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Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input

ABSTRACT: Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input

PROVIDER: PRJNA510931 | ENA |

REPOSITORIES: ENA

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Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input

Project description:Library preparation is a key step in gene expression quantification. There are considerable advantages to both strand specific sequencing and the ability to sequence samples with very small amounts of starting material. Until recently there was no kit available that allowed both simultaneously. The standard Illumina-TruSeq stranded mRNA Sample Preparation kit requires abundant starting quantity while the Takara Bio-SMART-Seq® v4 Ultra® Low Input RNA kit allows for ultra low starting quantities but sacrifices strand specificity. Recently a kit that can do both, SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian by Takara Bio, has become available. Evaluating the performance and effects of these sample preparation kits is a critical determinant for selecting the appropriate sequencing protocol, but a comprehensive comparison is currently missing. To address this we performed a detailed comparative analysis of sequencing libraries prepared with the three kits. We prepared a set of samples representing two experimental conditions with each kit, allowing for comparison of the kits in a standard realistic differential expression analysis. We find substantial differences in the levels of alignment and differential gene expression. Using differential expression analysis we show that using Pico results in identifying 55% less differentially expressed genes than TruSeq. Nevertheless, using gene pathway enrichment analysis we find similar results for all three kits, indicating that ultimately comparable functional results can be reached.

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

Project description: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.

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Project description:Advances in Next Generation Sequencing (NGS) have made available a wealth of information that had previously been inaccessible to researchers and clinicians. NGS has been applied to understand genomic, transcriptomic, and epigenomic changes and gained traction as a significant tool capable of accelerating diagnosis, prognosis, and biomarker discovery. However, these NGS assays have yet to be practical methods for patient stratification or diagnosis because of the gap between the tiny quantities of biomaterials provided by a clinical sample and the large DNA input required by most of these assays. Current library preparation methodologies typically require large input amounts of DNA and a long and complicated manual process. Here we present a microfluidic reactor system for NGS library preparation, capable of reducing the number of pipetting steps significantly, automating much of the process, while supporting extremely low DNA input requirement (10 pg per library). This largely automated technology will allow for low-input preparations of 8 libraries simultaneously while reducing batch to batch variation and operator hands-on time.

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