Automated Illumina NGS Sample Preparation: TruSeq
Automated Illumina NGS Sample Preparation:TruSeq Stranded mRNA on the HamiltonMicrolab STARApplication NoteAuthors: Susan Lee, Hamilton Robotics; Tim Hill, IlluminaAbstractThe Illumina TruSeq Stranded mRNA Sample Preparation kit provides reagents and a workflow for converting the mRNA intotal RNA into a library of template molecules of known strand origin, which is then suitable for cluster generation and DNAsequencing. The kit has been verified for use on the Microlab STAR liquid handling workstation and the automation protocol hasbeen Illumina Qualified*. Implementation of both the low throughput (LT) and high throughput (HT) sample preparation kits on theSTAR have resulted in high yield and high quality libraries.IntroductionIllumina’s sample preparation kits produce high quality libraries for DNA and RNA sequencing. Hamilton offers the robustMicrolab STAR line of robotic liquid handling workstations for automating sample preparation. Together, the automation ofthe TruSeq Stranded mRNA sample preparation protocol on the STAR gives the user hours of reliable, unattended librarypreparation.Any number of samples between 1 and 96 can be processed. All steps of the protocol are done on-deck with the exception ofone centrifugation step and the PCR thermal cycling. The programmed method is compatible with any index combination orpooling scheme.To verify the capability of the STAR to perform the TruSeq Stranded mRNA sample preparation, two tests were run. One testran the LT kit, with adapters in vials. The other test ran the HT kit, with adapters in the RNA Adapter plate (RAP). After librarypreparation, the samples were run on the HiSeq 2500 System. Positive results of the test have led to the designation of theMicrolab STAR method being Illumina Qualified* for TruSeq Stranded mRNA sample preparation.Figure 1:The Hamilton Robotics Microlab STAR Automated Workstation
System Deck Layout:Figure 1: STAR deck layout for TruSeq Stranded mRNA Sample Preparation protocol.System and Materials:System Requirements8 1ml channels4 Tip carriersiSWAP plate handler2 5x50ml reagent carriersAutoload for barcode reading1 24-tube carrier (for kit reagents in 4ml tubes)Liquid waste system1 Multiflex carrier with 2 tip modules, 2 PCR96 modules, and 1MIDI plate module5 Hamilton heater shakers (HHSs) for on-deck incubations andmixing2 32-tube carriers with vial insertsXX2 with custom PCR plate heat blocksXX1 for kit reagents in 0.5ml and 2ml vialsXX2 with custom MIDI plate heat blocksXX1 for adapters in vialsXX1 with a flat surface for plate mixing1 Inheco CPAC for plate coolingInvitrogen magnetic stand 96 (Life Technologies, PN AM10027)1 Stacker carrier for on-deck storage of the many plates used inthe process1 base plate for 2 MIDI HHSs and a custom module for themagnetic stand1 base plate for 2 PCR HHSs, 1 flat HHS, and the CPAC
Required ConsumablesHamilton Part Number50ul filtered tips, Hamilton235948300ul filtered tips, Hamilton2359031000ul filtered tips, Hamilton23590550ml reagent tubs, Hamilton56694-0196-well PCR plates, Bio-Rad, PN HSP-9601HSP-9601Illumina Sample Preparation KitsIllumina Part NumberTruSeq Stranded mRNA LT Sample Prep Kit – Set A, IlluminaRS-122-2101TruSeq Stranded mRNA LT Sample Prep Kit – Set B, IlluminaRS-122-2102TruSeq Stranded mRNA HT Sample Prep Kit, IlluminaRS-122-2103Other Required Materials and Equipment96-well storage plates (“MIDI” plates), Fisher ScientificAgencourt AMPure XP beads, Beckman Coulter GenomicsEthanol, absolute, for molecular biology, Sigma-AldrichSuperScript II Reverse Transcriptase, InvitrogenMineral oil, molecular biology grade, SigmaPart NumberAB-0859A63881E702318064-014M866296-well thermal cycler with heated lidSample Prep WorkflowFigure 2 illustrates the steps of the workflow. In addition to the indicated automation steps, qPCR set-up is also automated.The start and stop points are indicated in the diagram, and these correspond to the optional stopping points in the Illuminaprotocol.Figure 2: The steps of theworkflow are shown here.The automated steps arecircled. The method allowsthe user to start/stop theprotocol at the pointsindicated by a star.
Automated MethodThe automated protocol allows for run customization. The user selects which sections of the workflow to run, whether to run theHT or LT protocol (adapters in RAP or vials, respectively), whether controls are to be used, the fragmentation time (to allow fordifferent fragment sizes), and a worklist that specifies how many samples and which adapters to use for each sample. See theFigure 3.All incubations are done on-deck with the Hamilton heater-shakers (HHSs) and the Inheco CPAC. The HHSs are fitted withcustom adapters for optimal heat exchange. For the 94C incubation, mineral oil is used to prevent evaporation.The automated method is based on the “High Sample (HS)” portion of the Illumina sample prep protocol, regardless how manysamples are to be processed (1 to 96).All reagents required for the desired run process(es) are loaded onto the deck at the start of the run. The reagents are nottemperature-controlled, with no adverse affects.Figure 3: This dialog appearsat the start of the run. The userselects the desired options.
Verification Run SetupTwo runs were performed to qualify the STAR. Table 1 shows the plate layouts. For both runs, controls were used and thefragmentation time was 8min.Table 1: Plate layouts for the 2 verification runs.Results and DiscussionThroughputThe STAR-automated parts of the workflow were done over 3 days. Typical total hands-on time is 3hr 45min, to preparesamples and reagents, load and unload the deck, put away samples and reagents at the end of the run, and performvery minimal (10 minutes) daily maintenance. The method is fully automated, with the exception of these offline steps: onecentrifugation step near the end of cDNA synthesis cleanup, PCR thermal cycling, BioAnalyzer and qPCR validation. The tablebelow lists the STAR and thermal cycling run time requirements for the 48-sample run, as well as for typical 16- and 96-sampleruns.Day 2:Adenylation, Ligation,and PCRDay 3:Normalizationand PoolingTotal Run Time4hr 30min5hr 5min10min9hr 45min485hr 40min6hr 15min15min12hr 10min966hr 45min7hr 15min20min14hr 20minNumber of SamplesDay 1: Purification,Fragmentation, andSynthesis16Table 2: STAR run times for the workflow processes, including off-line thermal cycling.Sample Quality and YieldSamples were run on the Agilent 2100BioAnalyzer to determine quality. See Figure4. In addition, qPCR results demonstratedconsistent yields. For example, the averageyield was 292nM for the 8 replicates of UHR and270nM for the 8 replicates of Brain.Figure 4: BioAnalyzer results.
Sequencing ResultsThe following figures and table represent data from the sequencing runs.Figure 5: Pass filter reads.Figure 6: Excellent correlation for technical replicates of Universal Human Reference RNA (UHR) and Reference Brain RNAdemonstrating reproducible library prep generation
Table 3: TruSeq Stranded mRNA Sequence Quality Metrics.Cross-contaminationAnalysis of the sequencing data showed no evidence of sample cross-contamination.ConclusionThe TruSeq Stranded mRNA sample preparation protocol has been successfully automated on the Hamilton Microlab STAR.The Illumina Qualified* automated method yields high quality libraries that produce comparable sequencing data results tomanual processing, ready for HiSeq or MiSeq sequencers.
*Illumina Qualified indicates that Illumina’s analysis of libraries prepared with the STAR automated method has shown the librariesto perform comparably to those prepared manually. This method is not an Illumina product, and Illumina does not support thisproduct. Illumina makes no representations or warranties with respect to this product. 2013 Hamilton Company. All rights reserved.Illumina and TruSeq are registered trademarks of Illumina, Inc.All Hamilton trademarks are owned and/or registered by Hamilton Company in the U.S. and/or other countries.Lit. No. L50154 Hamilton Company — 1/2014 QTY: 250 Printed in U.S.A.Web: www.hamiltonrobotics.com800-648-5950USA:Email: infoservice@hamiltonrobotics.comUnited StatesTel: 1-775-858-3000ChinaTel: 86-21-6164-6567United Kingdom & IrelandTel: 44 (0)121-717-0199FranceTel: 33 (01) 69751616BrazilTel: 55 (11) 9677-4093ItalyTel: 39-39-689-33-93Denmark, Norway,Sweden, FinlandTel: 45-70-26-4499Germany, Switzerland,Austria, BeneluxTel: 49 (089) 552649-0To find a subsidiary or distributor in your area, please visit hamiltonrobotics.com/contacts.
The Illumina TruSeq Stranded mRNA Sample Preparation kit provides reagents and a workflow for converting the mRNA in total RNA into a library of template molecules of known strand origin, which is then suitable for cluster generation and DNA sequencing. The kit has been verified for use on
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