views

How to find Exons in mRNA Computationally

I'm having trouble finding a method to find exons in the original DNA sequence used to create the mRNA, even given the sequence of the mRNA, as I cannot find a way to reliably identify the beginning ...
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1answer
12k views

meaning of the “reads” keyword in terms of RNA-seq or next generation sequencing

I'm an undergraduate student at computer science and currently, I'm interested in bioinformatics. Today, I've started to read a paper about clustering and classification of non-coding RNAs can be ...
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1answer
88 views

Genes-of-interest analysis between organisms

I am interested in identifying the differential expression between several genes-of-interest of different organisms. I am trying to assess the benefits of RNA sequencing over microarrays. Both ...
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1answer
146 views

Is it possible to sequence only the 16S rRNA component? If so, how?

There have recently been several papers on using 16S rRNA as a way of identifying species (here, and here). I'm wondering if it's possible to sequence either just that subunit of the ribosome or just ...
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5answers
216 views

Why analyse transcriptome instead of proteome?

Analysing the transcriptome (RNA-Seq, microarrays, qPCR etc) is probably the most widely used technology to assess gene expression and dynamic cellular processes. The results are then extrapolated (...
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1answer
96 views

determining meaning of basic biological keywords about C. elegans

First of all I have to say that I have no biology background since I'm a undergraduate computer science student. Nowadays, for my research I need to use some of the databases related with ...
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2answers
686 views

GO terms for non-model organisms

I have a list of differentially expressed genes from an RNA-Seq experiment in Xenopus laevis that I'm looking to functionally annotate with GO-terms. As X.laevis is not listed in DAVID it seems I have ...
4
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1answer
65 views

RNA-seq Data on domestic animal with different environment

I want to find the RNAseq data available on domestic animals in different environments. If microarray data is available, it would be more useful. If you know I would appreciate to inform me.
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1answer
637 views

How could I identify whether given RNA-seq data is paired end or single end

I need to have a RNA-Seq dataset and therefore, I've visited the following site NCBI-geo C. Elegans In the Supplemantary file part, I clicked the SRP/SRP051/SRP051702 ftp and downloaded sra file. ...
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2answers
256 views

New GO terms after TopGO enrichment? [closed]

I ran an enrichment analysis with custom annotations using TopGO and surprisingly I obtained new GO terms inside the significant GO terms. Is that possible? The only reason I can imagine this is ...
4
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1answer
108 views

How can chromatin state be measured?

I have some RNA-Seq data and I'd like to align it to the physical genome and see which sections of chromatin are geometrically open and being transcribed. The data are already sequence-aligned, and ...
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2answers
290 views

Current state of direct RNA sequencing

I had a colleague ask me recently whether mRNAs could be sequenced directly. I found this Nature paper[1] published by Helicos in 2009, in which they describe their developments in the area. It's been ...
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2answers
126 views

ChIP-seq for histone modification not in agreement with RNA-seq for expression

I have ChIP-seq for H3K79me2 and H3K36me3 and RNA-seq data for treated and untreated samples. Those two histones mark active genes. Lets say, hypothetically, a peak caller finds differential sites at ...
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1answer
96 views

correction of read counts for spikes

I have 4 RNA-seq samples (1st is control and the other 3 are treatments) and 5 spikes. I used different concentrations for 5 spikes but over different samples, I used similar concentration. for ...
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2answers
1k views

What is the most appropriate way to normalize gene expression data?

This question comes because reading a paper about normalization of gene-expression data, is not clear if the method for normalize the data is just for RNA-Seq data or could be applied also for ...
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1answer
242 views

Should the sum of RPKMs be constant over experiments

I have cufflinks data from 18 different RNA-seq experiments. I've noticed that if the sum all the RPKM values for a particular experiment, are vastly different between experiments. How could this be? ...
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1answer
3k views

RNA-seq analysis - q-values in cuffdiff

I'm using cuffdiff 2.1.1 to look for differential gene expression between two conditions. Each condition has 2 biological replicates. The results I get look promising from a log fold change ...
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1answer
280 views

How to predict a mRNA secondary structure with a large sequence?

When I use some web servers to predict a mRNA secondary structure, I find they always required in a small size sequence. If I use a long sequence and cut it into small parts, do these small parts ...
3
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1answer
315 views

What is meant by single molecule sequencing?

When sequencing papers refer to single molecule sequencing, what is their definition of a "molecule". Are they saying base by base? The entire DNA chain in a chromosome can also be though of as a "...
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1answer
5k views

What is the purpose of Y-shaped adapters in Illumina sequencing?

Y adapters different sequences to be annealed to the 5' and 3' ends of each molecule in a library. The arms of the Y are unique, and the middle part, connected to the DNA fragment, is complementary. ...
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Data analysis of transcriptome sequencing data [closed]

I want to learn more about the data analysis and statistics on transcriptome sequencing data. I would like to read some important papers of the field and books and maybe some MOOCS, if they are ...
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0answers
161 views

Good poly-A filtering rules or tools

I am aligning a large number of ESTs. It seems poly-A tails show in many different ways. In addition to occurring at the very end, they can be flanked by the cloning sequence one one end, or have ...
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2answers
933 views

Using RNA-seq to compare gene expression across patients instead of between Control and Experimental conditions

I am working with RNA-seq data from the Cancer Genome Atlas TCGA and I have been reading about how people have compared gene expression levels measured by RNA-seq. Many of the papers I have read talk ...
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1answer
22 views

Metatranscriptomics Extraction

I am attempting to perform metatranscriptomics analysis of a cleanroom. The DNA input is known to be rather low (<1pg). Despite this, I want to still attempt to try it. I am thinking it might be ...
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2answers
91 views

How much tissue is required to do RNA-seq analysis on a single organism?

How much tissue would be required to do RNA-seq analysis on a single organism? More specifically, if a person wanted an RNA-seq analysis of expression for a single organ, how much tissue would they ...
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1answer
163 views

Questions on adding a protein to a DNA library [closed]

Two questions regarding finding the DNA sequence of a amino acid sequence (AA): 1) If you are able to find out the mRNA sequence of an AA, then don't you automatically know the DNA sequence? 2) ...
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1answer
441 views

Differential gene expression analysis between species

I have RNA Seq data from mouse and human skin ( 2 replicates each) and want to compare the expression of the orthologous genes to find any which are differentially expressed. I have quantile ...
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2answers
103 views

In genome research, what is the problem in Mapping that may be caused by reads being too short?

In the following scenario: You were given short sequence reads of plant RNA obtained from a next-generation sequencing machine (fragments of 20–30 nucleotides in length). You attempt to map them back ...
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1answer
1k views

Problems with analysis of small RNAseq data - Adapter trimming

I have always faced a problem while analyzing small RNAseq data, at the step of adapter trimming. Overview of small RNAseq (Illumina) RNA is size fractionated using columns or PAGE 3' and 5' adapter ...
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2answers
223 views

Which information can be extracted from time course RNA-Seq Data?

So I am very new to the area of biology so I am sorry if this is a stupid question. I have RNA-Seq data carried out over a span of 100 days and I have gene expression data in the following format. ...
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1answer
155 views

Do all microRNA isoforms need to be known and sequenced to obtain microRNA expression?

From what I understand, microRNA are short "hairpin" shaped nc structures. When sequencing and counting miRNA, isoform miRNA are "found" which are subsections of varying length and possibly with some ...
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2answers
73 views

Is it possible to identify a particular gene sequence from next gen sequencing techniques (especially RNA-seq )?

I have to check the expression of a gene in a fish whose sequence is not known in the fish in question. Sequence is known in an another fish (zebrafish) but the gene has 10 isoforms. The genome of ...
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1answer
529 views

How do carrier RNAs increase yield in sequencing experiments?

I would like to know how carrier RNAs increase yield in RNA/DNA sequencing experiments. Is their main function in the precipitation steps of each protocol (i.e. small quantities are difficult to ...
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2answers
390 views

During bridge amplification of DNA sequences, why aren't sequences amplified in both orientations?

During bridge amplification, when sequences attached to adapters on the surface form "bridges" and are replicated, it seems like sequences with either end attached to the surface will be created. For ...
2
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1answer
547 views

Regarding the difference between cDNA library and RNA sequencing (Biochem. technique)

I was wondering why establishment of cDNA library required the step of reverse transcription (i.e. turning the sequence into DNA) why not directly using the extracted RNA for sequencing and converting ...
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0answers
64 views

RNA contains Uracil in place of Thymine [duplicate]

Thymine does not present in RNA but DNA containing thymine why