15.14: RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.

Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based techniques had drawbacks such as limited coverage and dependency on existing knowledge of the genome, Sanger sequencing has limitations, such as low-throughput, high cost, and inaccurate results. In contrast, RNA-seq is a next-generation sequencing (NGS) technology that provides relatively higher coverage and higher throughput. It also generates additional data that can help discover novel transcripts, understand allele-specific information, and identify alternatively spliced genes.

The RNA-seq process can be divided into several steps. The first step is the extraction and isolation of RNA of interest from the sample, followed by the conversion of this RNA to complementary DNA. This ensures the molecule's stability, easy handling, and ability to be put into an NGS workflow. Next, sequences known as adapters are attached to the DNA fragments to enable sequencing. The most widely used NGS platforms for RNA-seq include SOLiD, Ion Torrent, and HiSeq. The depth to which the library is sequenced varies depending on the end-goal of the experiment. For example, sequencing can involve single-read or paired-end sequencing methods. Single-read sequencing that sequences the DNA only from one end is a cheaper and faster technique, while the paired-end method that involves sequencing from both ends is more expensive and time-consuming. Additionally, additional information about which DNA strand was transcribed can also be retained through a strand-specific protocol.

The sequencing data is then aligned to a reference genome and used to generate a corresponding RNA sequence map. Depending on the nature of the analysis, different bioinformatic tools can be used to process data. For example, BitSeq and RSEM can help quantify expression level, whereas MISO can be used to quantify alternatively spliced genes.

RNA sequencing, or RNA-seq, is a high-throughput sequencing technique that identifies and quantifies RNA sequences in a sample. 

It can be used to analyze total RNA or specific RNA populations such as mRNA, tRNA, rRNA and miRNA. The technique has diverse applications in transcriptome analysis, differential gene expression analysis, and RNA editing.

In the case of the analysis of specific populations, the RNA type of interest needs to be isolated from the other RNAs. mRNAs can be isolated using oligo dT probes that are complementary to the poly A tails present on  mRNA transcripts. MicroRNAs,  which are usually around fifteen to thirty nucleotides long, are isolated by size-based extraction methods.

Alternatively, contaminating RNA, such as ribosomal RNA, can be removed using oligonucleotides complementary to the contaminant and covalently linked to magnetic beads. The hybridized ribosomal RNA is separated from the sample using a magnet, leaving behind the RNA of interest.

The purified RNA is used as a template by the enzyme reverse transcriptase to create cDNA, which is further amplified using PCR to create a library for sequencing.

Sequencing can be carried out through one of several available technologies. In one of the most common, cDNA fragments are ligated with short oligonucleotide sequences known as adaptors, which serve as primer binding sites for PCR amplification. Adaptors  may also have unique sequences, called barcode sequences, that are used to tag and identify each cDNA strand.

The library is then amplified using PCR. Following this it is diluted to a low concentration and denatured to single strands with heat, before immobilization on a sequencing chip consisting of oligonucleotides complementary to the adaptors. 

Once attached, the single stranded DNA is cloned using processes, such as bridge amplification, to form clusters of DNA  with the same sequence. This ensures that the strands from an area on the chip are from a single source and emit a uniform signal during sequencing.

Sequencing can be strand-specific or non-strand-specific. In the case of strand-specific protocols , the complementary strand is washed off and the other is used for sequencing.

Fluorescently labeled nucleotides are then added to the strands on the chip to create a new complementary strand. A characteristic fluorescence is emitted on each addition which can be read by a detector. 

Several million clusters of distinct cDNA fragments can be sequenced simultaneously using these methods.The resulting data can then be aligned to a genome of reference and assembled to produce an RNA sequence map for analysis.