Transcriptome (RNA-Seq)

 

RNA-Seq (RNA sequencing), also called whole transcriptome sequencing (WTS), uses next or third-generation sequencing to reveal the presence and quantity of RNA in a biological sample at a given moment in time.

RNA-Seq is used to analyze the continually changing cellular transcriptome. Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5′ and 3′ gene boundaries.

 

RNA Sequencing Summary

Application
Method
Feature
Sequencing Platform
mRNA
Selection using poly‐T oligo
Strand specific

Hiseq 2000/2500&Ion ProtonTM

Total RNA (Whole Transcriptome)
rRNA Depletion
Strand specific mRNA and various non coding RNA (lincRNA,snRNA, snoRNA) analysis
Small RNA
Size selection(20~70bp)
Separate analysis of non coding RNA and micro RNA

Whole Transcriptome Sequencing

Whole transcriptome sequencing is a major advance in the study of gene expression compared to traditional microarray-based approaches in that it provides a comprehensive view of a cellular transcriptional profile at a given biological moment enabling not only qualitative but also quantitative analysis of the transcriptome. It also provides information about diverse variations occurring at RNA level of post-transcriptional modifications such as splice variants and isoforms.

Expression Profiling Sequencing

Taken apart the quantitative aspect of whole transcriptome sequencing, expression profiling sequencing gives most comprehensive insight about the transcription level of individual mRNAs which is then further related to the expression level of proteins. It is a very cost-effective way of understanding the transcriptions in that it only sequences a small part of mRNA (normally 3’ end with poly (A) tail) and counts the number of sequences. It provides virtually unlimited dynamic range for highly abundant transcripts as well as not missing rare transcripts.

Isoform Sequencing

Empowered by the unique advantage of long reads from PacBio systems, isoform sequencing (Iso-Seq) reveals the splice variants of the transcripts providing the most comprehensive landscape of a transcriptome. The splicing junction of mRNA can be pin-pointed and this can be aligned back to the genome data to show what kind of splice variants are produced, further giving insights about the relation between splicing and a certain phenotype or diseases.

 

 

Single-cell RNA Sequencing

Traditional sequencing results are actually the average value from cells of a cluster, but in some very delicate researches we need to understand the nature at single cell level. Single-cell RNA sequencing is designed for this kind of approached.

Small RNA Sequencing

Small, non-coding RNA, or microRNAs are a short, 18-22 bp nucleotides which is known to play a critical role in the gene regulation and expression.