Forensic Genomics Flashcards
How has DNA analysis changed across the years?
- Transitioned from 16 to 24 autosomal STRs for normal profiling- aids cross-border NDNAD searches
- Introduction of rapidly mutating Y-STRs to Y-STR sets has enhanced identification of male relatives
- Big increases in sensitivity now require likelihood-based interpretation of complex (mixed) profiles
- Tissue-source identification is moving from cytology to RNA analysis (can be co-extracted with DNA)
- SNPs and INDELS are increasingly being added to STR/mtDNA typing to analyse very degraded DNA
- Contact traces not matched to NDNAD records can be tested for extra genetic data (ancestry, appearance, age)
- The roll-out of dedicated MPS systems has taken place in the last 10 years
A sequencing-Genomics historic timeline
A sequencing-Genomics historic timeline continued…
How are human chromosomes arranged?
Human chromosomes are arranged as p=short arm, q=long
– the only species with this pattern
Tell me about the strands in DNA
What are short genetic variations and define each one?
- SNPs and INDELs used for identification purposes like ancestry and in a phenotypic concept
- SNPs, INDELs and STRs are short genetic variations all bought together in DNA database
SNPs: Single nucleotide polymorphisms, frequently called SNPs (pronounced “snips”), are the most common type of genetic variation among people. Each SNP represents a difference in a single DNA building block, called a nucleotide. For example, a SNP may replace the nucleotide cytosine (C) with the nucleotide thymine (T) in a certain stretch of DNA.
INDELS:Indel is a molecular biology term for an insertion or deletion of bases in the genome of an organism.
STRs:Short tandem repeats (STRs), also known as microsatellites or simple sequence repeats, are shorl tandemly repeated DNA sequences that involve a repetitive unit of 1-6 bp, forming series with lengths of up to 100 nucleotides
Nucleotide substitutions occurs in sequences, and they become SNP polymorphisms in what situation?
When they are fixed in a population
INDELS
What does the dbSNP carry information on?
dpSNP: The Single Nucleotide Polymorphism Database is a free public archive for genetic variation within and across different species developed and hosted by the National Center for Biotechnology Information in collaboration with the National Human Genome Research Institute
dbSNP continued
How do we know where each of the chromosomes are?
Two main current human genome assemblies onto which all human sequence variation is mapped and has ‘map coordinates (Chr Pos)’
* All chromosome builds start with chromosome 1 and nucleotide 1
* Different in end chromosome as seen above just accounts for newly discovered things like indels, transitions etc
* Chromosome 14, SNP has different chromosome position between each build, each coordinate applied in different scenarios, useful for different position you may be interested in
We have now moved to more simplified mapping of SNPs…
When was the first human SNP map published?
2001 by the Human Genome mapping project
What are the different types of SNPs for forensic applications and what are the sub-groups?
Identity testing SNPs
- II-SNPs
- ID-SNPs
Lineage informative SNPs
- LI-SNPs
- Microhaplotypes
Ancestry informative SNPs
- AI-SNPs
- AIM-SNPs
Phenotype informative SNPs
- PI-SNPs
- EVC-SNPs
What can SNPs be amplified from?
Much shorter DNA fragments
How mant STRs are known to give global uniqueness
24 STRs give unique profiles
Low level DNA is better amplified by STRs or SNPs?
STRs
(however we now have MPS which is much better)
Are SNPs fast?
Yes
Are STRs good for mixtures?
STRs are multiple alleles so better opportunity to de-convolute mixed profiles as dealing with polymorphisms as only 2 alleles
SNPs are uninformative for mixed profiles but STRs are better for mixed DNA
The 911 victim ID was the pilot program for short-amplicon tests. Tell me about this test
- 72 SNP loci used
- Amplified fragments of roughly 100bp
- Small multiplexes of 6 SNPs
- SNPs given 12 extra identifications
- Most remains now fail to work with any analytical technique
- 46% of victims still not identified and 63% of remains
Whats a main CE system?
SNaPshot main CE system- a single base extension reaction
-
ExoSAP= remove primers from PCR
* SAO= remove unattached nucleotides with dyes as causes noise in Electropherogram - Size and extended fragments show different sizes in SNPs, indels are the same height
For places which may not have access to expensive and complex MPS systems, what can be used to identify bases?
Non-human pigtails
- Standard technique for those who don’t have access to expensive and complex MPS systems
- Simple primer at SNP site, dye link for different bases, attach to SNP site as single base extension of annealed primer, terminate sequence, dye show what base is attached to the SNP alleles
- *Get better steps for this
- To separate fragments: primers (hard after 24 primers), or non-human pigtail (alters fragment size, allows for short and long pigtails to separate fragments based on oligo sequences)
How can indels also be analysed?
Do SNP genotyping tests work well with highly degraded DNA?
What are the two techniques which can be used for DNA preparation
MiSeq and Thermofisher
Explain the MiSeq method for DNA preparation
Explain the Thermofisher method for DNA preparation
Tell me about Forensic DNA phenotyping (FDP) with SNPs
Its found that hair and eye colour mainly vary in what reigon?
Europeans
Tell me about the HIrisPlex-S system
HIrisPlex-S DNA test system (S for skin) for the simultaneous prediction of eye, hair, and skin colour from trace DNA. This FDP system consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin colour predictive SNPs and the previous HIrisPlex assay for 24 eye and hair colour predictive SNPs, 19 of which also contribute to skin colour prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye colour prediction based on 6 SNPs, the previous HIrisPlex model for hair colour prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin colour prediction based on 36 SNPs.
Are there now strong-effect SNPs for Skin, Hair and eye colour?
Yes
- Look at SNPs with strong association to melanin production pathways (for skin, hair and eyes)
- MC1R has the strongest effect (not necessarily coding SNPs but can turn on/off)
FDP has also been looked at beyond pigmentation, what is starting to be looked into ?
For facial prediction, FDP concentrates on what primarily to help to do this?
Determining sex, ancestry and age enhances EVC/physical characteristic prediction
Basic and enhanced tools for EVC-SNPs and phenotyping
How are the Y-Chr and mtDNA both useful for forensic ancestry analysis with SNPs?
The Y-Chr and mtDNA are both very informative
* Y-chromosome STR typing was developed shortly after autosomal STR sets were introduced for analysis of male DNA in sexual assault cases
* Mitochondrial DNA provide a system for analysis of hairs as evidential material, and is the best-preserved DNA in burnet remains
* However, Y-chromosome loci and mtDNA are single markers
How are STRs useful for forensic ancestry analysis?
- Some variation in frequencies between populations which can be exploited
Tell me about the first forensic SNP ancestry test
- The likelihood of an individual with a SNP genotype is from a particular population equates directly to the likelihood of that genotype occurring in that population
- Fixed SNPs with frequencies near 1 or 0 are most informative
- The duffy SNP (rs2814778) is a key African-informative markers- for a good reason
- Not all African populations have the T variant at a detectable frequency- so they would be misclassified using this SNP alone
o The Khoisan are a more deep-rooted human population that lacks resistance to malaria- so using this SNP alone leads to an erroneous non-African ancestry inference
What theorem can be used in forensic applications
Bayes theorem
Bayes theorem provides a way to calculate the probability of a hypothesis based on its prior probability, the probabilities of observing various data given the hypothesis, and the observed data itself
The principle of PCA as a type of multi-dimensional scaling
Verogen UAS ancestry SNP summary
MPS systems and their SNP ancestry analysis software
MPS systems and their SNP ancestry analysis software
When ancestry is combine with EVCs we get ‘synergy’
Snipper
Snipper v1.2 (released 09/30/2011) is a program for quickly looking up information from public databases on genes near SNPs of interest.
What can microhaplotypes only be typed with?
MPS
Without sequencing data the SNP phase is not known
Phase and the haplotype combinations are only obtained by sequencing the whole segment with SNPs
Most haplotypes represent a novel base change on an established allelic background that rises in frequency
Microhaplotypes can combine the benefits of short amplicons with increased variation
- USC microhaplotypes panels are components or form a large scale stand-alone MPS panel for identification purposes
- Microhaplotypes can be as short as SNPs- but usually have more variation
- Some MH loci are very informative, but rare variants predominate
- Many microhaplotypes are more akin to tri-allelic SNPs
- Microhaplotypes also have some ancestry informativeness
IGG: investigative genetic genealogy
Buckskin girl
IGG: investigative genetic genealogy
The Golden state killer
What were the critical steps to identifying the golden state killer suspect?
Identifying the golden state killer
Steps 2-3 were DNA analysis
Identifying the golden state killer
Steps 4-5 used genetic genealogy
Identifying the golden state killer
DNA tests applied genotypes very high number of SNPs
What are the different types of sequencing methods which can be done?
Whole-genome sequencing
MPS using conventional forensic PCR
Hybridisation capture methods
Whole Genome SNP arrays
Hybridisation capture methods
Tell me the stages to whole-genome sequencing
Tell me the stages to MPS using conventional forensic PCR
Tell me the stages to hybridisation capture methods
Tell me the stages to whole-genome SNP arrays
Tell me the stages to hybridisation capture methods
IGG goes from pairwise comparisons of kinship tests to one-to-many comparisons
SNP imputation was used and is now widely applied
Forensic STR-based familial searches finds a group of close relatives
Often pedigrees only share common ancestors after more than 8-10 generations
All commercial testers adapted illumine SNP arrays- GEDmatch compared their genotypes intersect
IBD segments can identify 2nd to 3rd cousins with reasonably good likelihoods, but less reliably for more distant relationships
Triangulation
Exploring segments- two different matches in different databases can initiate triangulation studies
Multiple individuals with matching segments allows triangulation
Conclusion
