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Related Concept Videos

RNA-seq03:21

RNA-seq

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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...
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Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA
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RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase.

Ryan M Nottingham1, Douglas C Wu1, Yidan Qin1

  • 1Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, 78712, USA Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, 78712, USA.

RNA (New York, N.Y.)
|January 31, 2016
PubMed
Summary

A new RNA sequencing method using thermostable group II intron reverse transcriptases (TGIRTs) offers improved accuracy and comprehensive transcriptome profiling. TGIRT-seq captures structured RNAs missed by conventional methods, enhancing RNA analysis.

Keywords:
TruSeqdiagnosticshigh-throughput sequencingsmall noncoding RNAtRNAtranscriptome

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Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Next-generation RNA sequencing (RNA-seq) is crucial for transcriptome analysis but faces limitations.
  • Existing RNA-seq methods exhibit biases and struggle to profile structured RNAs like tRNAs and snoRNAs comprehensively.
  • Variability between RNA-seq methods arises from differences in sample preparation, reverse transcription, and adapter ligation.

Purpose of the Study:

  • To introduce and evaluate a novel strand-specific RNA sequencing method utilizing thermostable group II intron reverse transcriptases (TGIRTs).
  • To compare the performance of TGIRT-seq with established Illumina TruSeq v2 and v3 RNA-seq methods using human RNA reference samples.
  • To assess TGIRT-seq's ability to profile a wider range of RNA molecules, including structured small non-coding RNAs.

Main Methods:

  • Development of a strand-specific RNA sequencing protocol employing TGIRT enzymes for cDNA synthesis and adapter addition.
  • Acquisition of TGIRT-seq data from well-characterized human RNA reference samples.
  • Comparative analysis of TGIRT-seq data against existing datasets generated by Illumina TruSeq v2 and v3 methods.

Main Results:

  • TGIRT-seq demonstrates comparable or superior recapitulation of RNA abundance compared to TruSeq v2 and v3.
  • TGIRT-seq exhibits enhanced strand specificity and eliminates biases associated with random hexamer priming.
  • TGIRT-seq provides more uniform 5' to 3' gene coverage and identifies more splice junctions, especially near mRNA 5' ends.
  • TGIRT-seq enables simultaneous profiling of mRNAs, lncRNAs, and structured small ncRNAs like tRNAs, which are typically missed by conventional methods.

Conclusions:

  • TGIRT-seq represents a significant advancement in RNA sequencing technology, offering improved accuracy, uniformity, and comprehensiveness.
  • The method overcomes limitations of existing RNA-seq protocols, particularly in profiling structured and small non-coding RNAs.
  • TGIRT-seq facilitates more complete transcriptome analysis, opening new avenues for biological discovery.