LGC Forensics

Jim Thomson STR DNA analysis of Cannabis Sativa

Background

• Cannabis cultivation in the UK has increased significantly over the last decade

• Much commercial growing is controlled by organised crime networks (OCN), often of Vietnamese origin, and frequently involved in other criminal activites.

• Grow rooms are highly efficient horticultural activities, often using hydroponic cultivation techniques

• Propagation is thought to be mainly achieved by taking cuttings from a productive mother plant, and distributions of cuttings within OCNs have been intercepted

Project outline

• Aim: To develop a DNA identification test for Cannabis capable of providing intelligence and evidence to link seizures and scenes (i.e. that two or more scenes have plants from the same genetic stock)

• Review of alternative DNA methods for identification of Cannabis samples

• Development and optimisation of Cannabis STR profiling method at LGC Forensics

• Confirmation of profiling method on known reference materials

• Population survey: stage 1. Collection and profiling of samples from local customer forces (Met, Surrey, Hants, Berks)

• Presentation to ACPO Drugs Committee November 2009• Population survey: stage 2. Collection and profiling of

samples from across the UK (England, Wales, Scotland).

STRs are the best choice

• Variable STR regions directly equivalent to well established human identification methods (e.g. SGMplus)

• Process, instrumentation and interpretation methods well established at LGC Forensics

• Many published loci in Cannabis which have been developed in a number of laboratories

• Alternative methods (AFLP, RAPD) are less robust.

Cannabis STR loci

1. Gilmore et al. For Sci Int 2003

2. Alghanim & Alimirall Anal Bioanal Chem 2003

3. Hsieh et al For Sci Int 2003

4. M. A. Mendoza et al. Anal Bioanal Chem., 2009

ANUCS303 (GTG)7 1ANUCS305 (TGG)10 1P17 (CTA)9 2P19 (TTG)9 2P24 (GTT)7 2P25 (ACG)7 2P14 (AAG)10 2ANUCS301* (TTA)15 1CS1 (CACCAT)40 3

RefP

has

e 2:

9 lo

cus

test

Ph

ase

1:

6 lo

cus

test

[4]

STR Locus Repeat

Example STR profiles:Mother plant and clone (cutting)

A B

CLONES HAVE IDENTICAL DNA

Project design

• Optimisation of DNA Extraction

– Mainly dried leaf samples– Some resin samples tested– LGC Genomics sbeadex® Plant DNA

kit

• Cannabis Samples

– Phase 1• Reference samples: 8 known mother

plants and clonal offspring from cuttings

• Seizures: 166 samples from 45 scenes in SE England

– Phase 2• Seizures: 422 samples from 115

scenes throughout England, Scotland and Wales

PROFILE C

Profile Designation

• Individual alleles were assigned in each profile

• Complete profiles were assigned a unique identifier (A-EE) to aid interpretation and matching

AK, 1

B, 7

C, 4

I, 2

Q, 1

S, 1

T, 1

Key Findings – Phase 1• 166 full profiles from 45 scenes

– Full profiles from all samples tested, including mouldy leaf samples

• 42 unique profiles observed • 38 scenes had 2 or more samples

– At 17 scenes, all plants from the scene shared the same profiles (indicative of clonal propagation of all plants from a common mother plant)

– At 12 scenes, all plants had one of two profiles (indicative of clonal propagation from two separate mother plants)

– At 9 scenes (with >2 samples), all plants had unique profiles, or at least 3 different profiles were observed (indicative of propagation by seed)

0

2

4

6

8

10

12

1 2 3 4 5 6 7 8 9

No of sample from a scene

No

of

scen

es

Geographical distribution of commonest profiles

Significance of findings

• Profile B was found at 15/45 (33%) of scenes and in 49/166 (30%) of plants tested. Does this mean that all these scenes obtained their plants from a common mother plant, or that this profile is common in the wider Cannabis population?

• Do we see different commonly shared profiles in different geographical areas, as we might expect if distribution networks operate locally?

• Will the improved discrimination power of the additional 3 give distinguish between apparently common profiles in a larger population?

• MORE DATA NEEDED

Project phase 2

• Integration of the 3 new tests with the 6 original to give a single test with 9 STRs. Validation of this new test.

• Collection of samples from UK police forces, supported by ACPO drugs committee– At least 4 plants from each scene where possible– Samples to be collected Jan – April 2010

• Genotyping project to establish feasibility of a nationwide intelligence service.

Nine STR multiplex developed

Scenes and Samples – May 2010Police force No of scenes No tested in study Police force No of scenes No tested in studyAvon and Somerset 6 6 North Wales 9 3

Derbyshire 9 3 North Yorkshire 14 2

Devon and Cornwall 1 1 Northamptonshire 5 1

Dorset 5 3 Nottinghamshire 13 6

Durham 5 3 Scotland - Fife 1 1

Essex 16 8 Scotland - Lothian & Borders 3 3

Gloucestershire 1 1 Scotland - Northern 2 2

Great Manchester Police 7 2 Scotland - Strathclyde 3 3

Hampshire 5 5 Staffordshire 1 1

Humberside 1 1 Suffolk Constabulary 12 7

Kent 2 2 Sussex 4 4

Lancashire 29 4 Thames Valley 7 4

Lincolnshire 6 4 Warwickshire 5 2

Merseyside 9 3 West Mercia 11 4

Metropolitan 46 11 West Midlands 2 1

Norfolk 8 4 West Yorkshire 73 11

Total 321 116

• Samples received from 32 different forces in England, Scotland and Wales

• A total of 1046 plants were sampled and submitted to LGC Forensics (av 3.2 plants/scene)

• 173 of the 321 scenes had 4 or more individual plants submitted

Sample Information Collected

• Sample Number

• Police force

• Scene address

• Town

• Police reference

• Any linked Scene

• Type of cultivation (e.g. Pot grown, hydroponic)

• No. grow rooms at the scene

• Room ID from which the sample was taken

• Additional note

Results

• 422 samples from 116 independent seizures from UK grow rooms were analysed. Samples were selected to give a wide geographical distribution and where possible from scenes where 4 individual plants were submitted.

• 110 distinct genotypes were detected; – 96 unique to single seizures– 14 shared among seizures.

• 3 genotypes (types B, C and X) were seen at multiple scenes (29, 23 and 12 different scenes respectively)

• 1 genotype (DN) was seen at 4 scenes (all in the Bristol area) • 10 genotypes were each seen at a 2 scenes only.

97 Scenes with 4 samples analysed

• Of the 97 scenes where 4 plants were submitted, 54 had all the plants from the scene sharing the same genotype

o152% B

16%

C29%

X11%DN

5%

AQ4%

BA4%

CE4%

O12%

O22%

O32%

O42%

O52%

O62%

O72%

O82%

O92%

O102%

O112%

O122%

O132%

O142%

Phase 2 – 54 scenes

UK-wide

Phase 1 – 17 scenes

SE England

41%

C, 24%DN

Scottish results19 plants from 9 different scenes

Scene ID Sample ID Force Scene Genotype

997 Northern Caithness DA998 Northern Caithness DB999 Northern Caithness DC

1000 Northern Caithness DD1003 Fife Dunfermline B1004 Fife Dunfermline B1001 Fife Dunfermline C1002 Fife Dunfermline C

73 1005 Northern Kirkwall, Orkney DE74 1027 Strathclyde Paisley DF75 1028 Strathclyde Paisley N76 1029 Strathclyde Paisley DG

1043 Lothian and Borders Edinburgh C1041 Lothian and Borders Edinburgh DH1042 Lothian and Borders Edinburgh DH1044 Lothian and Borders Edinburgh B1045 Lothian and Borders Edinburgh B1046 Lothian and Borders Edinburgh C1047 Lothian and Borders Edinburgh DH

78

79

72

77

71

Seizures in England and WalesCommon genotypes seen at > 2 scenes106 different scenes

Profile B – 23 scenesProfile C – 29 scenesProfile X – 12 scenesProfile DN – 4 scenes

Seizures in England and WalesAll genotypes106 different scenes

Profiles seen once or twice only

Paired genotypes seen twice only in the survey

Evidence or IntelligenceWhat is the significance of a match?

Likelihood ratio: Pr(match if they are cuttings from the same motherplant [Hp])

Pr (match if they are “unrelated” [Hd])

• Consider three scenarios:– Two scenes, nominated as possibly linked, both have all plants with

profile C, which has been previously observed at 25% of all crime scenes investigated. WEAK SUPPORT FOR Hp?

– Two scenes, nominated as possibly linked, both have all plants with profile DN, which has not previously been observed in 110 other scenes from around the UK. STRONG SUPPORT FOR Hp?

– Two scenes, nominated as possibly linked, have no profiles in common between them VERY STRONG SUPPORT FOR Hd?

Difficulties in estimating significance

• Human DNA – Population frequencies can be

estimated from nominally unrelated populations

– Substructure within populations is reasonable well characterised and can be accounted for

– All individuals are the result of sexual mating with 2 parents

– Individual tests are shown to be independent (on different chromosomes)

• Cannabis DNA– The populations available

through seizures are not unrelated

– Substructure (different distribution networks, different propagation techniques) is complex and unknown

– Both sexual (seeds) and asexual (cuttings) methods of propagation

– No knowledge of linkage of individual tests

At present, we cannot calculate a reliable estimate to express the significance of a “match”

Further work

• 624 samples remain to be tested– Improved data set, but still does not answer the difficult

population questions

• Combining the results already obtained from phases 1 and 2 will provide a better picture for the South East of England

• A dense regional map of West Yorkshire may serve as a useful blueprint for future localised distribution maps– 284 samples from 73 scenes– Potentially the best model for future use – work with individual

forces (or regions) to establish real distribution data.

Acknowledgements

King’s College London MSc Forensic Science students• Katherine Bache • Vivian Yuen Yam

• All participating forces