Abstract
With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation.
Highlights
Recent advances in RNA-sequencing (RNA-Seq) have provided a means for rapid characterization and quantification of transcriptomes
To compare our new High-throughput RNA-seq (HTR) library preparation method with the standard Illumina (IL) protocol, for each protocol, we evaluated RNA-Seq library reads generated from all four biological replicates of S. lycopersicum and S. pennellii
READ QUALITY A comparison of several statistical parameters on the data generated from HTR and IL protocol libraries showed that our HTR method produced RNA-Seq data is of similar quality to that of the IL protocol
Summary
Recent advances in RNA-sequencing (RNA-Seq) have provided a means for rapid characterization and quantification of transcriptomes. Even though sequencing capacity continues to increase, protocols for sample library preparation, being laborious, time consuming, and expensive, remain a limiting step. Illumina introduced a high-throughput method (TruSeq RNA sample preparation kit) replacing these purification steps with solid-phase reversible immobilization (SPRI) magnetic bead reaction cleanup methodology (Hawkins et al, 1994; Lennon et al, 2010). Using this method, a single technician can make 96 libraries from total RNA in 3 days. Similar improvements can be seen in the protocols by Zhong et al (2011) and Wang et al (2011a)
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