Lecture 7 - New Sequencing & Forensic Applications

What are some applications of HTS?

• Complex disease genomics

  • Not all diseases arise from a mutation ot a single gene

  • HTS can detect genetic variants associated with disease using multiple samples, faster than previous technologies

• Forensic genomics

  • More markers, DNA phenotyping

How does HTS work in forensic genomics?

• Forensic profiling of DNA databases

  • Easily expand sequencing runs to include more markers - all markers are sequenced simultaneously

• Use a single workflow on one system to complete all critical database tests for immediate or archival use

• Provide the turnaround and ease of use labs need

• Benefit from minimal sample preparation time - often just a library needs to be made

What is a genomic library?

• Contains fragmentary inserts of DNA generated from a variety of processes

• Would also contain elements required for sequencing

How does HTS work with mtDNA?

Offers clearer heteroplasmy (multiple copies exist in the same cell) analysis and greater resolution of mixture samples

What are the different methods for HTS using mtDNA?

• Target enrichment

• Sequencing platforms

• Data analysis

What is the subcellular mtDNA sequencing workflow?

1. Nanobiopsy of mitochondrial subpopulations

2. mtDNA library preparation and amplification

3. Illumina Sequencing

4. Sequencing analysis

How can mtDNA from skeletal remains be sequenced?

• DNA collected is typically highly degraded, and the poor quality of the sample limits the success of traditional typing techniques

• mtDNA sequencing can be useful:

  • Present in hundreds of copies/cell compared to nDNA
    Variation in mtDNA haplotypes

What is an example of a workflow when analyzing skeletal remains using mtDNA sequencing?

1. DNA extracted

2. a. Amplify four regions of the D-loop/control region b. Barcode + adapters

3. Pool individuals

4. Sequence on MiSeq system (Illumina & SBS)

How can you predict human appearance?

• Forensic DNA Phenotyping (FDP): predictions of human appearance from forensic samples

• Aims to infer the unknown sample donor’s externally visible characteristics from DNA directly from the biological material left behind

  • Considered complex traits, where several genes may contribute to the phenotype

• Developed to guide police investigations in cases without known suspects

What is IrisPlex?

• A sensitive DNA tool for accurate prediction of blue and brown eye colour

• The human eye is a highly polymorphic phenotype in people of European descent

• Developed to predict eye colour from genetics

• Used the 6 most eye colour-informative SNPs that previously revealed prediction accuracies of over 90%

• Allows prediction of blue and brown eye colour

• Future research into the genetic basis of non-blue and non-brown eye colours will need to show if such colours can be predicted with similarly high levels of accuracy

What is HIrisPlex?

• A system for simultaneous prediction of hair and eye colour from DNA

• Includes a single multiplex genotyping assay for 24 eye and hair colour predicting SNPs

• Two prediction models

• Hair summarized in 4 colours + 2 shade

What is the future of predicting human appearance?

Ongoing work to identify the underlying genes and develop predictive DNA markers for several others: skin colour, body height, male baldness, and face

What is the Illumina MiSeq FDx System?

• PCR cycles link the tags to copies of each target to form a DNA template consisting of the regions of interest flanked by universal tag primers

• The tags are used to attach indexed adapters (barcoded), which are then amplified using PCR, purified, pooled into a single tube, and then squeezed

• The index sequences allow the sequencing system to separate and isolate the data generated from each sample (sample multiplexing)

• A targeted approach where sequences are targeted through an amplicon-based workflow

What are the different acronyms associated with the Illumina MiSeq FDx System?

• iiSNPs → identity-informative SNPs (ideal fr degraded samples)

• piSNPs → phenotypic-informative SNPs (estimate eye colour and hair colour)

• aiSNPs → ancestral-informative SNPs (estimate biogeographical ancestry)

What are the 4 basic steps of the Illumina MiSeq FDx System?

1. Library Preparation (PCR)

   • Sequence-specific/universal-tagged primer PCR for each forensically relevant target sequence in the DNA sample

   • Indexes and adapters are incorporated into the amplicons

   • Amplicons are then purified, pooled, and linearized

2. Cluster Generation

   • Fragments are bound to surface oligos complementary to the library adapters on the flow cell

   • Each fragment is then amplified in distinct clonal clusters through bridge amplification

3. Sequencing by Synthesis

   • Method that detects single bases as they are incorporated into DNA template strands

4. Data Analysis

What is DNA-based facial composites?

• Facial similarity between identical twins, clear facial resemblances within families, distinctive facial features associated with particular genetic conditions, and facial similarities within geographic populations and within the sexes

• Suggest that inter-individual variation in facial morphology is to some extent determined by genetic variation

• In theory, a DNA-based facial composite should be possible given the compelling evidence that facial features are under genetic control

• Generally identified with Genome Wide Association Studies

  • Phenotype = Genotype + Environment

  • Shift in allele frequencies correlated with phenotype

What are the basics of how DNA-based facial composites work?

• First effort at generating facial composites from DNA in 2014, based on 24 SNPs

• Physical accuracy of the facial predictions in terms of overall similarity is mainly determined by sex and genetic ancestry

• Used genomic ancestry (based on 68 different SNPs) and sex to create a base-face, which is an average ancestry/sex matched face

• The effects of 24 individual SNPs that have been shown to have significant effects on facial variation are overlaid on the base-face, forming the predicted-face in a process similar to image blending

• The SNP effects increase the distinctiveness of the facial predictions

What is the two-step procedure to DNA-based facial composites?

1. Genomic ancestry and sex are used to create a “base-face”

2. Effects of the 24 significant SNPs are overlaid onto the base-face, forming the “predicted-face”

What are the differences between the base-face and predicted-face produced by the DNA-based facial composites?

• Individuality map

• This addition to the predicted face results in a boosted predicted-face is desired, which is a process akin to facial caricaturing

• The opposite face is illustrated as well, in contrast to the actual face

  • The opposite face is male instead of female, elongated instead of short, concave instead of convex

What are the challenges of DNA-based facial composites?

• Extract information from the evidentiary DNA sample

• Convert this information to values

• Create shape transformations from these values

• Combine multiple shape transformations into a single facial composite

What are some future avenues of DNA-based facial composites?

• Expanding knowledge on the genetic architecture of facial morphology

• Improving the predictive modelling of facial morphology

• Perceptual interpretation of the results

What are some current limitations of studies using whole genomes?

• Low-frequency variants that have larger effects

• Discriminate interregional admixture on a finer level

• Not permitted in court as science, not well established

• Creates a suspect population and onus on individuals to provide their DNA to prove innocence