A Crash Course in NGS for GI Pathologists. Sandra O Toole

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1 A Crash Course in NGS for GI Pathologists Sandra O Toole

2 The Sanger Technique First generation sequencing Uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc) These are molecules that resemble normal nucleotides but lack the normal -OH group. Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops.

3 1800 Sanger Sequencing & Fragment Analysis G1 Sequencer capillary electrophoresis

4 First generation sequencing Sanger sequencing via capillary electrophoresis around 1990 Revolutionary at the time, up to 384 reactions Slow and labour intensive expensive 4

5 First generation sequencing Cost of human genome project $2.7 billion 13 years to complete ( ) Pooled sequence from several individuals Craig Venter first published individual human full genome in 2007 $100 million using 1 st generation automated sequencers (Sanger) Craig Venter 5

6 The Next Generation 6

7 Mycoplasma sequenced Fragments clonally amplified Uses pyrosequencing, Life sciences platform 7

8 Next generation sequencing James Watson s DNA first published human whole genome sequence by next generation sequencing In 4 months and for $1.5 million 8

9 Next generation sequencing Generic term for high throughput sequencing massively parallel sequencing MPS Different platforms and chemistries Leaders are illumina and Thermofisher (prev Life technologies) Others Roche and SoLID 9

10 Next Generation Sequencing X10 45 genomes in a day approx $1000 USD

11 NGS principles Vast numbers of short reads are sequenced in a single stroke 1. Library preparation amp or ligation to custom linkers adapters 2. Amplification on a solid surface (bead or flat silicon) 3. Sequencing step by step detection of base incorporation by each library frag 4. Data analysis s to s of reactions per instrument massively parallel

12 Sequencing by synthesis Sample must be fragmented into shorter fragments Illumina use reads bp Fragments are ligated to generic adaptors Annealed to slide using these adaptors Unique sequences bar codes used to label individual samples that are then pooled Rounds of PCR result in a spot with many copies of the same fragment read Separated into single strands to be sequenced

13 Sequencing by synthesis The slide is flooded with fluoro labelled nucleotides and DNA polymerase Terminators so one base added at a time Photo taken in between each round

14 Sequencing by synthesis Computers covert optical signals into a sequence

15 Ion Torrent sequencing (Ion proton/ OGM) The chip is the machine, semiconductor chip

16 Data analysis 1. Sequence alignment to genome NGS Sequencing Technologies Elaine Mardis 2014 JHU

17 Data analysis Sequence alignment Human genome around 48% repetitive sequences NGS Sequencing Technologies Elaine Mardis 2014 JHU

18 Data analysis Clean up quality scores, mark duplicates Variant calling (SNPs, genotypes, structural variation) evaluate coverage What does it all mean?

19 What do I need to know as a pathologist? These platforms have in-built errors Each has different strengths Sample quality very important Tumour content ideally >20% Amplification can be affected by fixation issues, pigment, blood, necrosis, decal 15/11/2017

20 Types of NGS Whole genome 30x Whole exome 2% of human genome Targeted eg x RNA-Seq Methyl-seq

21 MD Anderson 2000 pts NGS panels 789 (39%) had an actionable mutation in 35 genes. MD Anderson is one of the largest oncology centers in the world but only 83 of these 798 patients (4%) could be treated in a genotype-matched trial targeting these alterations As we transitioned from the 11-gene Sequenom panel to the 46- and 50-gene Ampliseq panels, there were only modest increases in patients with potentially actionable alterations Clinically useful panels for most GI routine molecular testing small panels

22 Take home message Histopathologists have a unique role.. due to their ability to incorporate the clinical, macroscopic, microscopic and molecular pathology information into a comprehensive morphomolecular diagnostic report Understand advantages and limitations of technology Actively seek to learn and influence how it is used in clinical practice Flynn et al Integrating Molecular Diagnostics into histopathology Training. J Clin Pathol 2014;67: