Dataset Information

A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq.

ABSTRACT: The emergence of NextGen sequencing technology has generated much interest in the exploration of transcriptomes. Currently, Illumina Inc. (San Diego, CA) provides one of the most widely utilized sequencing platforms for gene expression analysis. While Illumina reagents and protocols perform adequately in RNA-sequencing (RNA-seq), alternative reagents and protocols promise a higher throughput at a much lower cost. We have developed a low-cost and robust protocol to produce Illumina-compatible (GAIIx and HiSeq2000 platforms) RNA-seq libraries by combining several recent improvements. First, we designed balanced adapter sequences for multiplexing of samples; second, dUTP incorporation in 2(nd) strand synthesis was used to enforce strand-specificity; third, we simplified RNA purification, fragmentation and library size-selection steps thus drastically reducing the time and increasing throughput of library construction; fourth, we included an RNA spike-in control for validation and normalization purposes. To streamline informatics analysis for the community, we established a pipeline within the iPlant Collaborative. These scripts are easily customized to meet specific research needs and improve on existing informatics and statistical treatments of RNA-seq data. In particular, we apply significance tests for determining differential gene expression and intron retention events. To demonstrate the potential of both the library-construction protocol and data-analysis pipeline, we characterized the transcriptome of the rice leaf. Our data supports novel gene models and can be used to improve current rice genome annotation. Additionally, using the rice transcriptome data, we compared different methods of calculating gene expression and discuss the advantages of a strand-specific approach to detect bona-fide anti-sense transcripts and to detect intron retention events. Our results demonstrate the potential of this low cost and robust method for RNA-seq library construction and data analysis.

SUBMITTER: Wang L

PROVIDER: S-EPMC3198403 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq.

Wang Lin L Si Yaqing Y Dedow Lauren K LK Shao Ying Y Liu Peng P Brutnell Thomas P TP

PloS one 20111019 10

The emergence of NextGen sequencing technology has generated much interest in the exploration of transcriptomes. Currently, Illumina Inc. (San Diego, CA) provides one of the most widely utilized sequencing platforms for gene expression analysis. While Illumina reagents and protocols perform adequately in RNA-sequencing (RNA-seq), alternative reagents and protocols promise a higher throughput at a much lower cost. We have developed a low-cost and robust protocol to produce Illumina-compatible (GA ...[more]

PMID: 22039485

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Project description:We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

Dataset Information

A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq.

Publications

A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq.

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