Comparing Novel Low-Input Total RNA-Seq Methods

Until recently, performing RNA-sequencing (RNA-seq) at low input often required specialized library preparation kits. The SMARTer® Stranded Total RNA-Seq Kit v3-Pico Input Mammalian workflow offered by Takara Bio is a popular option for generating high-quality libraries from low-input material. Recently, two RNA-seq library preparation kits, the Watchmaker RNA Library Prep Kit with Polaris Depletion by Watchmaker Genomics and the sparQ RNA-Seq HMR Kit by Quantabio, that offer a larger input range became available. These kits may offer a more accessible and affordable workflow for low-input RNA-seq; therefore, to determine their suitability for low-input applications, a comparative evaluation of the kits was performed using the market standard Takara Pico kit for benchmarking. To do this, two replicates of three total RNA samples of varied quality were prepared at three inputs (250 pg, 1 ng, and 10 ng) using each workflow. Paired-end 50 bp sequencing was performed on a NovaSeq 6000 to obtain 13 million reads per library. While both novel kits generated next-generation sequencing libraries at all inputs tested, neither workflow was specifically tailored for low inputs. We observed variability in library diversity, proportion of duplicate reads, types of transcripts detected, sensitivity of detection, and proportion of nuclear rRNA reads. This suggests that further optimization is required to obtain high-quality libraries from 250 pg to 10 ng input amounts using the novel workflows.