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Systematic comparative analysis of strand-specific, cost-effective, RNA-seq library preparation methods for low input samples

ABSTRACT: Despite the precipitous decline in the cost of genome sequencing over the last few years, library preparation for RNA-seq is still laborious and expensive for high throughput screening for drug discovery. Limited availability of RNA generated by some experimental workflows poses an additional challenge and typically adds to the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that also maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies – Swift and Swift Rapid – using the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics, as our reference. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark differential gene expression in these kits, at input quantities ranging between 10 ng to 500 ng. Read quality and alignment metrics revealed high mapping efficiency and uniform read coverage through genes for all samples across all three kits. Normalized read counts between all treatment groups were in high agreement, with pairwise Pearson correlation coefficients >0.97. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits are cost effective and offer shorter workflow times enabled by their patented Adaptase technology. Furthermore, the Swift RNA kit allows for a relatively broader (and lower) input range, producing consistent results across diverse samples. The Swift Rapid RNA method is the fastest and most cost effective NGS workflow that is best suited for higher RNA yields, with the exact same RNA input range as the Illumina TruSeq kit. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways attributable to input mRNA concentration.

ORGANISM(S): Homo sapiens

PROVIDER: GSE167300 | GEO | 2021/02/23

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples) Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested in triplicate by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For RNA-sequencing, each sample was tested by two library contruction kits: SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System with three input quantities: 50pg, 250pg and 2ng of total RNA, except for the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. RNA-seq based on poly(A) tail enrichment was considered as the reference and was done for the two FF samples at high amount (1µg of total RNA) . Results obtained with the two tested kits were compared to those based on poly(A) tail enrichment. To determine which is the best kit suitable for RNA-seq, low-input and low-quality RNA samples, we performed RNA-seq control quality metrics, principal component analysis, and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, library kit and method of sample preservation (FF or FFPE). Results: The Ovation SoLo NuGEN RNA-seq System library kit are recommended for quantification of gene expression of FFPE and FF samples at low-input (down to 50pg) whereas the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian is only suitable for FF samples from 250pg of total RNA.

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Systematic comparative analysis of strand-specific, cost-effective, RNA-seq library preparation methods for low input samples

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