Bobby L LaRue, Andrea Moore, Pamela Marshall, Jonathan King, Bruce Budowle
Institute of Applied Genetics; Department of Forensic and Investigative Genetics; University of North Texas Health Science Center; 3500 Camp Bowie Blvd., Fort Worth, Texas 76107
Automated rapid DNA typing systems provide a unique potential to deploy STR-profiling systems outside of
a traditional laboratory setting and into areas such as police stations or mobile laboratories without a
dedicated laboratory infrastructure, such as pre and post amplification areas. This type of forward
deployment has the capacity to increase the utility of arrestee DNA typing by reducing the time between
sample collection and profile generation. The RapidHit System (IntegenX; Pleasanton, CA) was
independently evaluated using primarily buccal swabs for reliability, reproducibility, sensitivity, concordance
, sensitivity to inhibitors, and both intra-run and inter-run contamination. The instrument analyzes swabs
that are added directly to a single-use micro fluidic sample cartridge where DNA extraction, normalization a
nd amplification occur. Capillary electrophoresis is performed utilizing an on-board capillary array, and data
are compiled in a standard .fsa file for analysis utilizing a separate computer. We also assessed instrument
performance against the backdrop of how samples are processed in a databasing laboratory (albeit with a
lower throughput). The results of our testing support that the reliability of the instrument mirrors that of
traditional methods used for generating profiles to be entered into a database. Additionally, reference
swabs processed using the instrument could be recovered for subsequent analyses in either another
RapidHit System run, or extraction and processing by more traditional STR-typing methods. The
instrument consistently generated high quality STR profiles that were concordant with standard methods
using the PowerPlex 16 HS (Promega; Madison, WI) chemistry, which is the current STR configuration of
the sample cartridges and instrumentation. The initial success rate was >90%. Re-analyses of a failed run
increases
the overall success rate and mirrors the process in the database laboratory.
Using an
alternating pattern of blank and buccal swabs, the instrument was tested for both intra-run and inter-run
contamination. No evidence of cross-contamination was detected over the course of several consecutive
runs completed over multiple days. In summary, the RapidHit System can reliably generate STR profiles
from reference samples. The automated single platform system performs comparable to that of standard
DNA-typing methods, and in the event of a failed result allows for further analysis of the same sample by
repeating the same test or by using other methods. The platform can be operated by laypeople with
minimal training which could potentially free analysts to focus on the more difficult tasks of data analyses
and interpretation.
Rapid DNA typing instruments have the potential to free DNA analysts from the laborious tasks of
processing biological samples. Additionally, if used for arrestee screening, collected reference samples
could be run by personnel outside of traditional crime lab environments with minimal training, and the
resultant data can be analyzed (via remote access) by a qualified analyst at a laboratory facility. Currently
the RapidHit System system has the capability of “going from reference swab to profile” in approximately 90
minutes using Promega PowerPlex16 HS chemistry.
The results of our studies demonstrate that the instrument delivers consistent, reproducible, profiles in
approximately 90 minutes with as little as 2 minutes “hands on” time from reference buccal swabs. The
instrument has similar failure rates to traditional STR typing methods, and samples typed by both the RapidHit
System and traditional PowerPlex16 HS assays were concordant. The assay is currently is calibrated towards
reference buccal cotton swabs, but our results suggest that a variety of collection substrates and analysis of
other sample types is possible. Finally, we have demonstrated that samples may be recovered and successfully
retyped following analysis.
3
RapidHIT Results
Sample
HSC 1
AMEL
CSF1PO
Penta_D
Penta_E
X
12
9,12
9,11
13,14
12,14
7,15
9.3
8
16,18
11,12
9,10
11,13
17
28,32.2
16
11,12
11,13
13,15
21.2,22
12,13
10,12
8
11
17,18
9,12
8,12
D13S317
10,12
D16S539
12,15
D18S51
28,31
D21S11
15,18
11,12
8,10
14
21,24
9
13,15
6,7
8,11
18
10,11
12
14,18
12
28,30
D3S1358
14,15
D5S818
11,13
D7S820
10
D8S1179
10,13
FGA
20,22
TH01
TPOX
vWA
HSC 2
HSC 3
X,Y
X
HSC 4
HSC 5
X,Y
10,13
X
11
13,14
19,25
8,12
10,12
7,9
8,9
14,18
HSC 6
HSC 7
X,Y
11,12
8,13
11,12
15,17
28,31.2
16,18
10,11
9,10
12,14
20,21
10,11
11,16
8,8.3
8,10
15,17
X
11,12
9,12
11,12
15,18
30,31
17
11,12
9,11
8,13
22
10,11
11,12
7,9
8
16
HSC 8
HSC 9
X,Y
10,11
12,14
10
13,14
29,32.2
15,18
12
7,10
12,13
21,24
9,12
12,21
6,9
9,10
16,18
X,Y
12
10,11
12
14,21
29,30
17
10,12
8,11
10,13
24,24.2
9,13
11,17
7,9
11
14,15
HSC 10
HSC 11
X
10,12
11,13
11,12
13,16
29,32.2
16,17
7,11
9,12
12,14
25,26
11,12
8,15
6
8
17
X,Y
10,12
11
12,13
12,18
30,31
15,18
12
10,11
13,14
23
8,13
5,8
6
8,11
17,19
HSC 12
HSC 13
X
10,13
11
9,14
12,17
28,30
14,17
11,12
8,11
14,15
23,25
11,13
10,11
6,9.3
8,9
19
X,Y
12
9,11
11,12
13,18
28,30
16,17
12,13
9
15,17
20,22
11
5,10
7,9.3
9,11
HSC 14
HSC 15
X
12
11
11,13
12,18
28,30
15,16
12
10,11
10,11
22,24
9,13
7,12
9.3
8
16
X
12
11,13
11,14
13,14
29,31.2
18
11,12
10,11
10,15
22
10,13
16
8,9
11
17,18
8,13
9,14
14,15
12,13
27,31.2
28,32.2
16,18
11,13
9,11
11,12
11,12
19,24
9,13
5,11
6,9.3
8
16,17
16,17
HSC 16
X
11,12
11
11
13,14
31,32.2
15
11,12
10
10,14
20,22
11,12
12,17
9.3
9,11
17,18
HSC 17
X
11,12
9,11
8,12
14,23
30,32.2
14,16
9,13
9,11
13,14
21,25
9,10
5,18
7,9.3
9,11
16,17
HSC 18
HSC 19
HSC 20
X,Y
12
8,12
8,11
X,Y
10,11
8,11
9
12,17
30,33.2
15,17
11,12
11,12
14
23,24
9
11,18
6,9
8
14
X,Y
12,13
8,10
9,11
14,16
29,30
16,17
12,14
10,11
12,15
22,23
9,11
12
7,9
5,8
14,17
D7S820
D8S1179
FGA
Penta_D
Penta_E
TH01
TPOX
vWA
15,17
30
15
12,13
11,12
10,14
22,26
9
7,10
6,9.3
8
14,15
PP16HS
Sample
HSC 1
AMEL
CSF1PO
X
12
9,12
9,11
13,14
12,14
7,15
9.3
8
16,18
HSC 2
HSC 3
X,Y
11,12
9,10
11,13
17
28,32.2
16
11,12
11,13
13,15
21.2,22
12,13
10,12
8
11
17,18
X
9,12
8,12
10,12
12,15
28,31
15,18
11,12
8,10
14
21,24
9
13,15
6,7
8,11
18
X,Y
10,13
10,11
12
14,15
27,31.2
14,18
12
9,11
11,12
19,24
9,13
5,11
6,9.3
8
X
11
8,13
9,14
12,13
28,32.2
16,18
11,13
11,12
13,14
19,25
8,12
10,12
7,9
8,9
14,18
HSC 4
HSC 5
D16S539
D18S51
D21S11
28,30
D3S1358
14,15
D5S818
11,13
10
10,13
20,22
16,17
HSC 6
HSC 7
X,Y
11,12
8,13
11,12
15,17
28,31.2
16,18
10,11
9,10
12,14
20,21
10,11
11,16
8,8.3
8,10
15,17
X
11,12
9,12
11,12
15,18
30,31
17
11,12
9,11
8,13
22
10,11
11,12
7,9
8
16
HSC 8
HSC 9
X,Y
10,11
12,14
10
13,14
29,32.2
15,18
12
7,10
12,13
21,24
9,12
12,21
6,9
9,10
16,18
HSC 10
HSC 11
Figure 2. Contamination Study
Representative electropherograms from a single sample channel in consecutive runs with a) a sample from
individual “a”, b) a blank swab in the next run, c) a sample from individual “b” in the third run, and d) a blank
swab in the final run.
D13S317
X,Y
12
10,11
12
14,21
29,30
17
10,12
8,11
10,13
24,24.2
9,13
11,17
7,9
11
14,15
X
10,12
11,13
11,12
13,16
29,32.2
16,17
7,11
9,12
12,14
25,26
11,12
8,15
6
8
17
12,13
12,18
30,31
15,18
12
10,11
13,14
23
8,13
5,8
6
8,11
17,19
9,14
12,17
28,30
14,17
11,12
8,11
14,15
23,25
11,13
10,11
6,9.3
8,9
19
13,18
28,30
16,17
12,13
9
15,17
20,22
11
5,10
7,9.3
9,11
16,17
12,18
28,30
15,16
12
10,11
10,11
22,24
9,13
7,12
9.3
8
16
11,12
10,11
10,15
22
10,13
16
8,9
11
17,18
11,12
10
10,14
20,22
11,12
12,17
9.3
9,11
17,18
9,11
13,14
21,25
9,10
5,18
7,9.3
9,11
16,17
X,Y
10,12
11
HSC 12
HSC 13
X
10,13
11
X,Y
12
9,11
11,12
HSC 14
HSC 15
X
12
11
11,13
X
12
11,13
11,14
13,14
29,31.2
18
HSC 16
HSC 17
X
11,12
11
11
13,14
31,32.2
15
X
11,12
9,11
8,12
14,23
30,32.2
14,16
9,13
HSC 18
HSC 19
HSC 20
X,Y
12
8,12
8,11
X,Y
10,11
8,11
9
12,17
30,33.2
15,17
11,12
11,12
14
23,24
9
11,18
6,9
8
14
X,Y
12,13
8,10
9,11
14,16
29,30
16,17
12,14
10,11
12,15
22,23
9,11
12
7,9
5,8
14,17
15,17
30
15
12,13
11,12
10,14
22,26
9
7,10
6,9.3
8
14,15
Figure 3. Concordance Study
Results from a preliminary concordance study comparing reference buccal swabs run on a) by the RapidHit
System and b) by standard methods utilizing PowerPlex® 16 HS System (Promega; Madison, WI).
Figure 7. Sample Type and Substrate Tolerance
Representative electropherograms from one individual showing successful typing of a variety of samples and sample
collection substrates. a) buccal on a cotton swab, b) buccal on a Copan Floq swab, c) buccal on an EasiCollect
punch, d) 10μL of blood on a cotton swab, e) blood on a Diomics X-swab, and f) blood on FTA paper. The system
typed most substrate sample combinations well, with the exception of the EasiCollect and fresh, neat blood on cotton.
Follow on studies will identify protocols to assist with these sample substrate combinations.
Before implementing such a system, it is incumbent upon the community to perform validation studies that
define the limitations of the process.
We have been validating the RapidHit System for reliability, reproducibility, evidence of contamination (both
intra-run and inter-run), and sensitivity of detection. In addition to these studies, we also ascertained the
impact of inhibitors on the system, the feasibility of using other types of samples and sample collection
substrates, and the capacity to reanalyze samples that have been run through the system.
1a
Channel:
1
2
3
Run 1
Buccal 1 Buccal 2 Buccal 3
Run 2
Blank
Blank
Blank
Run 3
Buccal 6
Blank
Buccal 7
Run 4
Blank
Buccal 9
Blank
Run 5
Buccal 11 Blank Buccal 12
Run 6
Blank Buccal 14 Blank
Run 7
Buccal 16 Buccal 17 Buccal 18
Run 8
Blank
Blank
Blank
4
Buccal 4
Blank
Blank
Buccal 10
Blank
Buccal 15
Buccal 19
Blank
5
Buccal 5
Blank
Buccal 8
Blank
Buccal 13
Blank
Buccal 20
Blank
6
Ladder
Ladder
Ladder
Ladder
Ladder
Ladder
Ladder
Ladder
7
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
8
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
b Channel:
1
2
3
4
5
6
7
8
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Run 7
Run 8
Buccal A
Buccal E
Buccal D
Buccal C
Buccal B
Buccal A
Buccal E
Buccal D
Buccal B
Buccal A
Buccal E
Buccal D
Buccal C
Buccal B
Buccal A
Buccal E
Buccal C
Buccal B
Buccal A
Buccal E
Buccal D
Buccal C
Buccal B
Buccal A
Buccal D
Buccal C
Buccal B
Buccal A
Buccal E
Buccal D
Buccal C
Buccal B
Buccal E
Buccal D
Buccal C
Buccal B
Buccal A
Buccal E
Buccal D
Buccal C
Ladder
Ladder
Ladder
Ladder
Ladder
Ladder
Ladder
Ladder
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Figure 1. Experimental Design for Contamination and Reproducibility Studies
a) A graphical representation of the pattern used on the instrument to test for intra-run and inter-run
contamination. Blank swabs are listed as “Blank” and individual buccal swabs were numbered to compare
to paired swabs from concordance testing. b) A graphical representation of the pattern used for the
reproducibility study. Runs 1-4 were performed on one day and runs 5-8 on the following day. This testing
was performed on two separate instruments to evaluate instrument to instrument reproducibility.
Figure 5. Re-analysis of Recovered Samples
Representative electropherograms from a sample
that was a) analyzed on the RapidHit System,
recovered, b) re-analyzed on the instrument, and
then c) the process was repeated for a total of
three runs utilizing the same swab sample.
Samples are retained for follow on testing using
traditional bench top methods.
Preliminary data suggest that the RapidHit System had a run failure rate below 5%, and a full profile
success rate of 100% (n=150) on successful runs with standard cotton buccal reference swabs. During
the contamination study, of the 20 negative swabs run, 19 showed no DNA results (95%). In the one
negative sample that did return a profile, the profile was inconsistent with any recorded profiles at
UNTHSC or IntegenX, Possibly the contamination was associated with the swab.
Figure 4. Sensitivity Study
Representative electropherograms of the same individual at the following quantities of DNA placed on swabs.
a) 480 ng, b) 202 ng, c) 86 ng, d) 63 ng, and e) 32ng.
Figure 6. Potential Inhibitors
Representative
electropherograms
showing
successful typing of buccal swabs collected from
individuals who had recently used potential
inhibitors.
a) and b) were collected from
smokeless tobacco users and c) was collected
from a coffee drinker.
Our results indicate that the RapidHit System is capable of producing full STR profiles from reference
buccal swabs reliably in a reproducible fashion. The profiles are generated in approximately 90 minutes
with as little as 2 minutes of “hands-on” time. The profiles generated are concordant with standard STR
analysis methods. The failure rate of the system is comparable to current STR-typing methods (our
preliminary results suggest ~3%). In the case of an instrument failure, the swab may be recovered for
re-analysis either via an additional RapidHit System run or traditional methods. No intra-run or inter-run
contamination was observed. In addition, common potential inhibitors and a variety of sample collection
types and samples did not affect the ability to obtain STR typing results. Future studies will focus on
increasing our sample size to more accurately determine the performance of the instrument, full profile vs.
partial profile results, evaluation of intra and inter locus peak heights and to explore expanding the use
of the system to non-reference samples.
We thank the following companies for materials support: IntegenX (Plesanton, CA), Diomics
CA), and Promega Corporation (Madison, WI).
(La Jolla,