LGC Forensics

Jim Thomson STR DNA analysis of Cannabis Sativa

BackgroundCannabis cultivation in the UK has increased significantly over the last decadeMuch 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 techniquesPropagation is thought to be mainly achieved by taking cuttings from a productive mother plant, and distributions of cuttings within OCNs have been intercepted

Project outlineAim: 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 samplesDevelopment and optimisation of Cannabis STR profiling method at LGC ForensicsConfirmation of profiling method on known reference materialsPopulation survey: stage 1. Collection and profiling of samples from local customer forces (Met, Surrey, Hants, Berks)Presentation to ACPO Drugs Committee November 2009Population survey: stage 2. Collection and profiling of samples from across the UK (England, Wales, Scotland).

STRs are the best choiceVariable STR regions directly equivalent to well established human identification methods (e.g. SGMplus)Process, instrumentation and interpretation methods well established at LGC ForensicsMany published loci in Cannabis which have been developed in a number of laboratoriesAlternative methods (AFLP, RAPD) are less robust.

Cannabis STR loci1. Gilmore et al. For Sci Int 20032. Alghanim & Alimirall Anal Bioanal Chem 20033. Hsieh et al For Sci Int 20034. M. A. Mendoza et al. Anal Bioanal Chem., 2009

Example STR profiles:Mother plant and clone (cutting)CLONES HAVE IDENTICAL DNA

Project designOptimisation of DNA Extraction

Mainly dried leaf samplesSome resin samples testedLGC Genomics sbeadex Plant DNA kit

Cannabis Samples

Phase 1Reference samples: 8 known mother plants and clonal offspring from cuttingsSeizures: 166 samples from 45 scenes in SE EnglandPhase 2Seizures: 422 samples from 115 scenes throughout England, Scotland and Wales

PROFILE CProfile DesignationIndividual alleles were assigned in each profile

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

Key Findings Phase 1166 full profiles from 45 scenesFull profiles from all samples tested, including mouldy leaf samples42 unique profiles observed 38 scenes had 2 or more samplesAt 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)

Chart5

1

7

4

2

1

1

1

Sheet1

SceneNo of samplesProfies presentbin

13T1No of samples from sceneNo of scenes

25B217

32Q3210

49B436

54B/C549

68I654

77C763

84B873

94J/K/L/M981

103S92

111N

121A

135C/B

142Y/D

156W/X/Z/AA

162E/P

173U/R/O

189F/H/V

197F/G/V

202F/V

211F

223I

237C/B

244B

256C

264I/O

271AC

285AD/AL/AM

292AE/AB

304AF/AG/AH/AI

316B/T

324C

332B

343B/I

354C/B

361AN

375B

383B/C

391AJ

404C

412AK

422B

431C

442O/AP

452C/B

Sheet1

No of scenes

No of sample from a scene

No of scenes

Distribution of 166 samples across 45 scenes

Sheet2

SceneNo of samplesProfies present

13TAK1

25BB7

32QC4

49BI2

68IQ1

77CS1

84BT1

103S17

223I

244B

256C

324C

332B

375B

404C

412AK

422B

Sheet2

Sheet3

Chart6

7

10

6

9

4

3

3

1

2

No of scenes

No of sample from a scene

No of scenes

Sheet1

SceneNo of samplesProfies presentbin

13T1No of samples from sceneNo of scenes

25B217

32Q3210

49B436

54B/C549

68I654

77C763

84B873

94J/K/L/M981

103S92

111N

121A

135C/B

142Y/D

156W/X/Z/AA

162E/P

173U/R/O

189F/H/V

197F/G/V

202F/V

211F

223I

237C/B

244B

256C

264I/O

271AC

285AD/AL/AM

292AE/AB

304AF/AG/AH/AI

316B/T

324C

332B

343B/I

354C/B

361AN

375B

383B/C

391AJ

404C

412AK

422B

431C

442O/AP

452C/B

Sheet1

0

0

0

0

0

0

0

0

0

No of scenes

No of sample from a scene

No of scenes

Distribution of 166 samples across 45 scenes

single profile at scene

SceneNo of samplesProfies present

13TAK1

25BB7

32QC4

49BI2

68IQ1

77CS1

84BT1

103S17

223I

244B

256C

324C

332B

375B

404C

412AK

422B

single profile at scene

0

0

0

0

0

0

0

2 profiles at scene

SceneNo of samplesProfies present

54B/CB/C3

135C/BC/B1

142Y/DY/D1

162E/PE/P1

202F/VF/V1

237C/BC/B1

264I/OI/O1

292AE/ABAE/AB1

316B/TB/T1

343B/IB/I1

354C/BC/B1

383B/CO/AP1

442O/AP

452B/C

2 profiles at scene

0

0

0

0

0

0

0

0

0

0

0

0

Geographical distribution of commonest profiles

Significance of findingsProfile 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 2Integration 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 committeeAt least 4 plants from each scene where possibleSamples to be collected Jan April 2010Genotyping project to establish feasibility of a nationwide intelligence service.

Nine STR multiplex developed

Scenes and Samples May 2010

Samples received from 32 different forces in England, Scotland and WalesA 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 NumberPolice forceScene addressTownPolice referenceAny linked SceneType of cultivation (e.g. Pot grown, hydroponic)No. grow rooms at the sceneRoom ID from which the sample was takenAdditional note

Results422 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 seizures14 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 analysedOf the 97 scenes where 4 plants were submitted, 54 had all the plants from the scene sharing the same genotype Phase 2 54 scenesUK-widePhase 1 17 scenesSE England41%C, 24%DN

Chart5

1

7

4

2

1

1

1

Sheet1

SceneNo of samplesProfies presentbin

13T1No of samples from sceneNo of scenes

25B217

32Q3210

49B436

54B/C549

68I654

77C763

84B873

94J/K/L/M981

103S92

111N

121A

135C/B

142Y/D

156W/X/Z/AA

162E/P

173U/R/O

189F/H/V

197F/G/V

202F/V

211F

223I

237C/B

244B

256C

264I/O

271AC

285AD/AL/AM

292AE/AB

304AF/AG/AH/AI

316B/T

324C

332B

343B/I

354C/B

361AN

375B

383B/C

391AJ

404C

412AK

422B

431C

442O/AP

452C/B

Sheet1

No of scenes

No of sample from a scene

No of scenes

Distribution of 166 samples across 45 scenes

Sheet2

SceneNo of samplesProfies present

13TAK1

25BB7

32QC4

49BI2

68IQ1

77CS1

84BT1

103S17

223I

244B

256C

324C

332B

375B

404C

412AK

422B

Sheet2

Sheet3

Scottish results 19 plants from 9 different scenes

Seizures in England and Wales Common genotypes seen at > 2 scenes 106 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 significanceHuman DNA Population frequencies can be estimated from nominally unrelated populationsSubstructure within populations is reasonable well characterised and can be accounted forAll individuals are the result of sexual mating with 2 parentsIndividual tests are shown to be independent (on different chromosomes)Cannabis DNAThe populations available through seizures are not unrelatedSubstructure (different distribution networks, different propagation techniques) is complex and unknownBoth sexual (seeds) and asexual (cuttings) methods of propagationNo knowledge of linkage of individual testsAt present, we cannot calculate a reliable estimate to express the significance of a match

Further work624 samples remain to be testedImproved data set, but still does not answer the difficult population questionsCombining 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 maps284 samples from 73 scenesPotentially the best model for future use work with individual forces (or regions) to establish real distribution data.

AcknowledgementsKings College London MSc Forensic Science studentsKatherine Bache Vivian Yuen Yam

All participating forces

***Profile C Commonly shared profile.**In scenes where more than one sample was tested. Only having one plant from each scene limits what you can sayAll samples had the same profile, indicative of clonal propagation. This was expectedUnique profile indicative of seed propagation distinctive DNA. More scenes than clonal so not as common. Possible because the seeds produce male and female plants and if pollination occurs, THC content decreases (unless feminised seeds)

At least 2 plants share a common profile and the other plants share a different profile. More than 1 mother plants, could confirm by testing the produce at the seeds to test if its a mixture of the profiles skunk extraction enable thisSeed propagation was confirmed in one case where suspect admitted to purchasing the seeds.