Next Generation Sequencing Flashcards
What is NGS?
- next generation sequencing (massively parallel DNA sequencing)
- utilises DNA sequencing technologies that can manage many DNA sequences at once
- allows forensic scientists to extract key information from small amounts of material
- efficient in cases involving degraded DNA samples - due to its high sensitivity and capability to small amounts of DNA
- even aged or compromised samples can yield accurate DNA profiles through MPS
NDNAD
- what is it
- who has access to it
- what info does it hold
- how does it work
- national DNA database
- only a small team in Home Office have access to it
- police forces own DNA profile records on database and receive notifications if they have a match (they do not have access to it)
- 6.6 million subject profile records (80% male) and 650,000 crime scene profile records
- after being reprogrammed in 2020, matching process takes seconds, operates 24/7 and is fully automated
Explain conventional DNA analysis e.g. DNA 17
What are the three pros of it?
But what if…
- analyse multiple regions in an individuals DNA where short sequences of DNA are repeated numerous times (STRs)
- DNA profile obtained is present graphically as a series of peaks that we can interpret
- works very well and is well established
- easy to upload DNA results and search NDNAD
- software available for statistical evaluation of results
- but what if there is insufficient information to search NDNAD
- or there is no match on NDNAD
What is MPS?
How does it work?
two pros?
- massively parallel sequencing (MPS)
- looks at not only the length of each DNA fragment but also 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 for potentially significant intelligence leads
Why is MPS better than DNA 17?
- it can work when there is insufficient information to search NDNAD or if there is no match on the NDNAD
What is the name of the first NGS instrument developed and validated for forensic genomics?
- MiSeq FGx platform
What is the kit used for preparing DNA samples for MPS?
- ForenSeq DNA Signature Prep kit (Verogen)
What are the steps for the ForenSeq DNA Signature Prep Kit (Verogen)
What information does it use?
- prepare
- ForenSeq DNA Signature Prep Kit
- sequence
- runs on MiSeq FGx instrument
- analyse
- ForenSeq Universal Analysis Software
- information:
- global automated STRs
- Y-STRs
- X-STRs
- identify SNPs
- biogeographical ancestry SNPs
- phenotypic SNPs
What can be said about ForenSeq DNA signature prep kit (Verogen) in terms of:
- launched in?
- primer sets needed
- DNA extract needed
- sensitivity
- markers analysed
- accredidation
- 2015
- two primer sets depending upon analysis required
- 5 uL of DNA extract
- 1 ng DNA is optional
- equivalent sensitivity to DNA 17
- up to 230 markers analysed in single test (results in approx 48 hours)
- ISO/IEC 17025 accredited (september 2021)
What are the three biological steps when using MPS for DNA typing?
- DNA extraction
- DNA quantitation
- PCR amplification of multiple loci
What are the two technological steps when using MPS for DNA typing?
- library preparation and sequencing of amplified product
- DNA sample genotype determination
What are the three applications when using MPS for forensic DNA typing?
- compare simple genotype to reference crime samples
- compare DNA profiles to population databases
- generate reports using statistics (RMP/LR)
Why is MPS more differentiating than DNA17?
- we are looking at 230 markers compared to 34 markers in DNA17
When using singular nucleotide polymorphism (SNP) sequencing:
- what are we utilising
- what are we doing
- age
- utilising next generation sequencing
- reading sequences in an individuals genome
- not new (20 years old) in biomed and research but new in forensics
How do we determine what type of SNP sequencing to use for our evidence sample?
- depends upon the quality and quantity of DNA from our biological evidence
