andrea m. cabibbe · next generation sequencing: definition “ngs” includes all the technologies...

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. CabibbeWHO Collaborating Centre in TB Laboratory Strengthening

TB Supranational Reference LaboratoryEmerging Bacterial Pathogens UnitOspedale San Raffaele, Milan, Italy

Outline

Sequencing workflows:

• DNA extraction

• Library Preparation

• Sequencing

• Analysis

• Practical considerations for country implementation

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 assembledinto longer contiguous sequences in order to reconstruct the original sequence in the absence ofa reference sequence or genomeResequencing: 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 coveredwith a certain depth

Supporting information:J. Besser et al., Clinical Microbiology and Infection 24 (2018) 335-341, PMID: 29074157

NGS general workflow for M. tuberculosis

Supporting information:Lee RS, Ther Adv Infect Dis (2016) 3(2) 4762, PMID: 27034776

NGS general workflow: laboratory workspace

Supporting information:World Health Organization; 2018 (WHO/CDS/TB/2018.19)

Supporting information:Cabibbe AM et al., Eur Respir J 2018; 52: 1801163, PMID: 30209198

NGS general workflow: TAT and outputs

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, OD260/280 1.8-2.0; OD260/230 2.0-2.2)• 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

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:

DNA extraction and purification

Promega Maxwell 16 System: automated

beads-basedpurification

Qiagen QIAcubeConnect: automated

column-basedpurification


DNA extraction and purification: summary

Supporting information:McNerney R et al., International Journal of Infectious Diseases 56 (2017) 130–135

Sample type DNA extraction Application

Clinical isolates(e.g. positive MGIT/LJ)

Genomic DNA WGS

Clinical specimens(e.g. sputum sediments)

Genomic DNA Targeted NGS

Clinical specimens(e.g. sputum sediments)

Genomic DNA WGS*

*optimization of protocols ongoing

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

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

Library preparation


Example: Illumina Nextera XT DNA Library Prep Kit

✓ 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 (ifdesired)

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


Sequencing: NGS instruments

Sequencing: Illumina

Science Squared

• Nova Seq, HiSeq, NextSeq, MiniSeq, Miseq, iSeq• 70% of the market

• Sequencing by synthesis (bridge PCR)

• Applications: genomics, transcriptomics

High platform cost (now broad range available)Low cost per outputHigh read accuracyShort read length (max 300bp)Long run time (9hours-3 days)

Sequencing by synthesis technology is used to detect each base by fluorescently labeled dNTPs


BGI platform

Sequencing: Thermo Fisher Scientific

• S5, Proton, PGM• 15% of the market

• Sequencing by synthesis-semiconductor(emulsion PCR)

• Applications: genomics, transcriptomics(targeted)

Medium platform cost (broad range available)Lower throughputLower read accuracy (homopolymers)Short read length (max 400bp)Shorter run time (3-24 hours)

Seq based on detection of H+ ions released duringpolymerazation of DNA. Change of pH


Sequencing: Pacific Biosciences

• Sequel, RS II• 5% of the market

• Single-molecule real-time

• Applications: genomics, epigenomics

Very high platform costLower throughputLow read accuracyLong read length (up to 60,000bp)Shorter run time (up to 20 hours)

Single DNA polymerase fixed on the bottom of a zero-mode waveguide (ZMW). When a nucleotide isincorporated by the enzyme, the fluorescent tag iscleaved-off and diffuses out of the observation area of the ZMW. Base call according to the correspondingfluorescence of the dye.


Sequencing: Oxford Nanopore Technologies

• PromethION, GridION, MinION

• Single-molecule real-time

• Applications: genomics, transcriptomics, epigenomics

PortableReduced library prepLow platform costLow cost per outputLow read accuracyLong 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 reducesspecifically the current


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

DNA samples extracted from MTB clinical isolates, biological and technical replicates

Median genome coverageSimilar

High GC content regionsMiSeq: coverage drops when GC >75%PGM: coverage drops when GC >69%, subjected to greater variability

Variant error ratesLow for bothMiseq: higher quality SNP callPGM: fewer SNP calls

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

Supporting information: 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.

Supporting information: Nakano et al., Human Cell (2017) 30:149–161

(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

Pacific Biosciences PacBio RS II

Oxford Nanopore Technologies MinION

Despite the high sequencing error rate, high accuracy genotyping of known SNPs/indels was achievable as described.

Supporting information: Votintseva et al., J Clin Microbiol. 2017 May;55(5):1285-1298

Oxford Nanopore Technologies vs Illumina

Supporting information: 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 99.92 %

- Able to resolve the GC-rich and highly repetitive PE/PPE gene families which were poorly resolved when Illumina reads were used

Estimated genotypic error rate of 5.3% identifying all the known drug resistance and compensatory mutations in concordance with in vitro susceptibility testing

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.

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)

Analysis: workflowBIOINFORMATICS!

Supporting information: Altmann et al., Hum Genet (2012) 131:1541–1554

AnalysisBIOINFORMATICS!

• WGS resequencing: Mapping to a reference genome

Alignment → reads aligned to M. tuberculosis H37Rv genome (NC_000962.3)

• 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_000962.3)

Analysis: resequencing - general stepsBIOINFORMATICS!

• 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

Supporting information: Peng Q, Next-Generation Sequencing: an Intro to Tech and Applications, 2013

Analysis: general stepsBIOINFORMATICS!


Analysis: some available bioinformaticspipeline BIOINFORMATICS!

Supporting information:Kohl TA et al., PeerJ 6:e5895

MTBseq

Supporting information:Ezewudo M et al., Scientific Reports, 2018, 8:15382

CPTR-ReSeqTB

Analysis: User-friendly tools for DRTB diagnosis by WGS

Supporting information:Schleusener V et al., Sci. Rep. 7, 46327 (2017) NO BIOINFORMATICS SKILLS!

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

- Laboratory spaceLab bench and clearances depending on instrument size; molecular biology (sample preparation, pre-and post-PCR)

- ElectricityPower specifications, safety measures, UPS

- Gas supply (instrument-specific)

- EnvironmentTemperature, humidity, elevation, air quality, ventilation, vibration

- Network and internet

- ComputingHardware, storage

- Consumables and equipment

- Staff

Practical considerations for country implementation: infrastructure

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

NGS platform-specific equipment/consumables: sequencing instrument and reagent kits

Supporting information: World Health Organization; 2018 (WHO/CDS/TB/2018.19)

Practical considerations for country implementation: equipment

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)

Practical considerations for country implementation: computational capacity

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

Practical considerations for country implementation: training


Practical considerations for country implementation: training

NGS platform manufacturer

- Local distributor- Prompt technical assistance- Installation and operation- Troubles and troubleshooting

Ideal: Core facility (in the Institute)

Practical considerations for country implementation: technical support

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

Practical considerations for country implementation: quality assurance

Practical considerations for country implementation: quality assurance


Practical considerations for country implementation: reporting

Supporting information: Crisan et al. (2018), PeerJ 6:e4218; DOI 10.7717/peerj.4218

TAT: h 48 / 72 Cost/sample: € 115 / 170

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

Practical considerations for country implementation: budgetingSupporting information: Rossen JWA, Clin Microbiol Infect. 2018 Apr;24(4):355-360.

Supporting information: Pankhurst, Lancet Respir Med 2016; 4: 49–58

✓ 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

Practical considerations for country implementation: advantages

Supporting information: Satta G, Clin Microbiol Infect. 2018 Jun;24(6):604-609.

McNerney R, Expert Rev Anti Infect Ther. 2018 May;16(5):433-442.

andrea m. cabibbe · next generation sequencing: definition “ngs” includes all the technologies...

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