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

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DNA and aminoacid sequence [on hold]

5’ ATG TCA ACT CGG GCA ACA CAT TGT CTG TAT GAC GAA TAA TTA ACG 3’ TAC AGT TGA GCC CGT TGT GTA ACA GAC ATA CTG CTT ATT AAT TGC Q1: What will be the resulting ...
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3 end RNA seq library construction

I have tried to make cDNA libraries from FFPE tumor blocks (highly degraded RNA) using the 3' end RNA seq protocol provided by West lab. Here is the link for the same. ...
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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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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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86 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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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 ...
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66 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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Is the default kmer 25 value in Trinity still ok for assembly of 150bp reads from Illumina HiSeq 2500?

I will soon be receiving Illumina HiSeq 2500 data (150bp PE stranded reads). In the past we have been using Trinity as our assembler of choice, but it uses a default kmer value of 25. I believe this ...
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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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69 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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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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Data analysis of transcriptome sequencing data

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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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 ...
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RT-qPCR of selected differential expressed genes: which dilutions in calibration curve?

I've seen in MIQE guidelines that calibration curves a needed when performing RT-qPCR. I'll use genomic DNA to perform these calibration curves. My question is that I don't know the range of ...
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37 views

Linearity with SuperScript® VILO™ Master Mix?

I would like to know your experiences with SuperScript® VILO™ Master Mix. According to de manufacturer, this kit maintains linearity from 1 pg to 2.5 µg in a 20 µL reaction. enter link description ...
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94 views

New GO terms after TopGO enrichment?

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 ...
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420 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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49 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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102 views

how to clone a gene fragment in two direction as sense and antisense in vector

I have a sequence ATG GGG CCC TTT AAA TAA and want to use it as antisense RNA in my vector. How should I clone it? I am confused with the direction of my clone. I looking for the direction and ...
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641 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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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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107 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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142 views

Can PolyA tails be within Expressed Sequence Tags?

Can polyA tails occur within (rather than at the end) of a sequenced tag? Consider, for example the following two sequences from NCBI: DY008075 ...
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880 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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688 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' ...
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179 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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233 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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67 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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823 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 ...
7
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66 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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120 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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153 views

Too few transcripts from transcriptome assembler Oases

I am trying to run Oases for transcriptome assembly. The result is far from expected, so I would like to ask whether I am running it in a right way? Thanks. Here is my running command: ...
7
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1answer
104 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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5k 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 ...