What does Strand Specific mean?

Strand specific libraries allow you to identify the coding DNA strand for the transcript you sequence, while the strand information is lost in what you call regular library protocols.

How do Strand specific RNA-Seq Kits Work?

Before sequencing, RNA samples have to be prepared using a protocol where the single-stranded RNA molecules are first converted into double-stranded complementary DNAs (cDNAs), followed by adapter ligation and PCR amplification. After sequencing, the generated reads can be mapped back to their transcripts of origin.

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.

What is the RNA strand?

An RNA strand has a backbone made of alternating sugar (ribose) and phosphate groups. Attached to each sugar is one of four bases–adenine (A), uracil (U), cytosine (C), or guanine (G). Different types of RNA exist in the cell: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).

How do you know if data 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 positive sense DNA?

An individual strand of DNA is referred to as positive-sense (also positive (+) or simply sense) if its nucleotide sequence corresponds directly to the sequence of an RNA transcript which is translated or translatable into a sequence of amino acids (provided that any thymine bases in the DNA sequence are replaced with …

What is the sense strand used for?

The sense strand has the information that would be readable on the RNA, and that’s called the coding side. The antisense is the non-coding strand, but ironically, when you’re making RNA, the proteins that are involved in making RNA read the antisense strand in order to create a sense strand for the mRNA.

Why are strand specific RNA seq libraries important?

Strand-specific (also known as stranded or directional) RNA-seq libraries substantially enhance the value of an RNA-seq experiment. They add information on the originating strand and thus can precisely delineate the boundaries of transcripts in regions with genes on opposite strands. There are several ways to accomplish strand-specificity.

Where does the ambiguity in stranded RNA-Seq come from?

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.

What are the shortcomings of RNA Seq technology?

While RNA-sequencing (RNA-seq) is becoming a powerful technology in transcriptome profiling, one significant shortcoming of the first-generation RNA-seq protocol is that it does not retain the strand specificity of origin for each transcript.

What happens when read 1 aligns to RNA strand?

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”. Sometimes sequences of read 1 align to the RNA strand; the other times sequences of read 2 align to the RNA strand. The library is “unstranded”.