What are the limitations of RNA-seq?

Limitations of RNA-seq Lack of standardization between sequencing platforms and read depth, equivalent to the percentage of total transcripts sequenced, can compromise reproducibility. Although RNA-seq has become increasingly affordable, its cost remains prohibitive for many laboratories.

What causes chimeric reads?

A chimeric read is a digital DNA sequence (i.e. a string of letters in a file that can be read as a DNA sequence) that originates from an actual chimera (i.e. a physical DNA sequence in a sample) or produced due to misreading the sample. The latter is known to occur with sequencing of electrophoresis gels.

What does RNA sequencing detect?

RNA sequencing (RNA-seq) allows for the detection of a wide variety of RNA species, including mRNA, non-coding RNA, pathogen RNA, chimeric gene fusions, transcript isoforms and splice variants, and provides the capability to quantify known, pre-defined RNA species and rare RNA transcript variants within a sample.

What can you do with RNAseq data?

RNA-seq can be used solo for transcriptome profiling or in combination with other functional genomics methods to enhance the analysis of gene expression.

What are chimeras sequences?

Chimeras are artifact sequences formed by two or more biological sequences incorrectly joined together. This often occurs during PCR reactions using mixed templates (i.e., uncultured environmental samples). Once created, the chimeric sequence is then further amplified in subsequent cycles.

What are chimeric molecules?

A chimeric molecule is an engineered construct in which two or more components are linked to form a novel biological agent. This concept describes a bifunctional molecule in which one component targets the molecule to a specific cell and the second component exerts a pharmacological activity.

How is RNA sequencing different from DNA sequencing?

While sequencing DNA gives a genetic profile of an organism, sequencing RNA reflects only the sequences that are actively expressed in the cells. To sequence RNA, the usual method is first to reverse transcribe the RNA extracted from the sample to generate cDNA fragments.

How does RNA sequencing RNA-Seq pipeline work?

RNA-seq involves conversion of a sample of RNA to a cDNA library, which is then sequenced and mapped against a reference genome. In addition to the ability to measure the level of gene expression, it provides further information on alternative splicing and non-coding RNA (such as microRNA) (Chaussabel et al., 2010).

What is the role of ATP Sulfurylase in Pyrosequencing?

ATP sulfurylase converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts as a substrate for the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount.