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
What are the three sequencing options?
1 - SNP microarray
2 - targeted kit: Kintelligence
3 - whole genome sequencing
What is SNP microarray?
- around 600,000 SNP’s
- around 200 ng
- most inexpensive
- not good with degraded DNA
What is kintelligence?
- around 10,230 SNPs
- 50 pg - 1 ng
- mid range cost
- future in house crime lab capacity
What is whole genome sequencing?
- more than a million SNPs (not the whole genome)
- more than 50 pg
- most expensive
- recommended for degraded DNA
What are the benefits of NGS?
- eliminate need for multiple STR kits
- interrogate 200 genetic markers using a single streamlined workflow
- access a wider range of informative SNPs (this provides valuable information not widely available with current technology
- superior analysis of challenging samples (complex mixtures or degraded DNA)
- obtain discriminating data from less than 100 pg of DNA
- multiplexing and rapid sample processing
- prepare up to 96 genomic libraries simultaneously using a single plate-based format and standard lab equipment
- works for both high and low molecular weights
What are microvariants?
- microvariant alleles are alleles that contain an incomplete repeat unit and often complicate the process of DNA analysis
What is beneficial about being able to see greater allelic detail when using MPS?
- can see if there are any microvariants occurring within the repeats
What does ForenSeq DNA signature prep kit allow for an inference to be made with respect to?
- (most likely) eye colour and hair colour
What must all SNPs be typed for predictions using?
- Verogen software
What is an alternative software?
- Erasmus HirisPlex webtool
What is hair and eye colour determined by?
- phenotyping
What are the main uses of MPS?
- DNA database searching in UK and overseas
- phenotyping
- ancestry
MPS cases to date?
- ancestry of murder victim
- dismembered human remains
- conventional DNA analysis - no match
- doubt as to where victim was from
- intelligence suggested east Asian
- MPS analysis on bone sample - individual ancestry was to be European (didn’t agree with intelligence)
MPS cases to data
- ancestry and phenotype prediction from human remains
- 1984 male body washed up in France
- locate international identified as possible link to drowning victim in east Anglia
- body exhumed and bone sample submitted
- conventional DNA analysis eliminated East Anglia link
- MPS used for ancestry, hair, eye colour prediction as part of joint effort at facial reconstruction
What is next for MPS?
- increased prediction capabilities (skin colour, ancestry, age of donor (blood, bone and semen
- investigative genetic genealogy
