What is directional RNA-Seq?

Paired-end sequencing means that you obtain two reads from a given DNA fragment, one from each end, that may or not overlap depending of its size and the length of the reads. Directional sequencing means that, after sequencing, you know from which of both DNA strands a given RNA molecule is transcribed.

What is stranded vs non stranded RNA-Seq?

The read ambiguity in stranded RNA-seq arises only from overlapping genes transcribed from the same strand. In contrast, for non-stranded RNA-seq, the ambiguity arises from both the overlapping genes on the same strand and also from the opposite strands.

How is mRNA sequenced?

Each group of three bases in mRNA constitutes a codon, and each codon specifies a particular amino acid (hence, it is a triplet code). The mRNA sequence is thus used as a template to assemble—in order—the chain of amino acids that form a protein. The codons are written 5′ to 3′, as they appear in the mRNA.

What method is usually used for mRNA in RNA-Seq?

Summary of RNA-Seq. Within the organism, genes are transcribed and (in an eukaryotic organism) spliced to produce mature mRNA transcripts (red). The mRNA is extracted from the organism, fragmented and copied into stable ds-cDNA (blue). The ds-cDNA is sequenced using high-throughput, short-read sequencing methods.

How do I know if my Rnaseq is stranded?

If sequences of read 1 align to the RNA strand, the library is “stranded”. If sequences of read 2 align to the RNA strand, the library is “reversed stranded”.

What is library preparation in RNA-seq?

Current RNA-seq library preparation methods comprise preparing rRNA-depleted or poly(A)-enriched RNA followed by RNA fragmentation, cDNA synthesis, adaptor ligation and multiple cleanup steps. These methods are generally time consuming, requiring about 1.5 d and significant hands-on time.

How do I know if my RNA-Seq is stranded?

Is RNA-Seq strand specific?

Strand-specific RNA-Seq allows for sense and antisense transcript structures to be predicted, overlapping regions of transcription can be identified exactly and expression levels of sense and antisense genes can be more accurately estimated.

What is the function of messenger RNA mRNA?

Specifically, messenger RNA (mRNA) carries the protein blueprint from a cell’s DNA to its ribosomes, which are the “machines” that drive protein synthesis. Transfer RNA (tRNA) then carries the appropriate amino acids into the ribosome for inclusion in the new protein.

What is the difference between RNA-Seq and microarray?

The main difference between RNA-Seq and microarrays is that the former allows for full sequencing of the whole transcriptome while the latter only profiles predefined transcripts/genes through hybridization.

What is the purpose of RNA-Seq?

RNA-seq (RNA-sequencing) is a technique that can examine the quantity and sequences of RNA in a sample using next-generation sequencing (NGS). It analyzes the transcriptome, indicating which of the genes encoded in our DNA are turned on or off and to what extent.