Next generation sequencing in diagnostic laboratories: opportunities and challenges

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1 Next generation sequencing in diagnostic laboratories: opportunities and challenges Vitali Sintchenko Marie Bashir Institute for Emerging Infectious Diseases & Biosecurity

2 Declaration No conflict of interest to declare 8

3 We can sequence everything. Does it matter? 8

4 Disruptive technology: NGS platforms Platform Output (Gb) Read length (bp) Reads (x10 6 ) Running time Error rate Illumina MiniSeq Illumina MiSeq Illumina NextSeq Illumina HiSeq x x x x / h 5-55 h h 1-6 days 0.1% Ion PGM Ion S5/S5 XL h 3-18 h 1% Oxford Nanopore MinION Up to 300, min-48 h 4-12% PacBio RSII PacBio Sequel Up to 60,000 Up to 60,000 55, ,000 30min-4 h 30min-6 h?

5 Relationship between quality and coverage depth Coverage depth = Number of times a particular base pair is covered by sequenced reads x7 Dallman et al. Microb Genet 2016

6 Genomics Paradigms Infectious disease genomics has been recognised as a subspecialty by NSW Health Pathology Applications of NGS Pathogen discovery Pathogen identification and characterization [e.g., detection of drug resistance associated substitutions (RAS)] Public health surveillance Outdated databases and RAS catalogues can be dangerous for business 20

7 Power of unbiased pathogen discovery 20

8 Power of unbiased pathogen discovery Dr Kerri Rose, NSW Wild Life Surveillance Program J-S Eden et al. Emerg Inf Dis

9 Pathogen Identification: From Pasteur to Watson Conventional Microbiology Clinical specimen WGS based examination Organism growth detected from culture Identification WGS identification & characterisation Characterisation/typing by reference laboratory (PFGE, MLST, VNTR etc) Identification of specific subtypes from WGS data Upload to reference DB with early warning of emergence or spread of virulent/resistant strains

10 Genome sequencing as a lab automation 20

11 NSW Health Pathogen Genomics Partnership Marie Bashir Institute for Emerging Infectious Diseases and Biosecurity (Tania Sorrell, Alicia Arnott) Centre for Infectious Diseases and Microbiology-Public Health (Jon Iredell, Lyn Gilbert) Pathology West-ICPMR (Dominic Dwyer, Sharon Chen, Peter Howard) NSW Health (Vicky Sheppeard, Kirsty Hope, Chris Lowbridge)

12 NSW Health TRG Translating Pathogen Genomics into Improved Public Health Outcomes: Prospective evaluation of the effectiveness of genome sequencing-guided investigation of outbreaks

13 Genomics-guided laboratory surveillance 8

14 Portable sequencing of Ebola outbreaks

15 Monitoring Ebola outbreaks Evolution of Ebola virus over the course of epidemic

16 Temporal phylogeny and EBOV dispersal events G Dudas et al. Nature 1 7 (2017) doi: /nature

17 Good Laboratory Practice: Wet lab NGS NA extraction Library preparation Picogreen against DNA standard (DNA normalisation protocol) Measure quantity and cell content Confirm amplification Sequencing Assess sequencing metrics Data analysis 8

18 Good Laboratory Practice: Wet lab NGS NA extraction Library preparation Picogreen against DNA standard (DNA normalisation protocol) Fragmentation of input NA PCR amplification and clean-up Measure quantity and cell content Check fragment size distribution Confirm amplification Sequencing Library normalisation and quantpcr Quantify library concentration Assess sequencing metrics Data analysis Fewer QC steps required as protocols being streamlined, less sample-sample variability and fewer purification steps FDA clearance of sequencing platforms as Class II Exempt devices 8

19 Good Laboratory Practice: Wet lab NGS NA extraction Library preparation Sequencing Picogreen against DNA standard (DNA normalisation protocol) Fragmentation of input NA Measure quantity and cell content Check fragment size distribution PCR amplification Confirm amplification and clean-up Organism Kappa (pm) Optimal Pass Fail Library normalisation and Mycobacterium Quantify library 500 concentration 60 <30 quantpcr Salmonella <150 Assess sequencing metrics Staphylococcus <300 Data analysis Fewer QC steps required as protocols being streamlined, less sample-sample variability and fewer purification steps FDA clearance of sequencing platforms as Class II Exempt devices 8

20 Assess sequencing run metrics 8

21 Monitoring Q30 95 Q30 (i.e. 99.9% base call accuracy) values for TRG sequencing Illumina requirement [VALUE] [VALUE] [VALUE] Courtesy of Rajat Dhakal and Qinning Wang 21

22 Automation Good Laboratory Practice: Dry lab NGS Primary analysis Base calling Data trimming Read length, % reads with ~Q30 or above (e.g. FastQC) (performance specifications established by manufacturer) FASTQ Secondary analysis De-multiplexing Alignment [to a reference] Assembly Id of sequence variants N50 No of reads mapped to reference Coverage % of SNPs in clusters Multiplicity and parsimony BAM and VCF files Tertiary analysis Annotation Filtering and classification Reporting and retention of lab results % of intact MLST genes Phylogenetic tree shapes Concordance among prediction programs Credentialing of pipelines Concordance among labs in PT Clinicalgrade VCF 8

23 Summary Pre-processing NGS output and TAT Pathogen/host reads ratio [Standard] Genomics Genomic DNA extraction & library prep 1-2 GB h Low Pathogen discovery (RNASeq) Additional host DNA depletion & microbial DNA enrichment 40 GB 14 days High Taxonomic accuracy Species and subtypes Species Differentiation of virulent/mdr strains Detection of pointsource outbreaks, estimation of transmission pathways + +/- +?

24 Thank You!