Genomics Application Notes

High Throughput TruSeq Stranded mRNA Library Construction on the Biomek FX P

Mary Blair Life Science Division Beckman Coulter, Inc. Indianapolis, IN USA

Zach Smith and Scott D. Michaels The Center for Genomics and Bioinformatics Indiana University Bloomington, IN USA

Abstract A collaboration between Beckman Coulter, the Center for Genomics and Bioinformatics at Indiana University, and Illumina, Inc. has resulted in the automation of the TruSeq Stranded mRNA sample preparation kit from Illumina on the Biomek FX P . This automation method generates high quality stranded mRNA sequencing libraries compatible with Illumina sequencers. Data has been generated on the Illumina HiSeq 2500 and MiSeq platforms confirming that libraries produced with this automation method yield target-specific, highly uniform, strand-specific sequence data, with no detectable cross contamination of template samples, while reducing the risk of use error. Introduction The continuing development of next generation sequencing (NGS) platforms is providing unparalleled levels of sequencing capacity at relatively low cost. This advance makes possible larger scale genomic studies to understand the genetic and epigenetic underpinnings of a wide range of biological processes, including development, behavior and disease. A significant bottleneck to fully realizing the capacity of Illumina’s sequencing systems is the manual preparation of large numbers of sequencing libraries, which can be labor-intensive and time-consuming. Manual methods also carry a higher risk of human error and inconsistency. The Biomek FX P Laboratory Automation Workstation puts every aspect of liquid handling required for automation of NGS sample preparation – including optimized pipetting for precious reagents and samples, and heating, cooling, shaking and thermocycler integration to maintain protocol-defined environmental conditions – into a single, automated system while limiting manual handling of any potentially hazardous chemicals. It has the power, flexibility and efficiency to consistently provide high quality template libraries at a throughput needed to take advantage of the high capacity of NGS, at a reasonable cost. TruSeq Stranded mRNA Sample Preparation kits provide the clearest, most complete view of the mRNA transcriptome by providing precise assessment of strand orientation, uniform coverage, high-confidence mapping of alternate transcripts and gene fusion events as well as the detection of allele-specific expression. In addition to enabling the discovery of these and other features without prior knowledge, it is a highly accurate and cost- efficient gene expression quantification tool with multiplexing enabled by 96 available unique index pairs. This application note describes the automation of the Illumina TruSeq Stranded mRNA kit on the Biomek FX P for transcriptome library construction. One to 96 samples can be run simultaneously, and setup requires only a few clicks of the mouse and one-time pipetting of the required kit components into the reservoirs. The pipetting tools and software improve the ease and speed of reaction setup. The method then runs automatically, providing purification and fragmentation of mRNA from total RNA, first strand cDNA synthesis, second strand cDNA synthesis, AMPure XP purification, 3' adenylation, and adaptor ligation, double AMPure XP purification and PCR enrichment. The end result is as many as 96 sequence-ready libraries in just two days, from as little as 50 1 ng of mRNA. The resulting high quality libraries display consistent insert sizes, minimal adapter dimers, low numbers of ribosomal sequences, and high fidelity to the reference genome, enabling a detailed survey of the transcriptome under investigation. All of this was accomplished with minimal hands-on time (see Table 1) and no detectable sample cross-contamination.

IB-18319A