Automated Differential Digestion Analysis Utilizing DNase I
Advancing Justice for Survivors of Sexual Violence
Separating sperm cells via differential digestion from sample swabs is a time-consuming effort. With the ever-increasing amounts of backlog from sexual assault evidence collection kits (SAECK), the need for automated differential digestion analysis has become a necessity, not a wish list item. Even with the addition of commercially available extraction instruments, sample processing capabilities still remain at a low threshold, Aurora Biomed used the VERSA 1100 to look at other methods which aimed to minimize the three following pain points:
- Elongated wash steps.
- Multiple time-consuming centrifugation steps.
- Low sample processing capabilities.
How Is This Achieved?
Automated differential digestion analysis of sperm cells utilizing DNase I workflow has been optimized to dramatically reduce and even eliminate these aspects. This, in turn, has allowed sample processing to rise exponentially whilst reducing the overall time of sample processing:
5 hours from start to finish
96 Samples per run
6x faster than conventional method
1 reallocated forensic scientist
The Poster
At ISHI34, Krina Carman and Monica Dyer from Oakland Police Department discussed their experience in processing 25000 Sexual Assault Evidence Kit (SAEK) samples with the help of VERSA 1100 in past 10 years.
The Published Report
This method is currently employed by crime labs—Oakland PD has published a vigorous validation on the VERSA 1100. Follow the links below for more information and to view the latest publication from criminalist Helena Wong:
Overall Workflow
Switching workflows is never an easy task, especially when there have been so many resources pooled into an existing method, however, when you look at the different pieces of the overall process, automation of selective degradation is far from outlandish:
What is the throughput?
It can be configured for 96-well format depending on instrument 4-channel head– actual throughput time will depend on the specific procedure and steps involved.
What's the time frame required for processing a plate of samples?
Processing a complete plate of 96 samples typically requires around 5 hours including incubation time on workstation.
What is a brief overview of the automated protocol on VERSA?
Automation begins with the addition of buffers to the spin basket, including samples. After 30 minutes of sample rehydration, there’s a 30-minute shaking process to loosen the swabs. The first pause step involves removing the plate to centrifuge the cells and pellet them. Afterward, the plate is placed back on the VERSA, which then removes the supernatant into 2ml tubes to separate the aqueous extract. The spin basket is then returned to the deck, with the samples off the deck, enabling the removal of the supernatant. Subsequently, the VERSA adds digestion buffer (Tween buffer) and Pro-K for a 30-minute digestion of epithelial cells at 65 degrees, taking place on the on-deck heater. Following the removal of the epithelial cell fraction, the digestion process targets sperm cells.
What are the key steps involved in the differential extraction process?
The process involves cell lysis, separation of sperm cells from epithelial cells, and DNase1 treatment to selectively recover sperm DNA while minimizing the victim’s DNA.
Does the VERSA1100 require specialized consumables or reagents for automating the differential digestion process?
The VERSA1100 require specific automation consumables and reagents tailored for differential digestion protocols, ensuring optimal performance.
How many VERSA instruments in Forensic labs are running online the differential digestion protocol?
Oakland Police Department and Contra Costa County but other forensic labs use VERSA for other applications such as NGS, NAP, PCR, SPE, and LLE.
Can the purification step be carried out on the VERSA workstation?
The purification step is a magnetic bead-based separation method and can be easily performed on the VERSA because it has a magnet stand positioned on the deck.
Has any work been completed on the use of DNase 1 with degraded samples?
Yes, a study has been done on a number of different types of degraded samples: samples subjected to multiple freeze-thaw cycles, samples subjected to 75 degree C and 80% humidity, and aged semen stains. Results show that even if the samples are degraded, the same DNA typing results were obtained as compared to the traditional manual differential digestion method.
What are the chances of contamination issues?
There were negligible contamination issues due to the VERSA’s built-in HEPA/UV system.
Can VERSA can automate the slide preparation?
YES, VERSA can automate the pre-digest and post-digest slides. After spinning down the plate to pellet the cells, the VERSA carefully removes the supernatant into a 2mL tube (this could be the aqueous or epithelial cell fraction, depending on which step this is) by using liquid level sensing and leaves behind ~50µL. The VERSA then re-suspends the pellet into solution and then takes 5µL and puts it onto an 8-well microscope slide that is on the deck. The microscope slides were custom-made so that the distance between the wells on the microscope slides fit the distance between each of the channels of the 4-channel pipettor head. The slides are then removed from the VERSA deck to be heat dried and stained.
Does VERSA has on deck heaters?
YES, VERSA has Heater/Shaker positioned on the deck. The temperature range is 4-90°C.
Can the manual agitation step be automated on the VERSA instrument?
This step is probably the most tricky to automate. Need to make sure the cells are fully homogenized. It is difficult with the robotic arm to prevent cross contamination. There are concerns with samples sticking to the pipette head, which then increase risk of contamination. So it was easiest to leave the step.