An automated method for efficient, accurate and reproducible construction of RNA-seq libraries

Background Integration of RNA-seq expression data with knowledge on chromatin accessibility, histone modifications, DNA methylation, and transcription factor binding has been instrumental for the unveiling of cell-specific local and long-range regulatory patterns, facilitating further investigation on the underlying rules of transcription regulation at an individual and allele-specific level. However, full genome transcriptome characterization has been partially limited by the complexity and increased time-requirements of available RNA-seq library construction protocols. Findings Use of the SX-8G IP-Star® Compact System significantly reduces the hands-on time for RNA-seq library synthesis, adenylation, and adaptor ligation providing with high quality RNA-seq libraries tailored for Illumina high-throughput next-generation sequencing. Generated data exhibits high technical reproducibility compared to data from RNA-seq libraries synthesized manually for the same samples. Obtained results are consistent regardless the researcher, day of the experiment, and experimental run. Conclusions Overall, the SX-8G IP-Star® Compact System proves an efficient, fast and reliable tool for the construction of next-generation RNA-seq libraries especially for trancriptome-based annotation of larger genomes.


Background
Deciphering the underlying determinants of transcriptional regulation in relation to cell differentiation, functional diversification, environmental signaling, and disease development remains a central question in biology today. Integration of expression data with knowledge on chromatin accessibility, histone modifications, DNA methylation, and transcription factor binding, has been instrumental for the unveiling of cell-specific local and long-range regulatory patterns, facilitating further investigation on the underlying rules of transcription regulation at an individual and allele-specific level. Current interest by large collaborative projects, such as the ENCODE [1], the NIH Roadmap Epigenomics Mapping Consortium [2,3], and the C. elegans and D. melanogaster modENCODE [4], has been placed on generating genome-wide gene expression maps to locate gene expression changes that accompany important developmental and disease development processes. The pairing of traditional expression assays with high-throughput sequencing (RNA-seq) has allowed the generation of genome-wide gene expression data with unparalleled specificity, throughput, and sensitivity delivering a detailed representation of the transcriptome.
However, full genome transcriptional gene characterization has been partially limited by the complexity and increased time-requirements of available RNA-seq library construction protocols. Here we report the successful application of the SX-8G IP-Star® Compact System (Diagenode) for the easy, rapid, and reproducible RNAseq library construction of five Mus musculus (mouse) samples. Use of the SX-8G IP-Star® Compact System significantly reduced the hands-on time for RNA-seq library synthesis, adenylation, and adaptor ligation providing with high quality RNA-seq libraries tailored for Illumina highthroughput next-generation sequencing. Generated data exhibited high technical reproducibility compared to data from RNA-seq libraries synthesized manually for the same samples. Obtained results are consistent regardless the  researcher, day of the experiment, and experimental run. Overall, the SX-8G IP-Star® Compact System proves an efficient and reliable tool for the construction of nextgeneration RNA-seq libraries especially for trancriptomebased annotation of larger genomes.

Methods
A schematic step-wise representation of the two tested protocols is presented in Figure 1. Specifically, we tested application of the SX-8G IP-Star® Compact System for the construction of RNA-seq libraries of five mouse (Mm_1-5_Auto) samples in comparison to a manual protocol routinely used in our laboratory. The two protocols were compared using the same thermocycling machines and reagents. Total RNA integrity value following isolation was measured using the Agilent  ) for the corresponding sample number and to set up the necessary reagents and consumables following the robot's user-friendly and simple interface. RNA-seq data generated using the manual and automated protocols were aligned against the Mus musculus GRCm38/mm10 genome using TopHat 2.0.7 [5]. Following

Results
Application of the SX-8G IP-Star® Compact System for the RNA-seq library construction of five mouse samples, significantly reduced the amount of hands-on time required for the most time-demanding stages of library synthesis, adenylation, and adaptor ligation including all related clean up steps. Specifically, manual library construction with the protocol routinely used in our laboratory typically takes an average of four hours of hands-on time whereas Diagenode automated library construction with the same reagents and samples required only 30 minutes. This corresponds to a 8-fold decrease in the amount of time the researcher has to be directly involved with the procedure, offering substantial flexibility for experimental multitasking.
Notably, generated data with the automated protocol exhibited high technical reproducibility compared to data from RNA-seq libraries synthesized manually for the same samples regardless operator and experimental run. Specifically, density distributions of FPKM values demonstrated high data concordance among samples and technical replicates (Figure 2). Correlation coefficient values r 2 obtained using the linear regression model in R for the five mouse samples and corresponding technical replicates ranged from 0.97-0.98, confirming that the SX-8G IP-Star® Compact System can be reliably used for the efficient and accurate construction of RNA-seq libraries (Figure 3). Cluster analysis illustrated tight clustering between samples and technical replicates, further supporting high technical reproducibility between the two tested protocols (Figure 4).

Conclusions
Overall, the SX-8G IP-Star® Compact System proves an efficient, reliable and accurate tool for the construction of next-generation RNA-seq libraries, especially for trancriptome-based annotation of larger genomes. We foresee that incorporation of this technology in Next-Generation Sequencing Cores or Genomics Laboratories will prove an indispensable tool for high-throughput RNA-seq library construction, significantly saving onhands experimentation time, related costs and errorprone manual steps. Added benefits of the automated protocol include ease of operation and generation of consistent data regardless of human variability and experimental run. Adaptation of this technology should support the unveiling of the mechanisms governing differential gene expression and transcription processing genome-wide, leading to a better understanding of genetic and epigenetic regulation and inheritance in a timeefficient manner.

Competing interests
The authors declare that they have no competing interests. Submit your manuscript at www.biomedcentral.com/submit