Things that can muck up a DNA Profile The list is long and distinguished. . . By Christine Funk, with a lot of help from her friends. . . Board of Public Defense Trial Team 651.539.4846

There are many, many ways to muck up a DNA profile Collection Muck Ups -Contamination during collection Interpretation Muck Ups – Mixed Samples Amplification Muck Ups Degradation Primer Binding Muck Ups - Peak Height Imbalance Capillary Muck Ups - Blobs and Noise Dye Muck Ups - Pull Up Spikes DNA Mutation Muck Ups - Tri-allelic Patterns; Off Ladder Alleles Machine Muck Ups - Signal Data Low Level DNA - Allelic Imbalance Interpretation Muck ups – Examiner Bias Math Muck Ups – Fun with Numbers!

Let’s have a giggle, shall we?

Collection Muck Ups: Typically, DNA in criminal cases has done a little traveling. . .

Primary Transfer Picture courtesy of Jennifer Friedman

Secondary transfer Clipping courtesy of Bob Blaiser

Collection muck up

The Leskie Decision “ 8. . .The match to the bib occurred as a result of contamination in the laboratory and was not an adventitious match. . ."

The Leskie Decision “ 8. . . The samples from the two cases were examined by the same scientist within a close time frame."

Mixture Interpretation How does one interpret a mixture, that is, which bands or peaks are alleles and which are artifacts or errors?

Sample Mixture Example Profiler Plus data 4 peaks at a single locus D5S818 D13S317 D7S820 D8S1179 D21S11 D18S51 Amel VWA FGA D3S1358 blue panel green panel yellow panel Relative Fluorescence Units Higher than expected stutter Imbalance in X and Y peak ratios “Stutter” on wrong side of allele

Who’s in the Mix? Care to take a guess?

Who’s in here? XY 11, 15 28, 30 12, 15

But wait! What if… XY 11, 15 28, 30 12, 15 XY 13, 15 32.2, 32.2 17, 19

Whoops! I meant to say… XY 11, 15 28, 30 12, 15

Perspective is everything

Alleles Present: 13, 17, 18 12, 12 12, 13 12, 14 12, 15 12, 16 12, 17 12, 18 12, 19 13, 13 13, 14 13, 15 13, 16 13, 17 13, 18 13, 19 14, 14 14, 15 14, 16 14, 17 14, 18 14, 19 15, 15 15, 16 15, 17 15, 18 15, 19 16, 16 16, 17 16, 18 16, 19 17, 17 17, 18 17, 19 18, 18 18, 19 19, 19

Alleles Present: 13, 17, 18 Possible Contributors to D3: 13, 13; 13, 17; 13,18;17, 17; 17,18; 18,18 SIX possible profiles in D3 12, 12 12, 13 12, 14 12, 15 12, 16 12, 17 12, 18 12, 19 13, 13 13, 14 13, 15 13, 16 13, 17 13, 18 13, 19 14, 14 14, 15 14, 16 14, 17 14, 18 14, 19 15, 15 15, 16 15, 17 15, 18 15, 19 16, 16 16, 17 16, 18 16, 19 17, 17 17, 18 17, 19 18, 18 18, 19 19, 19

But Wait!! There’s more. . . *ladders not to scale

Alleles Present: 16, 17, 18, 19 11, 11 11, 12 11, 13 11, 14 11, 15 11, 16 11, 17 11, 18 11, 19 11, 20 11, 21 12, 12 12, 13 12, 14 12, 15 12, 16 12, 17 12, 18 12, 19, 12, 20 12, 21 13, 13 13, 14 13, 15 13, 16 13, 17 13, 18 13, 19 13, 20 13, 21 14, 14 14, 15 14, 16 14, 17 14, 18 14, 19 14, 20 14, 21 15, 15 15, 16 15, 17 15, 18 15, 19 15, 20 15, 21 16, 16 16, 17 16, 18 16, 19 16, 20 16, 21 17, 17 17, 18 17, 19 17, 20 17, 21 18, 18 18, 19 18, 20 18, 21 19, 19 19, 20 19, 21 20, 20 20, 21 21, 21

Still MORE contributors: 18, 18; 18, 19; 19, 19 Alleles Present: 16, 17, 18, 19 Still MORE contributors: 18, 18; 18, 19; 19, 19 Possible Contributors: 16, 16; 16, 17; 16, 18; 16, 19; 17, 17; 17, 18; 17, 19 11, 11 11, 12 11, 13 11, 14 11, 15 11, 16 11, 17 11, 18 11, 19 11, 20 11, 21 12, 12 12, 13 12, 14 12, 15 12, 16 12, 17 12, 18 12, 19, 12, 20 12, 21 13, 13 13, 14 13, 15 13, 16 13, 17 13, 18 13, 19 13, 20 13, 21 14, 14 14, 15 14, 16 14, 17 14, 18 14, 19 14, 20 14, 21 15, 15 15, 16 15, 17 15, 18 15, 19 15, 20 15, 21 16, 16 16, 17 16, 18 16, 19 16, 20 16, 21 17, 17 17, 18 17, 19 17, 20 17, 21 18, 18 18, 19 18, 20 18, 21 19, 19 19, 20 19, 21 20, 20 20, 21 21, 21

10!! 10 Possible Profiles in vWA!!!

60 profiles so far. We have 14 loci to go. . . 10 profiles from vWA 6 profiles from D3 60 profiles so far. We have 14 loci to go. . .

Why this is hard: You don’t know how many people are in the mix You don’t know when and where their alleles overlap You don’t know if you have alleles that have dropped out due to low copy numbers You don’t know if you have a triallelic pattern or stutter or primer binding site mutation

Amplification Muck up

STR Alleles with Stutter Products DNA Size (bp) D8S1179 D21S11 D18S51 Allele Relative Fluorescence Units Stutter Product 6.3% 6.2% 5.4%

Stutter in Pictures – First, let’s review how it’s supposed to happen. . . G T C C A G 1 2 3 4

Stutter in Pictures – how amplification is supposed to happen G T C C A G 1 2 3 4

Stutter in Pictures: amplification as it should be. . . G T C C A G G T C C A G 1 1 2 2 3 3 4 4

Stutter in Pictures – Amplification gone horribly awry. . . 1 2 3 4

Stutter in Pictures G T C C A G 1 2 3 4

Stutter in Pictures G T C C A G 1 2 3 4

Stutter in Pictures G T C C A G 1 2 3 4

Stutter in Pictures G T C C A G 1 2 3 4

Stutter in Pictures G T C C A G C A G G T C 1 1 2 2 3 3 4 4

Stutter in Pictures G T C C A G C A G G T C 1 1 2 2 3 3 4 4

St… St… Stutter!!

Stutter Happens So does teeny tiny contribution

Pop Quiz Why do we care about stutter?

A funny thing happened on the way to the forum. . .

Degradation Ahem. DNA degradation.

Degradation

Things that are insulting to DNA

Degradation: I know it when I see it!

Bonus points: How could increasingly smaller DNA sample size be a problem in a criminal case?

When degradation isn’t degradation

What causes inhibition? Hint: Monty Pyton wouldn’t know. . .

Size matters

Behavior patterns of degraded DNA To paraphrase Joe Walcott, “the bigger they are, the faster they fall.” Welterweight champion of the world, 1901 – 1904.

Behavior patterns of degraded DNA AmpFlSTR® Identifiler™ (Applied Biosystems)

Behavior patterns of degraded DNA AmpFlSTR® Identifiler™ (Applied Biosystems) AmpFlSTR® Identifiler™ (Applied Biosystems)

Behavior patterns of degraded DNA AmpFlSTR® Identifiler™ (Applied Biosystems) AmpFlSTR® Identifiler™ (Applied Biosystems)

What does this have to do with a ‘ski slope?’ AmpFlSTR® Identifiler™ (Applied Biosystems) D8S1179 D21S11 D7S820 CSF1PO The smaller stuff is over here TH01 D13S317 D3S1358 D16S539 D2S1338 D19S433 VWA TPOX D18S51 The Bigger stuff is over here!! D5S818 AMEL FGA Slide courtesy of John Butler

Option 1

Option 2 Slide courtesy of Carll Ladd

New Topic: Peak Height Imbalance

Heterozygous peak height balance as a function of signal intensity in RFU units. PHB 50-150 150-300 300-1000 > 1000 rfu’s Observed minimum 48% 57% 58% 78% Theoretical minimum Mean -3 SD mean 40% 80% 54% 85% 63% 88% 75% 92% N 145 140 233 209 Data from Holt, et al., J. Forensic Sci. 2002; 47(1):66-96.

So what?

Primer Binding Site Mutation

Capillary Muck Ups: Meet my friends ‘Noise’ and ‘Blob’

Noise Happens

What’s the problem with noise? You tell me. . .

Blobs

So what’s the problem? Bonus points if you can tell me how a blob could muck up a DNA profile.

Pull Up

Questioned Sample

New Topic: Spikes

Spikes

Can you tell me how a spike could impact a DNA profile?

More mutations that can mess things up. . .

Triallelic Pattern Known blood sample: 3 band profile at TPOX Positive control sample

Sneaky off ladder alleles: D3 Variant Allele Known blood sample: D3 profile 17,>19.

Machine Muck Ups

Good EPT Data

Bad EPT Data

Good Raw Data

Bad Raw Data

Good Rox

Bad Rox

Allelic Imbalance Impress your friends: Learn the term “stochastic effects” What happens when you have a small amount of DNA?

Allelic Imbalance Amplifications from same dilution tube (~60 pg). (2-012802 12.59 PM) Allelic imbalance present. “Different DNA profiles” @ THO1 and CSF1P0.

Allelic Dropout Reference sample Evidence sample

Biased Interpretation of results It is human nature to want to ‘solve’ the crime. The use of reference samples ‘assists’ the scientist in this process.

Blue Loci* Defendant 17 15, 17 25 Does he match? D3 vWA FGA Defendant 17 15, 17 25 Does he match? *Note the RFU values. This lab uses a ’50 RFU cutoff.’

Defendant 17 15, 17 25 Is he excluded? -- No, because additional alleles at D3 and FGA are “spurious”. The “12” is below the threshold and the FGA alleles are off ladder.

Defendant 12, 17 15, 17 20, 25 Is he excluded?

Defendant 12, 17 15, 17 20, 25 Is he excluded? --No, because stochastic effect or preferential amplification explains peak height disparity at D3; spike (pull-up) at FGA hides 20 allele.

Special Thanks Bob Blaiser, Attorney John Butler, National Institute of Science and Technology Simon Ford, Lexigen, Inc. Jennifer Friedman, LA Public Defender’s Office Jason Gilder, Forensic Bioinformatics Services Ted Kessis, Applied DNA Resources Dave Knudsen, Minnesota State Public Defender’s Office Dan Krane, Forensic Bioinformatics Carll Ladd, Connecticut State Crime Lab Carrie Roland, Forensic Bioinformatics Services Mike Sganga, Senior Scientist, Attorney, Law Office of Michael Burt Bill Thompson, UC Irvine