Automation of library preparation for RNA -Seq applications

The production of DNA libraries in sufficient quantity and quality is not only the actual sequencing of the essential step in order to achieve meaningful results in accordance with Next Generation Sequencing (NGS ) process . Automated methods must be able to adjust not only to the great variability of the methods used , but also on the common borderline material (quantity and quality). Also, there is a confusion about the term “fully automated ” . Here it is understood in the sense of ” walk away” . After loading the device with the total consumption of material samples and starting the system a manual intervention is necessary only after completion of processing. Even to the limited space in the laboratory be taken into account and only found in the contemplation of a “single deck” system into account , in this case for the production of RNA -Seq libraries. An important requirement is the capacity for the production of libraries of up to 96 RNA samples with optional modules for the reduction of the ribosomal RNA and / or consideration of the strand specificity using Illumina TrueSeq kits.


Figure 1: Biomek FXp – Overview

The method was implemented on a Biomek system FXp (Beckman Coulter) . Use is this a draft of the design of a generalized NGS Library workstation (Fig. 1) with a unified deck layout combines that was already in the generation of libraries using samples from genomic DNA chip DNA and Exome has proven to capture material : Two Pippetierköpfe , one for 96 disposable tips and a gripper , the second with eight adapters for disposable tips allow both parallel autonomous actions and the additional optimization for “Single Tip” addressing and Plate Processing in one step. The high-density deck has 23 positions that can be occupied up to four times. An already occupied U- shaped region encloses an assignable area . The Fixkonfiguration contains an automated PCR device for amplifications and fully evaporationskontrollierte incubations , two Peltier adapter for tunings , including one with additional shaking , an orbital shaker for Schütteloperationen large amplitude as well as positions for loading tips , hints a wash station with sample-specific washing channels a waste station . Thus, there are 10 positions for max. 40 plates or other functions in the disk format ( magnetic , reservoirs , etc.) freely available .

For the RNA process described here, the deck is configured as shown in Figure 2a . The Peltier positions are equipped with deep well plates for appropriately scaled master mixes and Ampure XP or Size Select ( Beckman) magnetic particles. Liquids , for which there is a greater need , such as ethanol and various washing buffer are kept in large volume reservoirs. Another position is reserved for a Mikrotiterplattenmagnet . The initial layout uses other deck positions for individual plates as input, output, index primers and PCR. Two positions are used to drive logistics, such as the parking of used plates. The last position is occupied by an ” arched lid” . Through the interaction of the high contact pressure of the PCR device as well as the elasticity of the prestressed metal lid and silicone inner coating is provided for sufficient closing pressure and thermal transfer in PCR and incubations .

The system has a variable capacitance 1-96 samples , without further manual intervention is necessary . The application software takes over the control of all additional hardware modules , the user performs a wizard through the setup of the device and shows in the Reagenzienkalkulator to be applied in volumes of the master mixes and their positioning.

Dr. Jürgen Zimmermann
Dinko Pavlinic
Dr. Vladimir Benes
Genomics Core Facility
European Molecular Biology Laboratory (EMBL)