1. An Introduction to molecular diagnostics
Dr Catherine Cargo
HMDS

2. Overview Basic molecular biology Mutations DNA sequencing
Sanger sequencing
Next generation/high throughput sequencing
Impact on haematology

4. What is Deoxyribonucleic acid (DNA)?
DNA is a nucleic acid that contains all of our genetic make-up inherited from each parent
Located in the nucleus of every (nucleated) cell
What are the DNA ‘building blocks’?
Double-stranded structure
sugar (deoxyribose) phosphate backbone and four nitrogenous bases forming a ‘genetic code’
Four nucleotide bases are the
Purines - adenosine (A) and guanine (G)
Pyrimidines - cytosine (C), thymine (T)
The double strands are held together by hydrogen bonds

5. Human Genome 3 billion base pairs
Arranged into 46 chromosome
Information carried in pieces of DNA called genes
Only 1.5% of human genome is protein coding (exons)
Human genetic variation
All humans are on average 99.5% similar to other humans
1000 genome project
“a typical individual genome differs from the reference human genome at 4.1 million to 5.0 million sites … affecting 20 million bases of sequence”
Mostly single nucleotide polymorphisms (SNPs)
some DNA sequences that do not code protein may still be involved in the regulation of gene expression or play structural roles in chromosomes

6. DNA sequence Protein Synthesis Transcribed Translated Unidirectional
Central dogma of genetics
Genes are simply the codes for making proteins
Unidirectional

7. Transcription Occurs in nucleus
RNA polymerase separates the DNA strands and synthesises a complementary RNA copy from one of the DNA strands
RNA complement strand includes the nucleotide uracil (U) in all instances where thymine would have occurred in DNA
- uracil is identical to methylated thymine
- this is because RNA is more short lived and any potential uracil related errors do not lead to lasting damage

8. Transcription DNA Transcription (RNA polymerase) Pre-mRNA
Exon 1 Intron 1 Exon 2 Intron 2 Exon3
Transcription (RNA polymerase)
Pre-mRNA
Exon 1 Intron 1 Exon 2 Intron 2 Exon3
5’ capping, RNA splicing, 3’polyadenylation
mRNA
AAAA( )
mRNA transported to cytosol where it is translated into protein

9. Translation Process in which cellular ribosomes create proteins
mRNA is decoded by a ribosome that reads the RNA sequence by base pairing the messanger RNA to transfer RNA which carried amino acids
Based on 3 letter ‘words’ called codons
Produces a specific amino acid chain or polypeptide
Polypeptide later folds into a active protein and performs its function in the cell

10. DNA damage
DNA is prone to damage from environmental insults and also during DNA replication
Protective mechanisms in place to eliminate detrimental abnormalities
DNA repair through various pathways
Proof-reading by DNA polymerase.
If these pathways fail
cell’s final ‘line of defence’ is apoptosis (i.e. cell death).
Survival advantage
Genes that regulate cell growth and differentiation are altered
e.g a gene that is part of the protective process (-a ‘tumour supressor’)
Cells may be open to more abnormalities as a result of its ability to evade protection becoming increasing unstable – ‘genetic instability’.

11. What is a mutation?
Permanent alteration of the nucleotide sequence of the genome
Result from
Inherited
Errors during DNA replication
Introduced during DNA repair
Induced mutations
Chemicals
Physical
radiation from UV rays/X-rays,
extreme heat
Damaging effects of a DNA mutation are observed in the protein
Germline
Somatic
Germline – can be passed on to descendants through their reproductive cells, present in all cells
Somatic – acquired, not inherited from a parent or passed on to offspring

12. What is a mutation?

13. How do DNA mutations cause malignancy?
Specific tissue
More than 1 abnormality is usually necessary for carcinogenesis
Steensma et al, Blood, 2015

14. Chromatin Modification
DNA Methylation
TET2 DNMT3A
IDH1/2
RNA Splicing
SF3B1 SRSF2 U2AF1 ZRSR2
Chromatin Modification
ASXL1
EZH2
Myeloid Malignancy
Signalling
KRAS NRAS FLT3 KIT
CBL
Receptors/ Kinases
JAK2
Transcription
RUNX1 BCOR
Hypermethylation of genes leads to transcription silencing – gene inactivation
Tumour Suppressors
TP53, WT1
Cohesin
STAG2
Adapted from Cazzola et al, 2013

15. Types of mutations THE CAT ATE THE RAT THE BAT ATE THE RAT
TSH ECA TAT ETH ERA
THE CTA TET HER AT A
3 main types of mutations
substitutions
insertions
deletions
Have varying effects on the codons ‘3 letter words’ read during transcription

16. Types of mutations No mutation Point Mutations Silent Nonsense
Missense
Conservative
Non-conservative
DNA level
TTC
TTT
ATC
TCC
TGC
mRNA level
AAG
AAA
UAG
AGG
ACG
Protein level
Lys
STOP
Arg
Thr

17. DNA sequencing
An essential tool in the molecular biology toolkit is the ability to read the base sequence of DNA molecules
Fred Sanger developed an elegant method to sequence DNA by using DNA polymerase enzyme
(for which he was awarded the Nobel Prize in 1980)
The Sanger method is also known as the chain termination method
Takes advantage of the process of DNA synthesis

19. Sanger Method Chain-terminator method
Key principle
Use of dideoxynucleotide triphosphates (ddNTPs) as chain terminators
DNA divided into 4 separate sequencing reactions containing – dATP, dGTP, dCTP, dTTP and DNA polymerase
To each reaction is added 1 of 4 dideoxynucleotides
ddATP, ddGTP, ddCTP, ddTTP
Results in DNA fragments of varying length
Heat denatured and separated by gel electophoresis
Autoradiography

21. Sanger vs. NGS Sanger sequencing NGS sequencing
One sample, one amplicon & one sequence
Multiple samples (48), hundreds of amplicons/fragments (361), millions of sequences (40,000,000 paired end reads) & 4 x 10(9) bases.

22. NGS = reduced sequencing costs

23. NGS: The basics Library Preparation Cluster generation / bead capture
Sequencing
Data analysis (Bioinformatics)

24. Library Preparation
The Library is a pooled tube of ALL the barcoded DNA fragments from ALL the patients.

25. Individual samples barcoded

26. NGS: The basics Library Preparation Cluster generation / bead capture
Sequencing
Data analysis (Bioinformatics)

27. The black box…..

34. NGS: The basics Library Preparation Cluster generation / bead capture
Sequencing
Data analysis (Bioinformatics)

35. Sample preparation assay Interpretation of results Low throughput
High throughput
Sample preparation
assay
Interpretation
of results

36. Analysis pipeline Extract DNA Library prep Sequence Coverage Alignment
Read- level QC
Variant detection
CNV
detection

37. Analysis pipeline Extract DNA Library prep Sequence Coverage Alignment
Read- level QC
Variant detection
CNV
detection

38. Burrows-Wheeler alignment
Each fragment/amplicon will generate its own SAM/BAM file

39. Coverage and depth
IGV coverage plots (pile up)

40. Detecting variation
Able to detect SNPs, INDELS and copy number variation (CNV)

41. Filtering

42. Potential applications of NGS in haematology
Diagnostic tool
Objective evidence of disease
Subclassification
Prognostic tool
Disease monitoring
Identify targets for therapy (in future!)

43. How will this impact on you?
Potential to greatly impact on patient care
Early diagnosis
Information on prognosis
Access to therapies
Early diagnosis = more patients in clinic
Patients will become increasingly aware of this
Ask questions on the significance of mutations
Personalised medicine
Quality results require quality samples!

44. Conclusions
NGS is set to become a key tool in the diagnostic armoury of the laboratory
NGS is cost effective, rapid, reliable and uses minimal amounts of clinical material.
Targeted sequence analysis of key myeloid and lymphoid driver mutations will form the basis of this testing
Guiding treatment based on the identification of some of these driver mutations is already happening in the clinic

46. Fluidigm Access Array system
-uses micro-fluidics

47. Fluidigm Principal

48. Library preparation 27 genes, 370 amplicons
TET2,
TET2,
DNMT3A, IDH1, IDH2
DNMT3A, IDH1, IDH2
Chromatin Modification
Chromatin Modification
ASXL1, EZH2
ASXL1, EZH2
Splicing
Splicing
SF3B1, SRSF2, U2AF1, ZRSR2
SF3B1, SRSF2, U2AF1, ZRSR2
Transcription Factors
Transcription Factors
NPM1, RUNX1, BCOR, WTI, TP53
NPM1, RUNX1, BCOR, WTI, TP53
Library preparation
27 genes, 370 amplicons
1 run will perform nearly 18 thousand reactions
this would take weeks to do by old technology…
Signalling
Signalling
FLT3, NRAS, KRAS, CBL, cKIT, JAK2, MPL,
FLT3, NRAS, KRAS, CBL, cKIT, JAK2, MPL,
CSF3R, STAT3
CSF3R, STAT3
Cohesin complex
Cohesin complex
STAG2
STAG2
Other
Other
SETBP1, CALR
SETBP1, CALR