NGS: Coming to a Lab Near You! An Introduction to Next Generation Sequencing (NGS) SNUG 2013

1. NGS: Coming to a Lab Near You!An Introduction to Next Generation Sequencing (NGS)SNUG 2013 Laurel Estabrooks, PhD, FACMG VP Genetics Business Development SCC Soft Computer

2. What is “DNA Sequencing”? Exon DNA sequencing involves the use of various methods for determining the order of the nucleotide bases — adenine, cytosine,guanine, and thymine— in a molecule of DNA. Intron Gene Exon

3. DNA Basics • Bases- In molecular biology and genetics, two nucleotides on opposite complementary DNA or RNA strands that are connected via hydrogen bonds are called a base pair. Adenine (A) forms a base pair with thymine (T) and guanine (G) forms a base pair with cytosine (C). In RNA, thymine is replaced by uracil (U), and therefore bonds to Adenine (A). • Genetic code - the set of rules by which information encoded in genetic material (DNA or mRNA sequences) is translated into proteins (amino acid sequences) by living cells. It is a triplet code in that three nucleotides (a codon) determine particular amino acids.

4. Basics of Transcription and Translation DNA mRNA Protein

5. Basics of Transcription and Translation Intron Exon DNA Intron information is not passed onto processed mRNA Transcription and mRNA processing mRNA Un-translated region Translation Protein Post-Translational Modification Active Protein

6. What isNext Generation Sequencing? • 1st Generation = Sanger Sequencing • 2 reads (forward & reverse) • 2nd Generation = Next Generation Sequencing • Millions of reads • 3rd Generation = Single Molecule Sequencing

8. What isNext Generation Sequencing? What isNext Generation Sequencing?

10. Major computations performed with NGS data • Data assembly with base calling at the level of individual reads • Alignment of the assembled sequence to a reference sequence • Variant calling

11. NGS Alignment Multiple, fragmented sequence reads must be assembled together on the basis of their overlapping areas.

12. NGS Technology Terminology • Read length - the average number of contiguous nucleotide bases in a polynucleotide sequence that are produced by a particular sequencing instrument (14-400) • Coverage – Number of times a nucleotide base is read (# followed by X: 300X) • Call – determination of a given base or base sequence by a sequencing instrument • Call Quality – accuracy of the call determination

13. Base Calling Accuracy

14. Q Scores • Base calling accuracy often measured by the Phred Quality Score (Q score) which assesses the accuracy of a sequencing platform. It indicates the probability that a given base is called incorrectly by the sequencer. • Logorithmic calculation • Q10 1/10 error rate • Q20 1/100 error rate • Q30 1/1000 error rate Example: Phred score of 30 (Q30) = probability of an incorrect base call 1 in 1000 times • Low Q scores can result in an increase in false positive variant calls

15. There are multiple types of DNA changes including: Substitution Inversion DuplicationTranslocation Insertion/Deletion (Indel) SNPs - Single Nucleotide Polymorphisms • Substitution change in more than 1% of the population • Considered a common variant CNVs - Copy Number Variations • Sections of DNA bases in our genomes that are commonly copied many times over • Number of copies may vary from person to person

16. Applications in Microbiology • Identifying the species of an isolate • Defining its properties, such as resistance to antibiotics and virulence • Monitoring the emergence and spread of bacterial pathogens

17. Phylogenic Map

18. NGS & Microbiology Case Study The NHS Rosie Hospital in Cambridge manages around 6,000 baby deliveries each year. All infants in its special care baby unit are screened for MRSA when admitted, and for every week while in the unit. This routine screening picked up MRSA in 12 infants. The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013

19. NGS & Microbiology Case Study The following was performed: • Bacteria was cultured from swabs and plated on selective media. • Antimicrobial susceptibility was tested against an array of antibiotics. • Sequencing libraries were prepared from each MRSA isolate, and amplified. • Whole genome sequencing was performed using the IlluminaMiSeq sequencer. The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013

20. NGS & Microbiology Case Study • All affected infants were treated • Unit was sanitized The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013

21. NGS & Microbiology Case Study Results • 14/17 infants had a new sequence type ST2371 • Only 20 SNPs varied among the 14 ST2371 isolates • ST22 is common MRSA sequence type in UK • ST2371 differs from ST22 isolate by an average of 550 SNPs

22. NGS & Microbiology Case Study • Short hiatus from outbreaks • Another outbreak • Tested all SCBU personnel The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013

23. Case Study Analysis

24. Case Study: NGS Benefit • Identification of asymptomatic carrier causing re-infections • Upon treatment of carrier, no further outbreaks The Lancet Infectious Diseases, Volume 13, Issue 2, Pages 130 - 136, February 2013

25. NGS in Human Genetics

27. Next Generation Sequencing Whole Genome -targets entire genome -incidental findings Whole Exome -targets entire coding region -incidental findings Targeted Panel -smaller target region -no incidental findings

28. Incidental Findings • Findings not associated with the original trigger for the testing • Currently under debate regarding whether to report • Recent guidelines published from American College of Medical Genetics and Genomics

29. Test Interpretation Next Generation Sequencing Test Ordering Unknown diagnosis Suspected diagnosis of disease with mutational heterogeneity Available variant data Patient clinical presentation Co-segregation of variant with clinical issue in family

30. Interpretation Categories Pathogenic Mutation A change that has been previously defined and is known to result in a given disorder, disease or phenotype.

31. Interpretation Categories Probably/Possibly Pathogenic Not a defined change, but there is additional evidence based on • the gene involved, • the gene position, • the type of the variation, or • the family history that lends greater likelihood that this could indeed be the origin of the patient’s clinical presentation/disorder.

32. How do you determine a variant is possibly/probably pathogenic? Use algorithms to assess how variation within a known gene would theoretically impact gene integrity, gene translation, or protein formation Example online tools: • PolyPhen 2http://genetics.bwh.harvard.edu/pph2/ (Polymorphic Phenotyping - predicts loss of function). PolyPhen-2 is a tool which predicts possible impact of an amino acid substitution on the structure and function of a human protein using straightforward physical and comparative considerations. • SIFT http://sift.jcvi.org/ (Sorting Intolerant From Tolerant, just computes >4.55 Mb deletions) SIFT predicts whether an amino acid substitution affects protein function.

33. Interpretation Categories Variant of Unknown Significance • Do not know the significance at this time • Incidence WGS>WES

35. Example of Result Tables

36. Excerpt of Interpretation Illustrating Interpretation Categories

37. Questions?