Examination papers

Question 1

What were the main ideas in Paper 1:
**Forensic Identity SNPs: characterisation of flanking region variation using MPS **

Answer 1

Typical STR typing
o Pros
 Separates alleles based on their size using CE which correlates to the number of repeat units found in the STR
 High power of discrimination
o Cons
 Number of loci targeted in one reaction
 Problems with successful amplifications of larger amplicons when analysing degraded DNA

**Identity SNPs **
o Pros
 SNPs used for identity and kinship applications in forensics with/without typical STR profiling
 SNPs have reduced amplicons size which helps to combat some of the limitations seen with STRs
 Lower mutation rate of 10-8 compared to STRs with 10-3- useful or kinship analysis
 Most SNPs are biallelic
o Cons
 Much larger number of loci targeted to achieve the same power of discrimination of STRs
 CE can be used for SNPs but… multiple analyses, time consuming, requires increasing sample volumes (not always possible in a forensic case)

Microhaplotype
o Pros
 Multiallelic nature
 Per-locus basis they are more informative then simple bi-allelic SNPs
 Low mutation rate
 Ease of interpretation
 Low stutter

**Flanking regions **
o Pros
 Inclusion of the flanking regions variation reduced the combined probability match
 Increases heterozygosity of some loci above that of the least useful STR loci
 Better enhanced analysis of currently targeted SNP markers for forensic applications
 The flanking regions of certain loci have a considerably higher propensity for extra variation than others
 Benefit of flanking region variability is masked when only traditional measures of marker utility such as heterozygosity, because there are numerous scenarios where identifying a shared rare allele can prove to be incredibly useful
o Cons
 Lots of additional flanking regions identified but after analysis have decided that those polymorphic sites selected by the UAS are the most generally beneficial
 Such a high proportion of the flanking region alleles are observed at a low frequency (<1%), the number of alleles gained per marker does not correlate with any observed increase in heterozygosity
Conversely, a number of loci show a considerable gain in heterozygosity when taking into account flaking regions by UAS
MPS
o Pros
 Multiple tests run concurrently
 Improved marker discrimination
 Better relationship resolution
 Better analysis of degraded DNA
 Enhanced resolution of mixtures
 Improved mitochondrial sequencing
 Metagenomics
 DNA intelligence tools
 Provides valuable sequence data for targeted regions which allows detection of additional variation seen in the flanking regions of amplicons
 Rapid sequencing of both SNPs and STRs within a single reaction
 Higher throughout (which is why preferred over traditional sanger sequencing)
 Additional variants observed with SNP amplicons that produces 3≤ distinct haplotypes= microhaplotype
 Additional sequence information can allow for the identification of rarer, often individual mutations which could be particularly useful in helping to distinguish between crime scene samples
 Increased implementation of MPS in data analysis allows this level of comprehensive genetic analysis to be more accessible in a forensic context

o Types
 MPS Forenseq DNA signature prep kit **
* Primer Mix A: Autosomal STRs, Y STRs, X-STRs
* Primer Mix B: Autosomal STRs, Y STRs, X-STRs, Identity SNPs, phenotypic SNPs and ancestry SNPs
* Been validated mainly using STRs
* Small number of studies also evaluated SNP performance
* Default analysis software: universal analysis software (UAS)
* Alignment of the amplicons shows a redundant primer for this marker has been included in the signature prep kit PCR primer mix- however this primer is targeting a potential C  T change in the reverse primer that is 2bp away from actual primer binding site
* The observed imbalance at these loci, indicates the need for caution when calling alleles, as there is an increased risk of falsely identifying a homozygous genotype
* Considerable variation seen in the SNP flanking regions
*
 MPS MiSeq FGx**
* When using primer mix A, allows simultaneous analysis of 27 autosomal STRs, 94 autosomal identity-informative SNP (iiSNP), 7 X STRs, 24 Y STRs and amelogenin
* Data analysis
o UAS and flanking regions
 UAS used demultiplex and process the sequencing data
 Used analytical and interpretation threshold which represent LOD and to ensure both alleles of heterozygous SNP are assigned, respectively
 UAS limitations: risk that loci displaying poor heterozygous balance may be falsely reported in cases where one allele is above the interpretation threshold and the other is below the analytical
o Identification of sequence variants
 UAS only highlights variation at pre-determined polymorphic sites within sequenced amplicons
 Genotypes reported based on nucleotide bases observed which helps to simplify reports
 Additional flanking region variations identified by comparing all the FASTA sequences on excel
o Statistical analysis
 GenAlEx used to calculate range of forensically relevant statistics from genotype data

Question 2

What were the main ideas in paper 2:
**Developmental validation of the PowerPlex Fusion 6C System **

Answer 2

PowerPlex Fusion 6C

**What it is **
 A 27 locus, 6 dye, multiplex that includes all markers in the expanded CODIS core loci and increases overlap with STR database standards universally.
 Contains: 23 autosomal STRs, 3 YSTRs (2x RM- Y STRs) and amelogenin
of the 23 Autosomal STRs= SE33, Penta D, Pent E
the 2 RM- Y STRs are DYS570 and DYS576
Both of these help to increase discrimination and also help with mixture analysis
 Genemapper used to analyse samples

**Pros **
 Capable of casework and database workflows, including direct amplification
 Improves robustness and efficiency of STR multiplex system
 Allows co-detection and fluorescent detection (same as MiSeq) of the 20 autosomal loci of the expanded CODIS core loci
 Direct amplification used 12.5µl volumes and extracted DNA 25µl
 Good reproducibility and repeatability
 Increased number of loci tested compared to other methods like identifilier
 When males were the minor contributor in a mixture study, they successfully genotyped greater numbers of unique minor contributor alleles, mainly difference due to Y-STR makers
 System is robust with withstand unintentional temperature variations
 System well developed for STR genotyping and human identification

**Cons **
 The primers included in the PowerPlex fusion 6C have been designed to amplify human DNA, however may also recognise site within non-human DNA samples
 DNA samples in lab may even contain trace or excess amounts of non-human DNA e.g., source of DNA, environment in which it was collected in, contamination from laboratory reagents and tubes
 Threshold of 175 RFU used for threshold for artifact peaks
 When 3 punches added, any inhibitors present in the sample began to overwhelm the amplification chemistry and only 77% of alleles called
 Examining the sensitivity of the powerplex system with chemical and environmental insults
* Presence of inhibitors led to less % of alleles being called at higher concentrations
 When looking at sizing prevision of this system is showed that the larger the allele size yielding the largest standard deviation from replicate amplification reactions, shows precision of assay is adequate to size and can distinguish alleles by as little as one base
 Increased time between sample taken and analysis led to decreased number of alleles seen
 Degraded samples had drop out
 Saliva stains had showed inhibition
 Touch samples produced lower peak heights and genotypes from multiple contributors
 Some disconcordance with genetic markers which is thought to be due to a 5 base deletion in the powerplex 6c system that is outside the primers used in other STR chemistries
 Mixture study
* Identify minor contributor at 1:2 ratio but this lessened as ratio increased to 1:19 to only 74%
* When males were the minor they successfully genotyped greater numbers of unique minor contributor alleles, mainly difference due to Y-STR makers
 Results of direct amplification saw greater variability with results from varying cycle number compared to extracted DNA
 Optimised annealing temp is 60˚c- peak height decreased as annealing temperature increased

Question 3

What were the main ideas in paper 3:
**Associations between forensic loci and expression levels of neighbouring genes may compromise medical privacy **

Answer 3

o 20 STRs used by US criminal justice system
o Markers for profiling were chosen in putative gene deserts (avoid protein coding genes) to avoid forensic profiling revealing protected medical information
o Investigating whether forensic genetic profiles reveal information about gene-expression variation or potential medical information
o Expression values based on transcriptome data from the lymphoblastoid cell line from individuals in the 1000 genome project

Pros
o Forensic genetic identification in US performed using 27 CODIS core loci (like paper 2)
 These are chosen markers as are highly polymorphic and provide identifying information about an individual
 13 of the CODIS core loci chosen due to their efficient PCR multiplexing while maximising identity information and minimising ancestry-based population differences and medically relevant information

Negative results in regard to profiling markers
o 11/27 CODIS loci are intronic (and therefore close to genes)
o STRs and gene expression
 Known that STR number can alter gene function and regulation, sometimes affecting phenotypes drastically e.g., HD gene linked to severe Huntington’s
 Non-coding STRs have been found to impact gene expression and result in trait variation e.g, causing Fragile X syndrome
 STR length variation can affect methylation as well as histone modifications (some of these changes associated with clinical traits)
 Somatic STR mutations linked to cancer
 De novo STR mutations and autism
o Linkage disequilibrium surrounding CODIS loci is strong enough to infer the genotypes of surrounding SNPs, phenotypic information may be inferable through CODIS genotypes
o Identified that CODIS loci significantly correlated with expression levels of neighbouring genes
o 6 CODIS STRS showed association with gene expression and these are found not to be caused by populations
o Small sample size used
o Seen some CODISeSTRS and tissue expression
o Identified CODISESTRS whose genotypes are correlated to expression of neighbouring genes in lymphoblast cell lines
o Results of studied showed:
 CODIS genotypes can convey trait information and be used to gain information about expression of these genes
 Some studies shown associated between medical conditions and CODIS loci including the CODISeSTRs

Study limitations
o Small sample size
o Only looked at 1,000 Genomes project data, where expression data was also available
o Expression data only available in lymphoblast cell lines
o Single cell type used means that analysis will miss genes that are not highly expressed or whose expression isn’t regulated by cis-elements in the lymphoblast cell lines specifically
o Data only available from select few subpopulations, 4/5 were European so didn’t give good representation
o Analysis can’t identify correlations specific to subpopulations not represented
o Errors in imputation of CODIS genotypes may erode powder to identify associations, particularly non-European populations where imputation as high error rates
o Approach for detecting associations is specific to linear relationships to STR allele length and expression levels
o Analysis gave significant correlations it is limited in scope and underpowered
o This would raise questions as to whether stronger correlations or lesser correlations would be identified on a larger, more representative sample size with direct STR genotyping