Massively parallel sequencing Flashcards
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?
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.
What is massively parallel DNA sequencing?
- 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.
The National DNA Database statistics
- 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.
Conventional DNA analysis
- 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.
Conventional DNA testing
- Works very well and is well established.
- Easy to upload DNA results and search NDNAD.
- Software available for statistical evaluation of results.
What does MSP allow?
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.
Forensic DNA typing sing MPS
Biology
- DNA extraction
- DNA quantitation
- PCR amplification of multiple loci
Forensic DNA typing using MPS
Technology
- Library preparation and sequencing of amplified product
- DNA sample genotype determination
Forensic DNA typing using MPS
Application
- Compare simple genotype to reference crime samples
- Compare DNA profiles to population databases
- Generate reports using statistics, RMP/LR
ForenSeq DNA signature prep kit
- 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)
When using SNP sequencing…
- We are utilising next generation sequencing.
- Reading sequences in an individuals genome.
SNP Microarray
SNP sequencing
- ~600,000 SNP’s
- ~200ng
- Most inexpensive
- Not good with degraded DNA
Targeted Kit: Kintelligence
SNP Sequencing
- ~10,230 SNPs
- > 50pg – 1ng
- Mid range cost
- Future, in house crime lab capacity
Whole genome sequencing
SNP Sequencing
- > 1 million SNP’s (not the whole genome)
- > 50pg
- Most expensive
- Recommended for degraded DNA
Benefits of MSP
- 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
