Sequencing workflows

Transcription

1 Sequencing workflows WEBINAR 3: February 19, 15:00-16:30 CET: Applications, protocols, and workflows Webinar Series 2019, Next-generation sequencing for drug-resistant TB Andrea M. Cabibbe WHO Collaborating Centre in TB Laboratory Strengthening TB Supranational Reference Laboratory Emerging Bacterial Pathogens Unit Ospedale San Raffaele, Milan, Italy

2 Outline Sequencing workflows: DNA extraction Library Preparation Sequencing Analysis Practical considerations for country implementation

3 Next Generation Sequencing: definition NGS includes all the technologies that generate high throughput, massively parallel sequence reads allowing DNA and RNA assembly in a short time and at relatively low cost (compared to classical sequencing). - Faster sequencing and greater coverage of genomes. Sequencing reads: sequence of a unique DNA fragment obtained at the end of the seq reaction. Library: a collection of DNA fragments prepared for sequencing Paired-end reading: sequencer starts reading DNA fragment at one end, finishes this direction at the specified read length, and then starts another round of reading from the opposite end of the fragment (e.g. Illumina) Output data: raw data generated by sequencing instruments (e.g. BCL, binary base calls for Illumina). These are converted to files that can be used for by analysis tools (e.g. FASTQ) FASTQ: text-based format for storing a biological sequence and its corresponding quality scores De novo assembly: during bioinformatics analyses, individual sequence reads are assembled into longer contiguous sequences in order to reconstruct the original sequence in the absence of a reference sequence or genome Resequencing: determination of the genomic variations of a sample in relation to a common reference sequence (e.g. H37Rv M. tuberculosis) Coverage depth: average number of reads that include a given nucleotide in the reconstructed sequence Coverage breadth: percentage of bases of a reference genome that are covered with a certain depth J. Besser et al., Clinical Microbiology and Infection 24 (2018) , PMID:

4 NGS general workflow for M. tuberculosis Lee RS, Ther Adv Infect Dis (2016) 3(2) 4762, PMID:

5 NGS general workflow: laboratory workspace World Health Organization; 2018 (WHO/CDS/TB/ )

6 NGS general workflow: TAT and outputs Cabibbe AM et al., Eur Respir J 2018; 52: , PMID:

7 DNA extraction and purification Key quality indicators and quality control considerations: Specimen/culture sample quality: adequate sample purity (with consideration for other organism DNA, human DNA or inhibitors in the sample) Specimen/sample quantity: Sufficient starting material (with consideration for targeted NGS or WGS application) DNA quality: Adequate DNA purity (spectrophotometer, OD 260/ ; OD 260/ ) DNA quantity: Sufficient starting material (fluorescent detection system, e.g. a Qubit fluorometer or qpcr); Adequate volume DNA size and integrity (agarose gel electrophoresis or microfluidic instruments, including Bioanalyzer) NOTES: DNA quantity and quality requirements are dependent on the sequencing application and platform. DNA Quantity/quality assessment must be always conducted after extraction step. DNA extraction procedures require adequate biosafety containment level when starting from direct specimens or cultured isolates, including appropriate safety equipment and work practices World Health Organization; 2018 (WHO/CDS/TB/ )

8 DNA extraction and purification Cell lysis: - Thermal (thermolysis 95 C), enzymatic (lysozyme, proteinase K), mechanical (sonication, bead beating) Purification: - Chemical (CTAB, alcohol precipitation), (para)magnetic-based, column-based NOTE: Automated systems can also be used. A couple of examples for gdna purification from TB samples: Promega Maxwell 16 System: automated beads-based purification World Health Organization; 2018 (WHO/CDS/TB/ ) Qiagen QIAcube Connect: automated column-based purification

9 DNA extraction and purification: summary Sample type DNA extraction Application Clinical isolates (e.g. positive MGIT/LJ) Clinical specimens (e.g. sputum sediments) Clinical specimens (e.g. sputum sediments) *optimization of protocols ongoing Genomic DNA Genomic DNA Genomic DNA WGS Targeted NGS WGS* McNerney R et al., International Journal of Infectious Diseases 56 (2017)

10 Library preparation This step aims at preparing a collection of DNA fragments for WGS or tngs. The general protocols include: DNA fragmentation (enzymatic or mechanical) of gdna or amplicons and tagging of fragments End repair (optional, NGS instrument dependent) barcoding (for multiplexing) PCR amplification (optional, NGS instrument dependent) Library purification and normalization World Health Organization; 2018 (WHO/CDS/TB/ )

11 Library preparation NOTES: The particular steps involved in library preparation will vary according to the NGS technology and desired application Laboratory technicians must critically follow the manufacturers directions for all methods It is critical to check the quality and quantity of the DNA before and after library preparation Automated systems are available (e.g. Eppendorf epmotion 5075) Considerations to select a library prep kit: - NGS instrument being used - Quality and quantity of DNA required - Application - Cost - Automation - Duration of library preparation - Compatibility with other NGS instruments (if desired) World Health Organization; 2018 (WHO/CDS/TB/ ) Example: Illumina Nextera XT DNA Library Prep Kit

12 Sequencing: some initial considerations Volume of data: benchtop (few Mb to 15Gb) or high-throughput sequencers (up to 6,000Gb) - Batching Cost: infrastructure, equipment, reagents, consumables, post-processing - Strategies: In-house or outsourced (NGS service) Read-length (<100bp to 100,000bp+) Duration: turnaround time Technology (chemistry) - Data quality, accuracy - Generally, short-read sequencing platforms provide higher quality data

13 Sequencing: NGS instruments World Health Organization; 2018 (WHO/CDS/TB/ )

14 Sequencing: Illumina Nova Seq, HiSeq, NextSeq, MiniSeq, Miseq, iseq 70% of the market Sequencing by synthesis (bridge PCR) Applications: genomics, transcriptomics World Health Organization; 2018 (WHO/CDS/TB/ ) High platform cost (now broad range available) Low cost per output High read accuracy Short read length (max 300bp) Long run time (9hours-3 days) Sequencing by synthesis technology is used to detect each base by fluorescently labeled dntps Science Squared

15 Sequencing: Thermo Fisher Scientific S5, Proton, PGM 15% of the market Sequencing by synthesis-semiconductor (emulsion PCR) Applications: genomics, transcriptomics (targeted) World Health Organization; 2018 (WHO/CDS/TB/ ) Medium platform cost (broad range available) Lower throughput Lower read accuracy (homopolymers) Short read length (max 400bp) Shorter run time (3-24 hours) BGI platform Seq based on detection of H+ ions released during polymerazation of DNA. Change of ph

16 Sequencing: Pacific Biosciences Sequel, RS II 5% of the market Single-molecule real-time Applications: genomics, epigenomics World Health Organization; 2018 (WHO/CDS/TB/ ) Very high platform cost Lower throughput Low read accuracy Long read length (up to 60,000bp) Shorter run time (up to 20 hours) Single DNA polymerase fixed on the bottom of a zeromode waveguide (ZMW). When a nucleotide is incorporated by the enzyme, the fluorescent tag is cleaved-off and diffuses out of the observation area of the ZMW. Base call according to the corresponding fluorescence of the dye.

17 Sequencing: Oxford Nanopore Technologies World Health Organization; 2018 (WHO/CDS/TB/ ) PromethION, GridION, MinION Single-molecule real-time Applications: genomics, transcriptomics, epigenomics Portable Reduced library prep Low platform cost Low cost per output Low read accuracy Long read length (100,000bp+) Shorter run time (30 minutes-up to 48 hours) ssdna passes through an opening in the membrane that conducts ionic current. As a given nucleotide pass through, it reduces specifically the current

18 Illumina MiSeq vs Thermo Fisher Ion Torrent PGM Phelan et al., Genome Medicine (2016) 8:132 DNA samples extracted from MTB clinical isolates, biological and technical replicates Median genome coverage Similar High GC content regions MiSeq: coverage drops when GC >75% PGM: coverage drops when GC >69%, subjected to greater variability Variant error rates Low for both Miseq: higher quality SNP call PGM: fewer SNP calls

19 Illumina MiSeq vs Thermo Fisher Ion Torrent PGM Phelan et al., Genome Medicine (2016) 8:132

20 Illumina MiSeq vs Thermo Fisher Ion Torrent PGM Phelan et al., Genome Medicine (2016) 8:132

21 Pacific Biosciences PacBio RS II Nakano et al., Human Cell (2017) 30: (1) long read lengths (half of data in reads >20 kb and maximum read length >60 kb; our best record is 92.7 kb as of Nov. 2016), (2) high consensus accuracy (>99.999% at 30x in coverage depth, free of systematic errors), (3) low degree of bias (even coverage across GC content), and (4) simultaneous epigenetic characterization (direct detection of DNA base modifications at one-base resolution). Mycobacterium tuberculosis Kurono. Hard-to-sequence regions: GC content of 80% region (2,000 bp), 117 sets of >1000-bp identical sequence pairs Elghraoui et al., BMC Genomics (2017) 18:302 The random error profile of this technology allows for consensus accuracy to increase as a function of sequencing depth. The coverage depth of our assembly corresponds to a Phred quality value greater than 60 (QV >60), which translates to fewer than four expected errors [11]. If such errors exist, they would most likely appear as single-base insertions or deletions unique to our assembly.

22 Oxford Nanopore Technologies MinION Votintseva et al., J Clin Microbiol May;55(5): Despite the high sequencing error rate, high accuracy genotyping of known SNPs/indels was achievable as described.

23 Oxford Nanopore Technologies vs Illumina Bainomugisa et al., Microbial Genomics 2018;4 - A XDR strain was de novo assembled into one contig using 238x read depth data from one flow-cell of the MinION, reaching an accuracy of % - Able to resolve the GC-rich and highly repetitive PE/PPE gene families which were poorly resolved when Illumina reads were used Plot of sequence similarity of 168 PE/PPE family genes identified from the assembly and their sequence and depth coverage from Oxford minion reads (left). Percentage breadth of coverage (black), and read depth (blue). Average depth of 238X. Plot of sequence and depth coverage for the assembly of 168 PE/PPE family genes using Illumina reads (right). Percentage breadth of coverage (red), and read depth (blue). Average depth of 46.3X. Estimated genotypic error rate of 5.3% identifying all the known drug resistance and compensatory mutations in concordance with in vitro susceptibility testing

24 Sequencing: take home-messages All NGS platforms can provide high quality data confirming their reliability and robustness, given appropriate: Depth of high genome-wide sequence coverage That is variable for each platform and depends on: single/paired-end and raw error rates NOTES Minimum sequencing coverage required by each NGS platforms will affect costs for generating sequencing data The platforms do not all perform to the same standard: need of quality monitoring (use of standard controls)

25 Analysis: workflow BIOINFORMATICS! Altmann et al., Hum Genet (2012) 131:

26 Analysis BIOINFORMATICS! WGS resequencing: Mapping to a reference genome Alignment reads aligned to M. tuberculosis H37Rv genome (NC_ ) WGS de novo: During bioinformatics analyses, individual sequence reads are assembled into longer contiguous sequences in order to reconstruct the original sequence in the absence of a reference sequence or genome. Targeted NGS: resequencing limited to a select set of genes or gene regions of the M. tuberculosis H37Rv genome (NC_ )

27 Analysis: resequencing - general steps BIOINFORMATICS! Demultiplexing Quality control Alignment reads aligned to M. tuberculosis H37Rv genome Alignment post-processing Quality Score recalibration Variant and Genotype calling Known SNPs calling Reporting Peng Q, Next-Generation Sequencing: an Intro to Tech and Applications, 2013

28 Analysis: general steps BIOINFORMATICS! World Health Organization; 2018 (WHO/CDS/TB/ )

29 Analysis: some available bioinformatics pipeline BIOINFORMATICS! MTBseq CPTR-ReSeqTB Kohl TA et al., PeerJ 6:e5895 Ezewudo M et al., Scientific Reports, 2018, 8:15382

30 Analysis: User-friendly tools for DRTB diagnosis by WGS Schleusener V et al., Sci. Rep. 7, (2017) NO BIOINFORMATICS SKILLS!

31 Practical considerations for country implementation Challenges: Infrastructure Equipment Procedures Computing Training Technical assistance Quality assurance Data analysis and interpretation Nomenclature and reporting Costs Capital investment Laboratory areas, power supply, environment NGS platform-specific, local distributors Development of SOPs Hardware/software, storage solutions Laboratory and bioinformatics Manufacturers and implementing partners Internal (validation, standards)/ External (PT) Standardization, user-friendly pipelines Standardization, supportive to clinical decisions Cost-benefit analysis, funding, sustainability

32 Practical considerations for country implementation: infrastructure - Laboratory space Lab bench and clearances depending on instrument size; molecular biology (sample preparation, preand post-pcr) - Electricity Power specifications, safety measures, UPS - Gas supply (instrument-specific) - Environment Temperature, humidity, elevation, air quality, ventilation, vibration - Network and internet - Computing Hardware, storage - Consumables and equipment - Staff

33 Practical considerations for country implementation: equipment Equipment/consumables for DNA extraction and quality/quantity assessment General: - Laboratory-grade water - Alcohol wipes - Laboratory tissues - Disposable gloves - Freezer - Refrigerator - Pipettes - (Micro)centrifuge - (Micro)centrifuge tubes - 96-well plates - Magnetic stand - Electrophoresis apparatus - 96-well thermal cycler - Heat-block - Vortex mixer - Shaker World Health Organization; 2018 (WHO/CDS/TB/ ) NGS platform-specific equipment/consumables: sequencing instrument and reagent kits

34 Practical considerations for country implementation: computational capacity For a safe storage and handling of raw sequencing data: - desktop PC (ideal requirements): memory 16GB, Hard Disk Mobility Solid State Drive 1TB, Unix-Like operating system, quad-core processor - external HD (size 1 TB) - Server- or cloud-based storage (data storage, sharing and protection)

35 Practical considerations for country implementation: training Laboratory technician for wet part: sample preparation, DNA extraction and sequencing Background (molecular biology) Training on: - Sequencing basics and principles - Laboratory workflow and protocols - Troubles and troubleshooting Bioinformatician for dry part: post-sequencing analysis and sequencing data handling Background (molecular biology and/or bioinformatics) Training on: - UNIX and Linux operating systems - Use of scripts and analysis pipelines; use of commercial or freely-available softwares - Storage of sequencing data - Troubles and troubleshooting

36 Practical considerations for country implementation: training World Health Organization; 2018 (WHO/CDS/TB/ )

37 Practical considerations for country implementation: technical support NGS platform manufacturer - Local distributor - Prompt technical assistance - Installation and operation - Troubles and troubleshooting Ideal: Core facility (in the Institute)

38 Practical considerations for country implementation: quality assurance Internal - Comparison of platforms - Quality control steps during the whole procedure (from sample preparation to post-sequencing analysis) - Use of standard controls - Data analysis: sequencing depth threshold; sequencing reads quality - Development of Standard Operating Procedures - Validation: accuracy and reproducibility External - Proficiency Testing programmes - Use of standardized protocols and analysis pipelines

39 Practical considerations for country implementation: quality assurance World Health Organization; 2018 (WHO/CDS/TB/ )

40 Practical considerations for country implementation: reporting Crisan et al. (2018), PeerJ 6:e4218; DOI /peerj.4218

41 Practical considerations for country implementation: budgeting Rossen JWA, Clin Microbiol Infect Apr;24(4): TAT: h 48 / 72 Cost/sample: 115 / 170 Pankhurst, Lancet Respir Med 2016; 4: Microcosting analysis conduceted in laboratories from Europe and North America Routine-diagnostic time: 9 days from culture for full analysis, median 21 days faster than conventional Routine-diagnostic cost: 481 per culture-positive specimen, 7% cheaper than conventional

42 Practical considerations for country implementation: advantages Time to result: diagnostic and surveillance pros Costs (considering: identification, full DST, typing, outbreak investigation) High sensitivity (improving) Potentially WGS from sputum specimens (tngs adopted yet): improved biosafety and logistics User-friendly interpretation of sequencing data (no skills, no IT infrastructure) Higher resolution for epidemiological analyses Research outcomes: discovery of new drug mechanisms and relevant mutations; studies on genetic variability (vaccines?) Data repository Multi-disease platforms Satta G, Clin Microbiol Infect Jun;24(6): McNerney R, Expert Rev Anti Infect Ther May;16(5):

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