Next generation sequencing (NGS) technology improves the speed, throughput and cost-effectiveness of RNA sequencing. An important application of NGS is whole transcriptome analysis, which is used to obtain information about gene expression, detect post-transcriptional mutations and quantify gene expression profiling in a particular pathway of disease development.
Biomek® FXP Laboratory Automation Workstation
A major hindrance to fully realizing the capacity of sequencing systems is the manual preparation of sequencing libraries. Scientists from Beckman Coulter have recently described the automation of RNA library construction using the Biomek TruSeq® RNA method on the Biomek® FXP Liquid Handler (BFXP).
Total RNA was extracted from tumor/normal pairs of archival FFPE prostate tissue and 500ng total RNA was used as the sample input for both the BFXP automated method and a manual construction method. The Agilent High Sensitivity DNA kit was used on the Bioanalyzer 2110 system to assess library quality.
Initially, library quality showed an average peak size for the amplified cDNA library at around 260bp. However, all samples also had a 126bp non-specific adaptor amplified peak, indicating adaptor-dimer contamination, which can lead to junk sequencing reads. An additional fragment of more than 400bp was also sometimes detected, which could be due to the presence of single-stranded library products that have self-annealed.
After implementation of a size selection clean up protocol, the pre and post-clean up libraries were compared for the manual versus BFXP automated method. Analysis of the post-cleanup plots showed that manually constructed libraries had a 126 bp peak that was eliminated in the libraries constructed using the Biomek automated method. The overall coverage was also superior when the automated method was used. Reproducibility was assessed by calculating the Pearson Correlation from transcript abundance values and both the BFXP automated libraries and the manually prepared libraries exhibited good concordance.
These findings demonstrate that the Biomek automated method for RNA library construction results in sensitive and reproducible sequencing data that can facilitate biomarker discovery in archival FFPE tissue. This method provides a viable alternative to manual RNA library construction, generating significantly superior libraries.
With the capacity to construct up to 96 libraries in seven hours, this automation solution offers users a high throughput workflow and a faster and more reliable way of generating libraries from precious clinical samples. Furthermore, the method can be used for library construction from fresh tissue or other sample types.
For more information about Biomek FXP automated RNA library construction, please access the following link: Automated TruSeq RNA Sample Prep
About Beckman Coulter
Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.