YFiler®Plus Verification Test Site Results
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Transcript of YFiler®Plus Verification Test Site Results
Lynne K. Burley Santa Clara County Crime Laboratory
San Jose, CA
Yfiler® Plus: Verification Test Site Results
Agenda
• Current limitations • Yfiler Plus design and configuration • VTS test site data
– Sensitivity – Inhibition – Mixtures – Non-probative casework – Discrimination of close relatives
Challenges to overcome Male relatives cannot be excluded
Adventitious matches in the database
Limited ability to overcome inhibition
Limited ability to amplify degraded samples
Lengthy amplification time
Allelic ladder does not cover full spectrum of known alleles
Taq
Yfiler Plus kit configuration
Streamlined workflow Reaction
component Volume per reaction (uL)
Yfiler Plus
Volume per reaction (uL)
Yfiler
Master Mix
10 9.2
Primer 5 5
Sample 10 10
Taq Included in master mix 0.8
Reduced amplification time Yfiler
95°C-11 min
94°C-1 min
61°C-1 min x 30 cycles
72°C-1 min
60°C-80 min
~3 hours
Yfiler Plus
95°C-1 min
94°C-4 sec
61.5°C-1 min
60°C-22 min
~1.5 hours
x 30 cycles
Expanded allelic ladder
Kit Alleles Virtual Bins Yfiler 137 72
Yfiler Plus 345 179
VTS plate setup
VTS sensitivity experiment
• Two-fold serial dilution of Control DNA 007 (0.1ng/uL*): 1ng, 500pg, 250pg, 125pg, 62.5pg, 31.25pg
• 10uL of each dilution added to reaction • Replicates of four • 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu * - 007 provided actually quant’d at 0.07ng/uL
Average total peak height - sensitivity experiment
0
5000
10000
15000
20000
25000
30000
35000
31.25pg 62.5pg 125pg 250pg 500pg 1ng
Concentration
rfu Yfiler Plus
Yfiler
Percent of alleles detected - sensitivity study
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 20 40 60 80 100 120 140
Concentration (pg)
% A
llele
s D
etec
ted
YfilerYfiler Plus
Intracolor balance - sensitivity study
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
250pg Yfiler Plus 250pg Yfiler 500pg Yfiler Plus 500pg Yfiler 1ng Yfiler Plus 1ng Yfiler
Concentration/kit
% b
alan
ce
ICB 6-FAM aveICB VIC aveICB NED aveICB TAZ/PET aveICB SID ave
Sensitivity study - baseline
VTS inhibition experiment • 1ng (10uL of 0.1ng/uL) control DNA 007
– 10ng/uL Humic Acid (25ng/uL?) – 100ng/uL Humic Acid (250ng/uL?) – 20uM Hematin (50uM?) – 200uM Hematin (500uM?)
• Replicates of four (Yfiler Plus) and two (Yfiler) • 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu
VTS inhibition study – hematin Yfiler
VTS inhibition experiment – hematin Yfiler Plus
VTS inhibition experiment – humic acid Yfiler
VTS inhibition experiment – humic acid Yfiler Plus
Average intracolor balance with inhibitors
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
hematin20-YFP
hematin20-YF
hematin200-YFP
hematin200-YF
humicacid 10-
YFP
humicacid 10-
YF
humicacid100-YFP
humicacid
100-YF
1ngYFP
1ng YF
Inhibitor and kit
% b
alan
ce
ICB 6-FAMICB VICICB NEDICB TAZ/PETICB SID
Inhibition – average peak height
500
1500
2500
3500
4500
5500
Ave
rage
rfu
Yfiler PlusYfiler
Hematin 20
Hematin 200
Humic acid 10
Humic acid 100
1ng
VTS Male:male mixtures • Four mixture ratios (1:1, 1:2, 1:4, 1:8) amplified
in triplicate (Yfiler Plus) and duplicate (Yfiler) • All samples had ~20ng/uL of female background
DNA (200ng per sample) • 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu
VTS Male:male mixtures • Major:minor consistent across all loci at 1:2, 1:4,
and 1:8 for Yfiler Plus • Major:minor flip-flopped at two loci in the Yfiler
1:2 mixtures
VTS Male:female mixtures
• Two male:female mixture ratios (1:1000 and 1:4000) in replicates of three (Yfiler Plus) and two (Yfiler)
• 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu
Intracolor balance male:female mixtures
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1ng_
YFP
1ng_
YF
MF 1:10
00_Y
FP1
MF 1:10
00_Y
FP2
MF 1:10
00_Y
FP3
MF 1:10
00_Y
F1
MF 1:10
00_Y
F2
MF 1:40
00_Y
FP1
MF 1:40
00_Y
FP2
MF 1:40
00_Y
FP3
MF 1:40
00_Y
F1
MF 1:40
00_Y
F2
% b
alan
ce
ICB 6-FAMICB VICICB NEDICB TAZ/PETICB SID
VTS male:female mixtures 1:1000 Yfiler
VTS male:female mixtures 1:1000 Yfiler Plus
VTS male:female mixtures 1:4000 Yfiler
VTS male:female mixtures 1:4000 Yfiler Plus
VTS male:female mixtures • Known artifact observed in TAZ dye
channel
VTS non-probative samples
• n=60 – NIST 2391b dilution series – Proficiency test samples – Adjudicated case samples with “anomolies” – Paternal references from six families (n=26),
half rxns • Amp target same as previous or 0.75ng • 3130 – 3kV / 5 sec injection • GMID/GMID-X analysis at 50rfu
Null allele and allele out of bin
Null allele and allele out of bin
Out of bin alleles
Out of bin alleles…and a duplication
Adjudicated sexual assault
• Case background • Quant of victim blood reference –
186ng/uL (human)/(6.4ng/uL) male • Autosomal results • Y-STR testing • Analysis of victim buccal swab
Adjudicated sexual assault
Adjudicated sexual assault
Adjudicated sexual assault
Family study
Family study
Family study
Major improvements Male relatives cannot be excluded
10 new Y-STR loci including 7 randomly mutating Y-STRS
Adventitious matches in the database Limited ability to overcome inhibition
Improved inhibitor resistance
Limited ability to amplify degraded samples
11 mini STRs below 220bp
Lengthy amplification time
Shorter amplification time
Allelic ladder does not cover full spectrum of known alleles
Expanded allelic ladder
Taq Eliminated