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9 votes
Accepted

Biological meaning of read length

The read length has absolutely nothing to do with what you are sequencing. It is a characteristic of the sequencing technology you use. NGS sequencing techniques typically produce this sort of short ...
terdon's user avatar
  • 12.9k
7 votes

Why is the GenBank entry for the genomes of RNA viruses like coronavirus written as DNA?

It would appear that the current policy at GenBank is to represent all genomic sequences as DNA, even though this is not made explicit in any of the easily retrievable documentation on their website ...
David's user avatar
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6 votes
Accepted

What is the best method to check for differential gene expression in large dataset? I want to identify aging differentially expressed genes

A standard pipeline for DGE would be Salmon for (pseudo)mapping + DESeq2 for statistical analysis. Salmon is one of a set of modern, fast and accurate mapping software. Requires a transcriptome to be ...
Greg's user avatar
  • 224
5 votes
Accepted

Tools to analyze RNA-seq data

You will likely need a tool to "map" the reads on the reference genome. You may find such a reference genome, together with annotations, here: ftp://ussd-ftp.illumina.com/. Mapping tools such as ...
bli's user avatar
  • 2,195
5 votes

What are the reasons for using oligo-dT instead of oligo-U to isolate mRNAs?

Note - This answer only covers the comparison of rA-dT (RNA:DNA) and rA-rU (RNA:RNA) duplexes, and not more exotic options involving deoxyuridine (dU) and ribothymidine (m5U). I can think of three ...
acvill's user avatar
  • 8,326
5 votes
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What is the difference between 4th generation sequencing and NGS?

I think that all of this is pure marketing and can be safely ignored. There is no basis for the generations of sequencing in chemistry or instrumentation, except possibly that second-generation (...
Maximilian Press's user avatar
4 votes
Accepted

Why analyse transcriptome instead of proteome?

Q: Why not just extract the proteins… A: It’s not just a question of extracting the proteins, you would need to separate them and then isolate each of them. There is currently no practical way to do ...
David's user avatar
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4 votes
Accepted

Confusion about read counts distribution in RNA-seq

Textbook example of multinomial distribution is multiple dice toss. The fair dice has following probabilities: ...
BagiM's user avatar
  • 583
4 votes
Accepted

What can be the issue during the total RNA extraction using QIAGEN kit?

As mentioned in the comments, RIN are about 2.2 - 2.4. These values are typically too low for RNA-seq, and indicate that the samples are quite highly degraded. One study by Reimann et al1 found that ...
bob1's user avatar
  • 12.5k
3 votes

Biological meaning of read length

My understanding is that if the reads come straight from the sequencing machine, they will all be the same length. That corresponds to the number of sequencing cycles that the machine was set to ...
bli's user avatar
  • 2,195
3 votes

Tools to analyze RNA-seq data

While I also agree @bli that R and Python (in particular Bioconductor) have more than enough packages for you to compare gene expression. You shouldn't align your ...
SmallChess's user avatar
  • 1,029
3 votes

16s rRNA Sequencing From Gut Microbiome (stool)

In general, you extract DNA, then PCR out the 16rRNA coding regions and finally sequence them. Here some links http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/ http://journals....
alec_djinn's user avatar
  • 3,108
3 votes

correction of read counts for spikes

The most common and simplest normalization method for spikes is the Total Count Normalization. It's actually what you're doing here, so what you're doing here is correct. TCM is a useful algorithm for ...
SmallChess's user avatar
  • 1,029
3 votes

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

Our DNA core wants 1.5 ug of RNA. I find that I can obtain that amount from a standard Trizol prep from about 1 mg of Arabidopsis floral tissue. I usually double or triple what I harvest to account ...
biologist's user avatar
3 votes

Correct structure of RNA

Both figures include A,G,T,C. Those are abbreviations for the bases adenine, guanine, thymine and cytosine. RNA does not contain thymine, but its unmethylated form uracil, abbreviated with an U. ...
Arsak's user avatar
  • 715
3 votes

If a person is infected with an RNA virus what percentage of the RNA in the blood of the person contains RNA of the virus?

I think your question is: Can you diagnose someone with an RNA virus by looking for that RNA in their blood? The use of nucleic acid tests, or NATs in diagnosis has been around for a long time. ...
De Novo's user avatar
  • 8,811
3 votes

Why analyse transcriptome instead of proteome?

Why not just extract the proteins and sequence them, quantify them, analyse and draw conclusions? Because proteomics is really hard. To provide one example, there is no protein equivalent of PCR. ...
Charles E. Grant's user avatar
3 votes

RNA sequencing and taxonomic analysis of gut microbiome

The article that you link to clarifies in the methods section that there are two different extraction protocols employed, one for 16S information (probably DNA extraction) and one for total RNA. In ...
Maximilian Press's user avatar
3 votes

snRNAseq vs scRNAseq in cancer

This is a very interesting question and I will give you an outline of the scenario I think is most likely. I'm using the same hypothesis to explain another common observation in single nuclei RNA-Seq: ...
PPK's user avatar
  • 196
2 votes
Accepted

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

Normalization of expression data is a big topic with new methods being published regularly. When approaching something like this you generally want look at people who have done similar things to what ...
Dermot Harnett's user avatar
2 votes

GO terms for non-model organisms

Updating this thread to include EnTAP, (Eukaryotic Non-model Transcriptome Annotation Pipeline) which wraps protein domain assignment, orthologous gene family assessment, Gene Ontology term assignment,...
geneticatt's user avatar
2 votes
Accepted

Iron metabolism in the brain

Many articles focus on the pro-oxidant function of iron (and globins in particular). It makes sense that levels may be higher in the CNS due to their role in respiration, which is increased secondary ...
Roby Vicary's user avatar
2 votes
Accepted

Sequence of ribosomal RNA

Unequivocally, YES. Before DNA sequencing was used to deduce the sequence of cloned or reverse-transcribed RNA molecules, slower and more cumbersome direct sequencing of RNA was performed using the ...
David's user avatar
  • 26.3k
2 votes

Tools to analyze RNA-seq data

I think that STAR is the preferred splice-aware aligner nowadays. STAR can output counts by gene or by transcript. Assuming you have Illumina data, you can try using the tools on Illumina's ...
swbarnes2's user avatar
  • 5,230
2 votes

Tools to analyze RNA-seq data

The answer @bli gave is great. I thought I would point out that Johns Hopkins also recently upgraded their tuxedo suite. Looks promising and has great instructions for use. Also, I've begun to grow ...
aaiezza's user avatar
  • 123
2 votes
Accepted

What factors should I consider when selecting a reference genome for mapping?

There are many reasons! Let's assume you are using the Human reference genome. The latest version is hg38 or GrCh38. This came out roughly three years ago (Dec 2013). Although now these same reasons ...
FoldedChromatin's user avatar
2 votes

the meaning of "Non-primary hits" and "tags"

"tag" is a weird term in this context. It used to mean "read", but RSeQC likes using it in an odd way. Suppose you have a read that aligns to a single contiguous (though possibly with InDels) stretch ...
Devon Ryan's user avatar
2 votes

where i can download reference MSU7 rice genome?

You can find this on the phytozome website. You will have to make a free JGI account to access it
mimat's user avatar
  • 1,425
2 votes
Accepted

Should I use RNAseZAP on my bench and pipettes before RNA extraction?

The common practice in my lab is to work in a laminar flow cabinet and clean it with alcohol, then spray it with the RNAseZAP. I've always heard from my professors that, while DNAses are more uncommon,...
LinuxBlanket's user avatar
  • 1,313

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