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Forensic DNA Processing Efficiencies using a Novel Consumable Device and Automated Workflow

Kevin Miller, Hamilton

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A significant contributing factor to forensic DNA sample backlogs is the manual nature of sample manipulation from sample collection through analysis. Each step introduces risks that may break a sample’s chain-of-custody or require retesting of the sample, which may be limited. Risk-contributing human errors may include documentation and labeling errors, cross-contamination, and lost samples that completely eliminate a crucial testing opportunity. Likewise, analyst subjectivity and performance variability, including that over time and among different analysts within the same laboratory, may introduce uncertainty that could be challenged in court.

Manual DNA evidence sample handling is also time-consuming, especially once the samples arrive at the laboratory. Sample throughput by manual means is limited by the analyst, who spends considerable time on low-value activities such as liquid pipetting.

A Novel Approach to Alleviate Forensic DNA Sample Backlogs

An automated workflow, incorporating unique AutoLys tubes and the AutoLys STAR assay ready workstation, both from Hamilton Company (Reno, NV), provides a low-risk, time-saving solution to reduce forensic DNA sample backlogs.

Figure 1:

Consumable AutoLys-A tubes (Figure 1) have a unique tube-in-tube configuration to facilitate hands-free forensic DNA sample processing. An inner tube with screw cap serves as a nucleic acid extraction unit, and may be used as a lysate filtration (spin basket) device using a non-DNA binding membrane for conventional evidence processing, or without a membrane for differential extraction of sexual assault evidence using conventional procedures. The outer tube is 2D barcoded on its base for easy scanning from the bottom of an AutoLys tube rack. The barcode creates a chain-of-custody to automatically and independently verify each sample (and reagent blank) and correlate it with the item number, case file, and assigned analyst. This saves time compared to manual sample verification and eliminates the risk of analyst transcription error.


The outer tube also receives cleared sample lysate for downstream processing. In addition to sample processing, the AutoLys-A tube may be used as a sample collection device, where a sample swab head, piece of fabric, cigarette butt, or other item is directly placed into, and stored within, it. This reduces the need for intermediate manual sample manipulations that may necessitate additional handling and documentation steps, as well as increase the risks of contamination, error, and mishandling.

The AutoLys STAR and AutoLys SAE STAR assay ready workstations are the only automated solutions specifically designed to work with AutoLys-A tubes. The AutoLys STAR is ideal for simultaneous processing of up to 96 biological samples, while the AutoLys SAE STAR is best suited for simultaneous processing of up to 48 mixed sexual assault samples using differential extraction methods. The foundation of both assay ready workstations is an automated liquid handler that achieves superior liquid measurement accuracy, precision and reproducibility across a broad range of volumes, from microliters to milliliters, by means of up to four independent air displacement pipetting channels.


When used with Hamilton Compressed O-Ring Expansion (CO-RE) tips, an air-tight seal is created to maximize liquid handling performance and minimize aerosol generation during tip ejection. The tight seal also enables precise sensing and traceable monitoring to ensure optimum performance when dispensing any type or class of liquid. Pressure-based and capacitive Liquid Level Detection (pLLD and cLLD, respectively) detect the liquid level, while Total Aspiration and Dispense Monitoring (TADM™) detects even slight deviations from pre-defined performance tolerances and Monitored Air Displacement (MAD) detects incorrectly filled wells due to clots or clogs in the pipette tip. Anti-Droplet Control (ADC™) reacts to real-time pressure changes when pipetting volatile organic solutions to prevent drop formation. In addition to liquid handling, CO-RE technology is used with on-deck gripper paddles to enhance functionality by automatically transporting sample plates and tube racks through the workflow steps.


The workstations are pre-configured with tools such as a centrifuge and heater/shaker to increase walkaway efficiencies and also feature AutoLys channels to automatically manipulate the AutoLys-A tubes. In addition to capping, decapping and transporting the AutoLys-A tubes around the deck, these specialized channels lift and lock the inner tube into a “spin basket” position, and also remove and replace it without the need for manual intervention. For laboratories preferring to use tube-based workflows instead of progressing to a microplate format during processing, the assay ready workstations may be optionally configured with the industry’s first FlipTube tool that opens and closes the lids of Hamilton’s FlipTube microtube without manual intervention.


The assay ready workstations may be integrated with any laboratory information management system (LIMS) for automatic evidence accessioning and/or worklist generation, and an autoload capability includes 2D barcode scanning to verify detailed sample information, including analyst and case number, along with correct positioning of the sample on the deck. Pre-programmed automated methods simplify use and also reduce the time and effort required to internally validate the automated workflow, while password-protected lockout protocols prevent unauthorized or accidental changes. In operation, user-friendly and interactive run setting dialogues from the integrated VENUS software guide users of any skill level through instrument setup and operation. Once the automated workflow is complete, a run file containing details on each sample type, location and action is sent back to the LIMS where it may be stored and output as a standardized form for use in a case file report.


By incorporating AutoLys-A tubes and the AutoLys STAR or AutoLys SAE STAR assay ready workstation into forensic DNA sample analysis workflows, risks of manually-derived errors, variability, and contamination are eliminated, sample integrity is strengthened, and sample processing throughput is increased. In addition to also reducing active analyst time during processing, the automated methods also reduce the numbers of analyst errors, and hence the amount of time spent on quality review. As active time devoted to sample processing is significantly reduced, analysts are free to focus on higher value activities. Beyond sample processing efficiencies, automated generation of run file details in a standardized output format reduces active time required during casework documentation review to further contribute to overall laboratory efficiencies.

Eliminating Bottlenecks in Multiple Applications

The automated workflow, using AutoLys-A tubes and the AutoLys or AutoLys SAE STAR assay ready workstation, is beneficial in any recent or cold case criminal investigation involving collection of forensic DNA, even among different sample types; and is especially useful in processing complex, mixed samples from sexual assault cases. In fact, when processing sexual assault evidence involving a mix of epithelial (victim) and sperm (subject) cells, the automated AutoLys workflow flexibility extends to multiple processing methods, including newer ones such as affinity capture of unique sperm proteins, nuclease-based digestion, and matrix derived separation, as well as traditional methods such as the differential extraction method first detailed by Peter Gill and colleagues in 1985¹.


Although the Gill method is commonly used in forensic laboratories, the variety of sample manipulation steps makes it a time-consuming and labor-intensive process. Slight modifications to the differential extraction method were recently published by Mark Timken and colleagues that improve efficacy, and automate the process, while including additional workflow steps²’³.

Applying the Automated Forensic DNA Typing Workflow

Here, we demonstrate performance of the automated workflow compared to manual processing in the context of differential extraction prior to analysis by autosomal short tandem repeat (STR) genotyping. Two sample types were processed and analyzed: mock sexual assault swabs prepared with semen and vaginal cells, and authentic post coital swabs collected at several time points.

In the automated workflow, the AutoLys SAE STAR software guides analysts through the modified Gill method setup, including where to place consumables and prepared reagents and also automatically calculates reagent volumes where indicated. Swabs are inserted into AutoLys-A tubes, loaded onto a 24-sample AutoLys rack, and placed onto the AutoLys SAE STAR deck. The AutoLys SAE STAR automatically decaps each tube, adds lysis reagent, recaps each tube, then moves each to an on-deck heater shaker. After heated incubation with shaking, each AutoLys-A tube’s inner tube is automatically lifted and locked, then the assembly is automatically transferred to an on-deck centrifuge to separate the swab from the liquid and pellet unlysed material consisting of mostly sperm cells. The supernatant, or non-sperm containing fraction (NSF; containing epithelial cell DNA) is transferred to a 96-well microplate, while the remaining pellet is resuspended in buffer as the sperm fraction (SF) then transferred to a 96-well microplate pre-loaded with lysis reagent. NSF and SF fractions may also be transferred to Hamilton’s FlipTubes instead of microplates according to laboratory-specific preferences. The SF microplate is automatically transported for heated incubation with shaking, then centrifuged to pellet the sperm cells and washed twice to remove any residual material. Sperm cells are then lysed, and SF DNA is transferred either to the 96-well microplate containing the corresponding NSF fractions, or to individual FlipTubes for further processing. In this example, capillary electrophoresis was performed on the sample fractions after processing, and they may also be prepared for use in massive parallel sequencing (MPS) workflows.

In the manual workflow, swabs are placed in tubes, lysis reagent is added, and the tubes are manually transferred to a heater shaker. Following incubation, each sample is manually vortexed, then each swab is removed, transferred to a separate spin basket and centrifuged to pellet unlysed material consisting of mostly sperm cells. The NSF supernatant is manually removed, and the SF pellet is washed multiple times, lysed, and collected for further analysis.

Figure 2A:

Figure 2B:

Per Figure 2, DNA quantification and STR typing analysis were performed on samples containing male and female cellular DNA that were processed manually (A) and robotically (B) using AutoLys-A tubes and the AutoLys STAR. STR profiles indicate comparable results among the automated and manual methods. The influence sampling and assay variation may be seen in the two manually processed samples, whereas, sample-to-sample consistency in DNA fragment peak heights was observed in STR profiles generated from those samples processed in the automated AutoLys workflow. Additionally, the automated method enables walkaway processing so that users can focus on higher value activities.

DNA quantification and STR typing were also performed on swabs collected up to 96 hours post-coitus and processed via the fully automated AutoLys workflow (Figure 3), further demonstrating robustness in the automated workflow when processing challenging samples.

Figure 3:

Summary

Unique AutoLys-A tubes and the AutoLys STAR or AutoLys SAE STAR assay ready workstations effectively support efforts to reduce forensic DNA sample processing backlogs. By fully integrating this novel automated system into the forensic DNA profiling workflow, sample throughput and accuracy/precision in processing is increased while manual labor involvement is significantly decreased. This imparts repeatability in high-quality results without the risks of analyst variability and errors or the burdens of quality incidents and retesting. Standardization in data, and the reallocation of analyst efforts towards data interpretation, report writing, and other high-value activities extends time-savings beyond sample processing to improve overall laboratory efficiency and reduce backlogs.


REFERENCES

¹ Gill, P.; Jeffreys, A.J.; Werrett, D.J. Forensic application of DNA ‘fingerprints’. Nature. 1985, 318, 577-579.

² Timken, M.D.; Klein, S.B.; Buoncristiani, M.R. Improving the efficacy of the standard DNA differential extraction method for sexual assault evidence. Forensic Sci Int-Gen. 2018, 34, 170-177.

³ Timken, M.D.; et al. Automation of the standard DNA differential extraction on the Hamilton AutoLys STAR system:A proof-of-concept study. Forensic Sci Int-Gen. 2019, 40, 96-104

Editor's Note:

Figure 1. AutoLys-A tubes on the AutoLys STAR assay ready workstation deck demonstrating (left) inner tubes in the lifted and locked position; (center) inner tubes being automatically transported around the deck; and (right) AutoLys-A tubes in AutoLys tube racks.

Figure 2. Green panel results for samples comprised of 0.033 µL semen on vaginal swabs that were processed (A) manually; and (B) robotically using AutoLys-A tubes and the AutoLys STAR.

Figure 3. Green panel results for 96-hour post-coital swab samples that were processed robotically using AutoLys-A tubes and the AutoLys STAR.