Breakthrough in forensic workflow automation, eliminating the

Breakthrough in forensic workflow automation, eliminating the
sample preparation and lysis bottlenecks with the AutoLys STAR:
Technology and Validation Study
Laurent Baron 1, Bas de Jong 2, Lois Tack 3, Maureen Antonie 2, Flurin Gallman 1, and Rainer Beckbissinger 1
1 Hamilton
Robotics, P.O. Box 26, CH-7402 Bonaduz GR, Switzerland, [email protected]
2 The Netherlands Forensic Institute (NFI), Postbus 24044, 2490 AA The Hague, Netherlands, [email protected]
3 Hamilton Robotics, 4970 Energy Way, Reno NV 89502, [email protected]
EAFS 2012
Introduction
AutoLys Tube™ Features
Forensic DNA sample processing frequently involves manual lysis and extraction to prepare
cleared lysates from various biological samples (swabs, cloth cuttings, etc.) prior to DNA
purification. Lysis steps are time-consuming, introduce gaps in sample workflows, loss of
traceability and can increase contamination risks and user errors. Sample preparation and lysis
steps are major bottlenecks in many forensic labs. We describe the Microlab® AutoLys STAR®,
a new automated lysis solution from Hamilton Robotics, and show validation data from the
Netherlands Forensic Institute (NFI).
Figure 3: AutoLys Tube side view
Figure 4: Tube inner diameter
Wide inner tube
diameter allows
efficient mixing during
lysis shaking step.
Cap
Inner tube with PTFE filter
Outer tube with 2D barcode
Inner diameter:
Automated Process


Figure 1: AutoLys system workflow
Process
evidence
sample
Match
barcode
Transfer
sample to
AutoLys
tube
Place
AutoLys
tubes in
racks
Load
racks and
reagents
on STAR
Samples
ready to
purify DNA
AutoLys
steps
AutoLys Tubes (Fig. 3) have been
designed specifically for the automated
lysis of forensic samples. The wide
opening of the inner tube (Fig. 4) allows
user to easily insert various materials
such as swabs, cigarette butts, fabrics
and tape.
AutoLys Tube = 11 mm
Standard 1.5-2 mL
microfuge tube = 8-9
mm
Figure 5: AutoLys Tube with
swab head
Cotton swab head
Integrated filter allows automated
separation of lysate from trace carrier.
The complete lysis workflow takes
place in a single vial, thus maximizing
yields, eliminating contamination risks
and drastically reducing operator time.
Lysis buffer (500 uL)
Swab sample is completely immersed by
liquid; add up to 500 uL of lysis buffer.
Figure 2: System deck layout and components – 96 sample processing
Tips
Tips
Tips
Tips
Tips
Tips
Tips
Tubes
Tips
Sample
Figure 6: AutoLys Tube workflow
Cap
Rack
AutoLys
Rack
Cap
Rack
Output
Rack
Output
Rack
AutoLys
Rack
Heater
Shaker
Balance
Racks
Balance
MAGNET Racks
AutoLys
Rack
AutoLys
Rack
AutoLys
Rack
Heater
Shaker
Add
Lysis
Buffer
Centrifuge
Heater
Shaker
Liquid
Waste
Heater
Shaker
Heater
Shaker
Lift
and
Lock
Incubate
and
Shake
2D BC
Reader
Spin
Clear
Lysate
Reagent
Reagent
Reservoirs Reservoirs
HHS Heater Shaker blocks
automate incubation &
shaking on the robot deck
Integrated 2D barcode reader
for total sample tracking
throughout the process
Integrated on-deck centrifuge
separates sample material from
cleared lysate
Figure 7: AutoLys Channel Functions
 Transport
 Capping and
individual
tubes to HHS
for incubation
decapping of
individual tubes
 Perform “lift and lock”
step, transforming
AutoLys Tube into
spin column
Results
AutoLys tube performance using the AutoLys STAR system was directly compared to manual processing
results using QIAshredder filter spin columns. Comparative yields were calculated and gave a “scoring
coefficient”. Subsequent DNA purification from all lysates was carried out manually following the NFIvalidated protocol. DNA samples were then quantified and analyzed by STR typing.
Sample Preparation
Blood (high): 5 uL undiluted blood spotted on cotton swab
Saliva (high): 40 uL undiluted saliva spotted on cotton swab
Blood (low): 5 uL (15x diluted) blood spotted on cotton swab
Saliva (low): 11 uL (3x diluted) saliva spotted on cotton swab
Lysis and DNA Extraction
Lysis on AutoLys STAR or manual:
450 uL lysis buffer and 50 uL ProK, incubation 4hr @56deg C.
Manual DNA extraction of all lysates using QIAamp® DNA Mini Kit (QIAGEN®):
Binding, washing, and elution in 2x 50 uL elution buffer
Downstream Processing
DNA quantification - Quantifiler® Duo DNA Quantification Kit (Life Tech.®)
STR amplification - AmpFlSTR® NGM Next Generation Multiplex Kit (Life Tech.®)
Fragment analysis - 5sec@3kV on ABI 3130xl™ Genetic Analyzer (Life Tech.®)
Scoring Coefficient
1
Autolys / QIashredder
Sample Type DNA Quantification
STR Peak Heights2
Blood (high)
118.1 %
124.2 %
Saliva (high)
120.8 %
116.2 %
Blood (low)
93.6 %
88.0 %
Saliva (low)
97.3 %
91.5 %
1
Scoring coefficient based on overall data from initial tests.
2 Average
of peak heights from DNA profiles, corrected for dilution
factor (i/a) and amount of input in PCR.
Developmental validation study
performed by NFI
Part 1 – Initial testing of
AutoLys Tubes and STAR system
Part 2 – Protocol optimization:
hardware and software
Part 3 – Testing of
mock crime scene samples
 Removes inner
tube after
centrifugation
Discussion
DNA extraction of crime scene samples involves multiple critical steps. Many labs choose to
manually perform DNA lysis and extraction of their precious samples due to concerns about yields
and quality. Although large portions of this process have been successfully automated, the most
critical steps are still done manually: (1) sample lysis, and (2) subsequent separation of the cleared
lysate from the trace carrier. These time-consuming steps have proven difficult to automate, nearly
always add gaps in traceability, with high risks of contamination and sample mix-up. They are
considered major bottlenecks for most forensic labs.
The AutoLys STAR is a fully automated lysis solution designed to overcome these essential crime lab
needs. The single instrument platform uses a unique 2D-barcoded AutoLys Tube and can process up
to four 24-tube racks at once. It carries out fully automated lysis & separation in a single tube without
user intervention. This includes all liquid handling, heated-shaking incubations and centrifugation
steps. The AutoLys drastically reduces contamination risk, sample-switching and other user errors.
New multifunctional AutoLys Channels are unique to the AutoLys platform. AutoLys, as a walk-away
robot, can run unattended overnight without need for assistance. Lysate purification (e.g., using
magnetic beads) can be on the same deck or transferred to another system for further processing.
The NFI is intensively testing the AutoLys STAR system, performing developmental validation testing
prior to its release. Initial validation data show that samples processed using the AutoLys give
equivalent or better results than the same samples processed manually. This innovative system is a
promising solution for forensic scientists to help process their ever increasing sample numbers while
maintaining quality, traceability and maximizing DNA yields.