Next Generation Sequencing

Question 1

What is NGS?

Answer 1

• utilises DNA sequencing technologies that can manage many DNA sequences at once
• high sensitivity
• capability to small amounts of DNA
• aged or compromised samples

Question 2

What is NGS also known as?

Answer 2

massively parallel DNA sequencing (MPS)

Question 3

The National DNA Database statisicts

Answer 3

• only a small team in the Home Office have access to it
• largest in Europe
• police will only get a notification if they have a match they do not have access to it

Question 4

How does MPS work?

Answer 4

• not only looks at the lengths 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

Question 5

Why is MPS better than DNA 17?

Answer 5

it can work when there isnt sufficient information to search NDNAD or if there is no match on the NDNAD

Question 6

What is the first next generation sequencing instrument called which is developed and validated for forensic genomics?

Answer 6

MiSeq FGx Sequencing System

Question 7

What are the biological steps in MPS?

Answer 7

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

Question 8

What are the technological steps when using MPS?

Answer 8

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

Question 9

What are the applications when using MPS?

Answer 9

• compare simple genotype to reference crime samples
• compare DNA profiles to population databases
• generate reports using statistics RMP/LR

Question 10

Why is MPS more differentiating than DNA 17?

Answer 10

looks at 230 markers compared to 34 markers in DNA 17

Question 11

How do we decide what type of SNP sequencing to use?

Answer 11

depends on the quality and quantity on DNA

Question 12

What are the three SNP sequencing options

Answer 12

1. SNP microarray
2. targeted kit: kintelligence
3. whole genome sequencing

Question 13

What is SNP microarray?

Answer 13

• ~600,000 SNP’s
• ~200ng
• most inexpensive
• not good with degraded DNA

Question 14

What is kintelligence?

Answer 14

• ~10,230 SNPs
• > 50pg - 1ng
• mid range cost
• future, in house crime lab capacity

Question 15

What is whole genome sequencing?

Answer 15

• > 1 million SNPs (not the whole genome)
• > 50pg
• most expensive
• recommended for degraded DNA

Question 16

What are the benefits of the ForenSeq DNA Signature Prep Kit (Verogen)?

Answer 16

• eliminate the need for multiple STR kits
• interrogate 200 genetic markers using a single, streamlined workflow
• access a wider range of informative SNPs
• multiplexing and rapid sample processing
• prepare up tp 96 genomic libraries simultaneously using a simple plate-based format and standard lab equipment
• great with complex mixtures or degraded DNA
• can discriminate data from less than 100pg

Question 17

What is a microvarient?

Answer 17

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

Question 17

What is the ForenSeq DNA Signature Prep Kit (Verogen)?

Answer 17

the kit used for preparing DNA samples for MPS

Question 18

What is benefical about being able to see greater allelic detail when using MPS?

Answer 18

• can see if there are any microvarients occuring within the repeats

Question 19

What is eye and hair colour determined by?

Answer 19

phenotyping

Question 20

What is next for MPS?

Answer 20

• increased prediction capabilities in skin colour, ancestry, age of doner (blood, bone, semen)
• investigative genetic genealogy