Fast and reproducible amplicon library preparation for HLA typing with the 454 sequencing system

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1 Fast and reproducible amplicon library preparation for HLA typing with the 454 sequencing system Application note Top 3 reasons for automation of this application HLA typing today benefits greatly from next-generation sequencing methods. The essential first step for the Roche 454 sequencing system is generation of an amplicon library specific for the target genomic region a multistep PCR procedure involving purification, quantification and normalization steps. Automation of these post-pcr steps is highly desired in order to avoid errors and to make it fast and robust. Prevent cross-contamination and increase reliability of the process. Increase speed compared to manual set-up. Increase reproducibility of the results compared to manual processing. Introduction Sequence based HLA typing resembles a necessity for a best-possible histocompatibility match in haematopoietic stem cell transplantation. The Roche 454 Genome Sequencer provides a next-generation sequencing platform that allows for clonal amplification and therefore ambiguity-free typing results become feasible (1, 2). Prior to sequencing a library has to be generated containing 17 amplicons per patient covering the loci HLA A, B, C, DQB, DRB and DPB presenting the bottleneck in the sequencing workflow due to critical but error-prone pipetting steps. Subsequent to a PCR each amplicon needs individual purification and quantification prior to equimolar pooling of all amplicons for one sequencing run (up to 680 amplicons): The quality of sequencing results depends on accuracy in pipetting. For the introduction of the high resolution HLA typing system automation of the purification, quantification and equimolar pooling of amplicons is needed. Figure 1: Microlab STAR

2 Fully automated amplicon library preparation speeding up the entire HLA typing process In this application, the Hamilton Microlab STAR platform is used for automating the steps between generating the amplicon library by PCR and clonal PCR using the Roche 454 REM e system. Amplicons are purified by innobilization on magnetic beads; purified amplicons are then quantified using a fluorescent dye and subjected to equimolar pooling. Method description Purification Up to 176 PCR products in two 96-well plates are loaded and diluted to 1:1.5 with water, followed by mixing with the prealiquoted AMPure XP beads in a 1:1 ratio in two 96-well U-bottom plates. Mixing cycles are performed during binding of PCR products to the beads. For immobilization of the beads each U-bottom plate is transferred to 96-well ring magnets. Recurrent wash cycles with 70% ethanol are conducted. After complete drying of beads at room temperature, purified amplicons are eluted from the beads by addition of 25μl 1 x TE and mixing, followed by subsequent bead immobilization. Purified amplicons are aspirated from the U-bottom plates and diluted (1:3.3) directly into a deep well plate, prefilled with 1 x TE. Finally, all purified amplicons are further diluted to 1:33 in 1 x TE for the quantification of purified amplicons (final dilution is 1:100). Quantification For the quantification of PCR products the Quant-IT dsdna Assay Kit is used and the PicoGreen dye is manually diluted to 1:200 with 1x TE and filled into a trough. Serial linear dilution of a DNA standard representing concentrations from 3.12 to 100ng/μl is carried out in 1.5ml tubes. In each quantification plate 100μl PicoGreen dye is pre-aliquoted and mixed with 100μl of standard dilutions and amplicons diluted 1:100. The plates are manually transferred to an external fluorimeter and measured. Concentrations are directly calculated in ng/μl from fluorescence signals and transferred to an Excel-file. On the basis of the standard concentrations the coefficient of determination is calculated and conduces as a critical validation criteria (r 2 > 0.98). Normalization and pooling For equimolar pooling of the 176 PCR products, the PCR product concentrations are taken from an Excel-file. This input is obligatory. On the basis of a configuration file that contains the amplicon lengths the concentrations of amplicons are converted from ng/μl to molecules/μl. From each amplicon 1x10 9 molecules are transferred to the pool. Due to the high variability in amplicon concentrations (0.5-60ng/μl), pre-dilutions for single amplicons with 1x TE are performed if necessary. The amplicon library pool, containing 1x10 9 molecules of each PCR product, is manually diluted to 5x10 5 molecules and used directly for emulsion PCR, followed by 454 s sequencing protocols for GS Junior or GS FLX. Technology The Microlab STAR pipetting workstation is equipped with monitored air displacement pipetting technology. It allows for highly accurate and failsafe pipetting, even of low volumes. It avoids use of tubing, pumps or system liquids, thus significantly reducing the risk of cross-contamination or contamination by bacterial growth. Cross-contamination is also prevented by the use of disposable tips with filters. Figure 2: Deck of the Microlab STAR for automated amplicon library preparation for HLA typing with the Roche 454 sequencing system.

3 System description The Microlab STAR for this application includes a CO-RE 96 Probe Head and eight individual pipetting channels (Figure 3). The deck is automatically loaded with reagents, disposable tips, elution plates and samples in 96-well PCR plates on appropriate carriers using the Autoload function of the robotic system (Figure 4). Figure 3: Microlab STAR equipped with CO-RE 96 Probe Head and 8x 1000μl pipetting channels. Figure 4: Microlab STAR deck loading for purification. The 96-well plates containing PCR products, deep well plates, assay plates for quantification and the pool tubes are labelled with barcodes. The barcodes are automatically scanned (Figure 5) and matched to user specified barcodes thus avoiding loading of wrong tubes. Figure 5: Microlab STAR Autoload. 1D barcode scanner is reading barcodes of deep well plates while carrier is pulled in by the Autoload function. Plate transfers are done by the CO-RE Gripper (Figure 6). Shaker modules for mixing and magnets for bead immobilization are mounted on two carriers that are fixed to the deck. The deck layout configuration enables processing of 176 amplicons per run. Using downholder frames, three different 96-well PCR plates can be employed. This allows flexibility of the PCR cycler platform. Figure 6: Transport of U-bottom plates with the CO-RE gripper to the 96- well ring magnets. Application software The methods were programmed using the Microlab VENUS Software. One method contains three different steps of the 454 amplicon library generation. It is subdivided into three modules which can be started individually or continuously: purification of PCR products, quantification of purified amplicons and equimolar pooling of PCR products.

4 Results Automated purification of PCR products by AMPure XP beads results in complete removal of primer dimers and further DNA fragments of up to 130bp. Minimal loss in quantity of the PCR product is observed as exemplarily depicted in Figure 7. Figure 7: Comparison of electropherograms of one PCR product (amplicon HLA C Exon 4) before purification (a) and after purification (b) on the Microlab STAR platform. PCR product purification by AMPure XP beads in a 1:1 volume ratio removes primer dimers efficiently.

5 Figure 8: Equimolar pooling accuracy of manual and automated approach by evaluation of sequencing data. Read counts per amplicon were plotted and shows an even distribution of reads per amplicon when pooling automatically. Figure 9: Equimolar pooling accuracy of manual and automated approach by evaluation of sequencing data. Graphical interpretation of the statistical evaluation by a box plot shows a higher median with a tight interquartile range for automated pooling. The validation of quantification by calculating the coefficient of determination exceeds the critical value of r 2 > 0.98 over 10%, thus emphasizing the reproducibility and precision of the automated pipetting by means of the serial dilution of the DNA standard. The advantage of the automated equimolar pooling setup over a manual pooling approach is shown in Figures 8 and 9. Whereas sequencing of automatically pooled amplicon libraries generates an even coverage of the PCR products, the coverage shows higher variability when pooled manually. Discussion Automation of the amplicon library generation resembles a necessity for amplicon sequencing by the Roche 454 pyrosequencing system for failsafe routine application enabling HLA high resolution typing for 10 patients within 3 days and for 40 patients within 4 days. Hands-on time could be reduced by 94% compared to manual set-up. Barcode labelling allows for sample tracking and LIMS integration. DNA concentrations and volumes used for pooling are logged per amplicon and sample, likewise the rest of the process is monitored and logged. Although this system was developed for HLA typing all kind of PCR products in 96-well plates undergo the automated pooling process highlighting the flexibility of the system. The modules can also be operated separately and thus the Microlab STAR can be applied in further post-pcr genomic approaches. Accordingly, the Microlab STAR presents a reliable and precise, as well as time-saving platform for generation of any amplicon library. The automation of pipetting steps assures contamination-free processes, provide error-free pipetting and prevent any mix-up of samples or plates. References: (1) Gabriel C et al.. Rapid high-throughput human leukocyte antigen typing by massively parallel pyrosequencing for high resolution allele identification. Hum Immunol Nov; 70(11): (2) Bentley G et al.. High-resolution, high-throughput HLA genotyping by next-generation sequencing. Tissue antigens Nov; 74(5):

6 System requirements Part number Microlab STAR, 8x 1000µl channels, CO-RE 96 Probe Head MPH, Autoload , , CO-RE gripper System dimensions: Width: 1990mm Height: 903mm Depth: 1006mm Labware requirements Part number / Provider LightCycler 480 Multiwell Plate 96 Roche MicroAmp Fast 96-Well Reaction Plate, 0.1ml twin.tec PCR Plate 96, semi-skirted U96 DeepWell plates U96 MicroWell plates, polypropylene, 0.5ml Corning 96 Well Black with Clear Flat Bottom Polystyrene NBS Microplate 50ml reagent reservoirs 120ml reagent reservoirs Reservoir, flat bottom Microcentrifuge tubes, extended capacity, 1.7ml 50ml tubes ABI Eppendorf NUNC NUNC CORNING / Hamilton / Hamilton NUNC Sigma GREINER Reagents and chemicals Provider AMPure XP Beads Agencourt Quant-iT PicoGreen ds DNA Assay Kit Invitrogen 1 x TE, Tris-EDTA buffer User-supplied H 2 O, molecular biology grade User-supplied 70% ethanol User-supplied Contact Österreichisches Rotes Kreuz, Landesverband OÖ, Blutzentrale Linz, Krankenhausstrasse 7, 4017 Linz, Austria, Tel. +43 (0) , Team Genomics: bz-genomics@o.roteskreuz.at 2012 Hamilton Robotics GmbH. All rights reserved. Lit. No. AN /00 QTY: 300, 11/12 Printed in Germany. Web: USA: infoservice@hamiltonrobotics.com United States Tel: United Kingdom & Ireland Tel: +44 (0) Brazil Tel: +55 (11) China Tel: France Tel: +33 (01) Italy Tel: To find a subsidiary or distributor in your area, please visit hamiltonrobotics.com/contacts. Denmark, Norway, Sweden, Finland Tel: +46 (0) Germany, Switzerland, Austria, Benelux Tel: +49 (0)