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

Questions?

Lynne Burley 408-808-6922

lburley@crimelab.sccgov.org