Analysis of stranded information using an automated procedure for strand specific RNA sequencing

Area of Science:

• Molecular Biology
• Bioinformatics
• Genomics

Background:

• Strand specific RNA sequencing is becoming the standard for transcriptome analysis, replacing conventional cDNA sequencing.
• Advancements in laboratory protocols and bioinformatics tools are crucial for its progress.
• Current protocols utilize the Illumina TruSeq library preparation kit for automated stranded library generation.

Purpose of the Study:

• To evaluate the robustness and reproducibility of a strand specific RNA sequencing protocol.
• To compare the effectiveness of different ribosomal depletion kits (RiboMinus and RiboZero).
• To compare the performance of two sequence aligners (Tophat2 and STAR).

Main Methods:

• Adaptation of the Illumina TruSeq kit for stranded library preparation.
• Utilizing freely available bioinformatics tools for quality assessment.
• Comparison of strand specific vs. non-stranded libraries for expression analysis.
• Evaluation of RiboMinus and RiboZero ribosomal depletion kits.
• Assessment of Tophat2 and STAR aligner performance.

Main Results:

• The adapted Illumina TruSeq kit reliably generates strand specific libraries.
• The RiboZero kit demonstrates high efficiency in ribosomal RNA depletion.
• The STAR aligner offers high mapping yield and speed.
• Strand specific data yields more detailed and accurate transcriptome information compared to non-stranded data.
• Strand specific data aids in transcript assembly and genome annotation refinement.

Conclusions:

• Strand specific RNA sequencing data enhances confidence in analysis and reduces the likelihood of false conclusions.
• Researchers are encouraged to adopt strand specific approaches for more reliable transcriptome studies.
• Awareness of the limitations of non-stranded data analysis is essential.