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

Despite the recent precipitous decline in the cost of genome sequencing, library preparation for RNA-seq is still laborious and expensive for applications such as high throughput screening. Limited availability of RNA generated by some experimental workflows poses an additional challenge and increas...

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Bibliographic Details
Published in:Scientific reports 2022-02, Vol.12 (1), p.1789-1789, Article 1789
Main Authors: Naphade, Swati, Bhatnagar, Rajat, Hanson-Smith, Victor, Choi, Irene, Zhang, Alice
Format: Article
Language:English
Subjects:
631/1647/514/1949
631/1647/514/2254
Automation
Biomarkers, Tumor - genetics
Comparative analysis
Gene expression
Gene Expression Profiling
Gene Library
Genomes
High-throughput screening
Humanities and Social Sciences
Humans
multidisciplinary
Neoplasms - genetics
Neoplasms - pathology
RNA, Neoplasm - analysis
RNA, Neoplasm - genetics
RNA-Seq - methods
RNA-Seq - standards
Science
Science (multidisciplinary)
Sequence Analysis, RNA - methods
Transcriptome
Transcriptomics
Tumor cell lines
Tumor Cells, Cultured
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Summary:Despite the recent precipitous decline in the cost of genome sequencing, library preparation for RNA-seq is still laborious and expensive for applications such as high throughput screening. Limited availability of RNA generated by some experimental workflows poses an additional challenge and increases the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies—Swift RNA and Swift Rapid RNA, presently offered by Integrated DNA Technologies (IDT) —alongside the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark gene expression in these kits, at input quantities ranging between 10 to 500 ng. We found normalized read counts between all treatment groups to be in high agreement. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits offer shorter workflow times enabled by their patented Adaptase technology. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways directly attributable to input mRNA amount.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-021-04583-z