Automated washing of FTA Card punches and PCR setup for reference samples using a LIMS-controlled Sias Xantus automated liquid handler

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. Punching and washing of buccal samples
  • 2.2. PCR
  • 2.3. Data analysis
• 3. Results and discussion
• 4. Conclusion
• Role of funding
• Conflict of interest
• Acknowledgements
• References
• Copyright

Abstract 

We have implemented and validated automated methods for washing FTA Card punches containing buccal samples and subsequent PCR setup using a Sias Xantus automated liquid handler. The automated methods were controlled by worklists generated by our LabWare Laboratory Information Management System (LIMS). The protocols were validated according to EN/ISO 17025 for use with STR amplifications kits AmpFℓSTR^® Identifiler^® and Y-filer^® (Applied Biosystems).

Keywords: Laboratory automation, LIMS, FTA, Reference samples

Back to Article Outline

1. Introduction 

Short tandem repeat (STR) typing of forensic genetic samples is an internationally recognized part of crime casework today [1]. Reference samples are in Denmark collected by police officers as buccal swabs on FTA Cards [2], [3]. FTA Cards are impregnated with chaotropic agents that destroy infectious agents and thus reduce the potential biohazards of the sample [2] and enable storage of FTA Cards at room temperature. Annually, 10–15,000 reference samples are processed in duplicate at the Section of Forensic Genetics, Department of Forensic Medicine at the University of Copenhagen.

Back to Article Outline

2. Materials and methods 

2.1. Punching and washing of buccal samples 

Buccal sample containing FTA Cards (GE Healthcare) were punched using BSD-600 Duet punchers (BSD Robotics, Queensland, Australia) into 96-well microtitreplates (twin.tec, Eppendorf GmbH, Hamburg, Germany) prefilled with 10μL autoclaved MilliQ water. FTA Cards were labeled with sample numbers in barcode format. The barcode was read by the puncher prior to punching of each FTA Card and the information was recorded in the puncher outputfile. Outputfiles were imported into the LabWare LIMS and used to generate punch batches. Based on one or more punch batches the LIMS generated a corresponding number of wash batches and worklists for a Sias Xantus (Sias, Hombrechtikon, Switzerland) automated liquid handler (ALH) controlling the number of microtitreplates to be processed and their individual content and location of samples to be processed. The Sias Xantus allowed for simultaneous processing of up to four microtitreplates. Following washing, the microtitreplates were completely dried prior to PCR setup controlled by another worklist.

2.2. PCR 

PCR master mix was prepared according to the manufacturer's instructions with modifications: For AmpFlSTR^® Identifiler^® PCR Amplification Kit and AmpFℓSTR^® Yfiler^® PCR Amplification Kit (Applied Biosystems (AB), Foster City, CA), the total reaction volume was reduced to 10μL. PCR conditions were according to the manufacturer's recommendations and amplifications were performed on Gold-plated 96-well GeneAmp^® PCR System 9700 (AB).

2.3. Data analysis 

Multicolor fluorescence capillary electrophoresis of amplified STR profiles was performed on ABI 3130xL Genetic Analyzers (AB). The results were analyzed using Genescan Analysis version 3.7 (AB) and allele calls were made using Genotyper version 3.7 (AB) macros.

Back to Article Outline

3. Results and discussion 

In LabWare LIMS, original samples were denoted “Parent samples” while aliquots from parent samples were denoted “Child samples”. All samples were uniquely identifiable by a sequential sample number. The FTA Cards were labeled with a barcode containing the associated parent sample number upon arrival to the department. Two child samples corresponding to two sample fractions and corresponding tests were automatically created by the LabWare LIMS upon sample registration of each FTA Card.

Previously, wash of FTA Card punches and subsequent PCR and capillary electrophoresis setup was performed using worklists and batches created manually from pick-lists controlled by an in-house developed sample management system. In LabWare LIMS, wash batches were created based on puncher outputfiles (Fig. 1). This enabled punching of both new samples and re-punching dictated by the LabWare LIMS in the same batch (Fig. 2A). The LabWare LIMS did not require samples to be punched in any given order resulting in a flexible punching process. Following import of the puncher outputfile, the status of punched samples was automatically updated within the LabWare LIMS and labels to microtitreplates were automatically printed.

• 
  • View Large Image
  • Download to PowerPoint

• Fig. 1. 

  Puncher outputfile. The outputfile contained among other information the name of the punching template used (IDFL_FTA), name of the batch (2009-0615_IDFL_P_HPE), well number and corresponding parent sample number. The sample with sample number “010355” in well D01 was not in queue for the IDFL test.

• 
  • View Large Image
  • Download to PowerPoint

• Fig. 2. 

  (A) Screendump from the LabWare LIMS displaying the batch layout. The batch view displays a view of the child samples (sample fractions) being processed. Hence, sample numbers were child sample numbers. Position 4 contained the sample automatically removed by the LabWare LIMS. Re-runs are indicated with either “DE” or “F”. (B) Worklist for the Sias Xantus automated liquid handler. The subsequent PCR batch was controlled by a worklist generated by the LabWare LIMS. Position 4 (well D4) contained the affected sample excluded from the batch. The “0” indicates wells where PCR master mix was not dispensed while “1” indicates wells into which PCR master mix was dispensed. The “TwintecPrefill_001” signifies the microtitreplate number 1.

If samples not in queue for punching were punched, affected samples were automatically excluded by the LabWare LIMS upon batch creation (Fig. 2A) preventing non-LIMS-controlled processing of samples. An error log containing affected sample numbers was automatically created and required manual intervention. Samples in queue were unaffected. The puncher software ensured that the same sample was not processed twice on one batch without an unmistakably warning was manually overridden. This ensured that two fractions from the same sample were processed on separate batches.

Samples excluded due to wash batch creation, were also excluded from PCR setup. Hence, PCR master mix was not dispensed into the wells of excluded samples (Fig. 2B). Combined, integration of the LabWare LIMS and a Sias Xantus automated liquid handler enabled processing of up to eight 96-well microtitreplates containing a total of 704 sample fractions within one working day.

Back to Article Outline

4. Conclusion 

We have demonstrated that washing of FTA Card punches and PCR setup for accredited, forensic genetic STR typing can be implemented on an automated liquid handler integrated with a LIMS leading to reduction of manual work as well as increased quality and easily scalable throughput.

Back to Article Outline

Role of funding 

None.

Back to Article Outline

Conflict of interest 

None.

Back to Article Outline

Acknowledgements 

None.

Back to Article Outline

References 

1. Morling N. PCR in forensic genetics. Biochem. Soc. Trans. 2009;037:438–440
   • View In Article
2. Dobbs LJ, Madigan MN, Carter AB, Earls L. Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing. Arch. Pathol. Lab. Med. 2002;126:56–63
   • View In Article
   • MEDLINE
3. Smith LM, Burgoyne LA. Collecting, archiving and processing DNA from wildlife samples using FTA databasing paper. BMC Ecol. 2004;4:4
   • View In Article
   • MEDLINE
   • CrossRef