9 votes
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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 ...
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8 votes

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

The Next-Gen sequencers cannot sequence a very long stretch of DNA with good reliability (~150 for the recent model- HiSeq2000; even less for older models such as GA (40), GA-II (70), GA-IIx (90)). ...
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8 votes
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How to convert FASTQ file format into GTF file format?

You should really read about both these file formats. As swbarnes mentioned, FASTQ and GTF hold different kind of information. GTF stores the annotation of a reference sequence. For example a GTF for ...
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7 votes
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Where to find E.coli gene expression data?

There are some databases in which you can search for E.coli gene expression data: GenExpDB: E. coli Gene Expression Database Many Microbe Microarrays Database (M3D): A resource of microbial gene ...
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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 ...
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6 votes
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meaning of the "reads" keyword in terms of RNA-seq or next generation sequencing

In RNA sequencing, the RNA is fragmented, DNA is synthesized complementary to the RNA fragments, which is followed by a complementary strand synthesis. Fragmentation can be done after the cDNA ...
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6 votes
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The meaning of RNA-seq data

RNA-seq data usually provides a snapshot in time of the transcriptome of that which is being sequenced. Single-cell sequencing is possible, but less common than RNA-seq on a sample (containing many ...
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6 votes
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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 ...
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5 votes
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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 ...
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  • 2,185
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 ...
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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 (...
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4 votes

How were the first primers made?

The process of sequencing was, and can be, assisted by cloning a DNA fragment into a known site in a plasmid designed to help sequencing. That cloning site is flanked by a known sequence(s) that can ...
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  • 796
4 votes

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

In Illumina sequencing, the DNA is (usually randomly) sheared into fragments. For paired end sequencing, fragments of a specific size range are selected and then sequenced from both sides. This ...
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  • 1,981
4 votes

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

Good question, a lot of this is still being figured out. Here's what's known so far: Fragmentation methods based on restriction enzymes aren't random. Reverse-transcription performed with poly dT-...
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  • 3,675
4 votes

Genes-of-interest analysis between organisms

In RNA sequencing, total RNA is extracted, then mRNAs are purified out from the sample using polyT columns (since mRNAs have a polyA tail, this will attach to a polyT DNA chunk that is attached to ...
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  • 3,078
4 votes
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determining meaning of basic biological keywords about C. elegans

Have you discovered wormbase yet? It will become your new best friend. In worms gene names reflect the loss-of-function (lf) phenotype. Daf means "abnormal DAuer Formation," so daf-2 was the second ...
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  • 3,422
4 votes

What is the frequency distribution of each base in a DNA sequence?

The frequency of the bases in the genome isn't equal to 0.25, the frequency depends on what kind of organism you mean. However let's take a look at some of them: bacteria, most of the time we can see ...
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4 votes
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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 ...
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4 votes
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Confusion about read counts distribution in RNA-seq

Textbook example of multinomial distribution is multiple dice toss. The fair dice has following probabilities: ...
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  • 562
3 votes

How to convert FASTQ file format into GTF file format?

The Tuxedo Suite (Tophat, Bowtie and cufflinks) used to to process RNA_seq data, assuming that is the origin of your .fastq files, should work for you. https://ccb.jhu.edu/software/tophat/index....
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How to convert FASTQ file format into GTF file format?

Your question is not very clear. What should the contents of your GTF file be? Typically, GTF files contain information about where exons are located in a set of DNA sequences. Determining the ...
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3 votes

How to convert FASTQ file format into GTF file format?

A fastq contains sequences. A gtf contains coordinates of where features like exons fall in a reference sequence. You can't interconvert them, that makes no sense.
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3 votes

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

messenger RNA has a poli A tail at its 5´ end. thus when the poly-dT hybridize the mRNA in the reverse transcription the cDNA will carry this poly-dT at its 3`end. Thus, as reverse transcription will ...
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  • 1,013
3 votes
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Should the sum of RPKMs be constant over experiments

I guess this arises because of the default cufflinks option --total-hits-norm in which it normalizes the FPKMs with total reads including the ones that are not ...
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3 votes
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What do HG and NA mean in Geuvadis project RNAseq sample labels?

If you google HG00105, among the first hits is geo accession GSM649517 with the title HG00105/NA12878. Channel1: ...
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  • 275
3 votes
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ChIP-seq for histone modification not in agreement with RNA-seq for expression

It is difficult to draw any conclusion without further experimentation. There may be many other factors that prevent the expression of the gene including factors like post-transcriptional regulators. ...
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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 ...
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  • 1,007
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 ...
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  • 1,007
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 ...
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  • 2,185
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. ...
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