Massively parallel sequencing

Question 1

DNA database matches; scene to scene, person to person and what else
DNA multiplex, in use at the launch of the DNA database, in 1995
Final stage of STR profiling, after separation and detection?
Single peak from appearing on a DNA, EPG
In contrast to STR’s – forensic, investigative, genetic genealogy focusses on?

Answer 1

DNA database matches; scene to scene, person to person and person to person
DNA multiplex, in use at the launch of the DNA database, in 1995 was SGM.
Final stage of STR profiling, after separation and detection is interpretation.
Single peak from appearing on a DNA, EPG is homozygous
In contrast to STR’s – forensic, investigative, genetic genealogy focusses on SNP.

Question 2

What is massively parallel DNA sequencing?

Answer 2

• Commonly referred to as next-generation sequencing (NGS), Massively Parallel DNA Sequencing serves as a pivotal innovation in the arena of forensic science
• The approach utilizes DNA sequencing technologies that can manage many DNA sequences at once, thereby allowing forensic scientists to extract key information from small amounts of material.
• MPS has demonstrated efficacy in cases involving degraded DNA samples, owing to its high sensitivity and capability to small amounts of DNA. Even aged or compromised samples, which posed substantial challenges for analysis previously, can yield accurate DNA profiles through MPS.

Question 3

The National DNA Database statistics

Answer 3

• NDNAD is run by the Home Office on behalf of the UK police force. A small team (less than 40) Home Office staff have access to it. Police forces own the DNA profile records on the database and receive notifications of any matches, but they do not have access to it.
• The database holds approximately 6.6 million subject profile records (80% male) and 650,000 crime scene profile records.
• The largest of its kind in Europe.
• The chance to crime profile, once loaded onto the database will match against the subject loaded onto NDNAD.
• Re - platformed in 2020, the matching process takes seconds, operates 24/7 and is fully automated.

Question 4

Conventional DNA analysis

Answer 4

• Analyse multiple regions in an individuals DNA where short sequences of DNA are repeated numerous times (STR’s)
• The DNA profile obtained it is presented graphically as a series of peaks that we can interpret.

Question 5

Conventional DNA testing

Answer 5

• Works very well and is well established.
• Easy to upload DNA results and search NDNAD.
• Software available for statistical evaluation of results.

Question 6

What does MSP allow?

Answer 6

A modern DNA analysis technique which allows us to:
* Look not only at the length of each DNA fragment making up the STR of interest but the specific DNA sequence each fragment contains
* Multiple DNA tests in one assay – no need to consider how to portion out DNA extract.
* Backwards compatible with NDNAD
* Allowed to potentially significant intelligence leads not available up till now.

Question 7

Forensic DNA typing sing MPS

Biology

Answer 7

• DNA extraction
• DNA quantitation
• PCR amplification of multiple loci

Question 8

Forensic DNA typing using MPS

Technology

Answer 8

• Library preparation and sequencing of amplified product
• DNA sample genotype determination

Question 9

Forensic DNA typing using MPS

Application

Answer 9

• Compare simple genotype to reference crime samples
• Compare DNA profiles to population databases
• Generate reports using statistics, RMP/LR

Question 10

ForenSeq DNA signature prep kit

Answer 10

• Launched in 2015
• Two primer sets depending on analysis required
• 5 µL of DNA extract.
• One ng DNA is optimal (works well down to 0.125 ng).
• Equivalent sensitivity to DNA 17.
• Up to 230 markers analysed in a single test (results in approximately 48 hours).
• ISO/IEC 17025 accredited (September 2021)

Question 11

When using SNP sequencing…

Answer 11

• We are utilising next generation sequencing.
• Reading sequences in an individuals genome.

Question 12

SNP Microarray

SNP sequencing

Answer 12

• ~600,000 SNP’s
• ~200ng
• Most inexpensive
• Not good with degraded DNA

Question 13

Targeted Kit: Kintelligence

SNP Sequencing

Answer 13

• ~10,230 SNPs
• > 50pg – 1ng
• Mid range cost
• Future, in house crime lab capacity

Question 14

Whole genome sequencing

SNP Sequencing

Answer 14

• > 1 million SNP’s (not the whole genome)
• > 50pg
• Most expensive
• Recommended for degraded DNA

Question 15

Benefits of MSP

Answer 15

• Eliminate the need for Multiple STR Kits
• Interrogate 200 genetic markers using a single, streamlined workflow
• Access a wider range of informative SNPs
• A dense set of forensically relevant SNPs provide valuable information not widely available with current technology
• Superior analysis of challenging samples
• Obtain highly discriminating data from less than 100 pg. of DNA, even with complex mixtures, or degraded DNA
• Multiplexing and rapid sample processing
• Prepare up to 96 genomic libraries simultaneously using a simple plate-based format, and standard lab equipment

Question 16

Microvariants

Answer 16

Microvariant alleles, defined as alleles that contain an incomplete repeat unit, often complicate the process of DNA analysis

Question 17

Forensic DNA phenotyping

Answer 17

ForenSeq DNA Signature Prep kit allows an inference to be made with respect to
* Most likely eye colour
* Most hair colour

Question 18

Least expensive way of SNP sequencing?
Using SNP we can generate greater allelic detail by looking at?
Estimating eye-colour is determined by (not genotype)
SNP sequencing option for degraded DNA?
Required to help duplicate the cells DNA?

Answer 18

Least expensive way of SNP sequencing? SNP Microarray
Using SNP we can generate greater allelic detail by looking at microvariants
Estimating eye-colour is determined by (not genotype phenotype.
SNP sequencing option for degraded DNA is MSP.
Required to help duplicate the cells DNA is DNA polymerase.

Question 19

MPS case

Answer 19

• 1984 male body washed up, Amblteuse, Northern France
• Locate international identified possible link to drowning victim in East Anglia, UK
• Body exhumed, bone sample submitted
• Conventional DNA analysis eliminated East Anglia link
• MPS used for ancestry, hair, eye colour prediction as part of a joint effort at facial reconstruction.

Question 20

Next steps for MPS

Answer 20

Increase in prediction capabilities:
* Skin colour
* Ancestry (more detailed)
* Age of donor (blood, bone and semen)

Question 21

What can MPS provide?

Answer 21

• A much more detailed look at the DNA recovered.
• Potential significant investigative leads (DNA database searching in the UK and overseas, phenotyping, ancestry)