Comparing Novel Low-Input Total RNA-Seq Methods.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Low-input RNA-sequencing (RNA-seq) traditionally required specialized kits.
• Takara Bio's SMARTer Stranded Total RNA-Seq Kit v3-Pico is a standard for high-quality low-input libraries.
• New kits from Watchmaker Genomics and Quantabio offer broader input ranges, potentially improving accessibility and affordability.

Purpose of the Study:

• To comparatively evaluate the suitability of the Watchmaker and Quantabio RNA-seq kits for low-input applications.
• To benchmark these novel kits against the established Takara Bio Pico kit.
• To assess library quality metrics across different input amounts (250 pg, 1 ng, 10 ng).

Main Methods:

• Comparative evaluation of three RNA-seq library preparation kits: Watchmaker RNA Library Prep Kit, Quantabio sparQ RNA-Seq HMR Kit, and Takara Bio SMARTer Stranded Total RNA-Seq Kit v3-Pico.
• Utilized three total RNA samples of varying quality across three input amounts (250 pg, 1 ng, 10 ng).
• Performed paired-end 50 bp sequencing on NovaSeq 6000, generating 13 million reads per library.

Main Results:

• Both novel kits successfully generated next-generation sequencing libraries at all tested input levels.
• Variability was observed in library diversity, duplicate read proportions, detected transcripts, detection sensitivity, and nuclear rRNA content.
• Neither novel workflow demonstrated specific optimization for low-input amounts (250 pg to 10 ng).

Conclusions:

• The evaluated Watchmaker and Quantabio kits can produce RNA-seq libraries from low input amounts.
• Further optimization is necessary for these kits to consistently yield high-quality libraries across the 250 pg to 10 ng input range.
• The market standard Takara Bio kit remains a reliable option for low-input RNA-seq.