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Bio and Microbiology Measurements as Support of Safety of Foods, Production, Trade and Quality of LifeDr. Denise OSullivanSimposio de Metrologa

Science for a safer world

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Health is our current focusCancerInfectious diseasesGenetics Surrogate BiomarkersSingle cell

Characterisation of nucleic acids

Nucleic acidTypeSequenceQuantity QualityModifications

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Targeted Nucleic Acid AnalysisPolymerase chain reaction (PCR)Quantitative PCRDigital PCRHigh-throughput PCRIsothermal amplification methodsGlobal Nucleic Acid AnalysisSpectrophotometry FluorometryElectrophoresis (Capillary)SequencingSanger sequencingNext generation sequencing

Molecular Biology Capability

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Split sample by dilution

dPCR 20 1 l reactionsqPCR 1 20 l reactions

Absolute DNA Quantification Digital PCR

Concept of digital PCR is not newRuano, Kidd & Stephens, 1990 (PNAS 87: 6296)Sykes et al., 1992 (Biotechniques 13: 444)Kalinina, Brown & Silver, 1997 (NAR 25:1999)Kinzler and Vogelstein, 1999 (PNAS 96: 9236)

PCR is the most versatile method avaliable for molecular biologyqPCR has demanded huge advances in PCR reagentsMuch of this knowledge can be directly transferred to dPCRCount is 10No need for standard curve

MIQE guidelinesTo enable authors to design, perform, and report dPCR experiments that have greater scientific integrityTo facilitate replication of experiments that are described in published studies in which these guidelines were followedTo provide critical information that allows reviewers and editors to measure the technical quality of submitted manuscripts against an established standard

Infectious diseasesWork packagesHigher order methodsMeasurement challenges associated with emerging methodologies.Near-patient testingDevelopment of a reference measurement framework to improve current clinical approachesModel diseasesHuman cytomegalovirus, Influenza, Tuberculosis and chronic obstructive pulmonary disease (COPD)

INFECT-MET : Standards for infectious disease diagnostics

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Next generation sequencing

Kahvejian et al., Nat Biotech (2008)

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Next generation sequencingTuberculosisInvestigating rare mutation detection in the development of drug resistance for MDR TB (deep sequencing)Assessment of whole genome sequencing for detection of novel drug resistance mutations and epidemiological analysis (WGS)Technologies are being evaluated for technical sensitivity, specificity and repeatability

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IntroductionMolecular Microbial Profiling

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Microbial Molecular Profiling characterises the structure of bacterial communities and their dynamics in the environmentEarly molecular profiling studies revealed that there are large groups of micro-organisms that cannot be cultured and thus cannot be sequencedMetagenomics (the study of metagenomes) can analyse the culturable and unculturable micro-organisms in an environment

Microbial Molecular Profiling9

Roche refer to 16S rRNA analysis as metagenomics.Conventional sequencing begins with a culture of identical cells as a source of DNA. However, early metagenomic studies revealed that there are probably large groups of microorganisms in many environments that cannot be cultured and thus cannot be sequenced. These early studies focused on 16S ribosomal RNA sequences which are relatively short, often conserved within a species, and generally different between species.Here are some examples of cases where rRNA surveys are referred to as metagenomics:Deep 16S rRNA metagenomics and quantitative PCR analyses of the premature infant fecal microbiota...- Wow -- rRNA as metagenomics even made it into the title here Paper: 16S rRNA metagenomics-based survey of oral biofilms in obese children...Poster Abstract Gastroenterology & Endoscopy News - Studies Link Composition of ...News Story Metagenomic study of the oral microbiota by Illumina high-throughput sequencing- paper is only about rRNA sequencing, not metagenomics Dr. Dag Harmsen publishes first 16S metagenomic study on the Ion PGM Sequencing ...a Youtube video highlighting a PLoS One paper Bacterial Community Shift in Treated Periodontitis Patients Revealed by Ion Torrent 16S rRNA Gene Amplicon Sequencing- PLOS One paper from the video above EUREKA GENOMICS | 16S metagenomic analysis service- a company pushing their services A Metagenomic Approach to Characterization of the Vaginal Microbiome Signature in Pregnancy ...PLoS One paper

I think the confusion arose when people started classifying their studies on where the DNA came from, rather than what they were looking for. Thus, anything from a community became 'meta' to differentiate it from axenic culture studies. I don't think it's anyone being deliberately misleading. Beyond the early studies (and now in some newer ones which enable improved quantitative analysis) I would say the field of metagenomics (as you define it) has been a mixed bag of success. Classifying a study as 'metagenomics' is as likely to be met with band-wagon glee as 'oh look, more basepairs' derision

Analysis of collective microbial genomes in a sampleMicrobiota = complex microbial communitiesOpportunity to analyse complex microbial populations without the need for culturingEnvironmental microbial communitiesHuman microbial communities {oral (1010 bacteria), skin (1012 bacteria), intestinal (1014 bacteria)

The Global Ocean Sampling Expedition (GOS) is an ocean exploration genome project with the goal of assessing the genetic diversity in marine microbial communities and to understand their role in nature's fundamental processes.The JCVI team and collaborators analyzed 7.7 million DNA sequences (6.3 billion base pairs) from data collected from Halifax, Nova Scotia through the Eastern Tropical Pacific. They discovered 6.12 million new genes and 1,700 unique protein families.

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Metagenomics for microbial analysisMetagenomics is the study of metagenomes

A metagenome is the microbial genetic material present within an environmental sample

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Microbiomes play a crucial role in health Implicated in many diseasesMicrobiome analysis will define:How they affect health and diseaseRole in Diagnostics/Prognostic monitoring Therapeutic manipulationHuman microbiome 8,000,000+ genes

Human genome23,000 genes>360 times-Oral (1010) bacteria-Skin (1012) bacteria-Intestine (1014) bacteria

The Human Microbiome Project11

Human Microbiome Project (GI tract, oral cavity, nasal cavity, skin and genital regions), will provide a reference set of microbial genome sequences and characterisation of the human microbiome, investigate the relationship between disease and changes in the human microbiome.Current estimates of oral and intestinal microbiota is that each is composed of 500-1000 different bacterial species.Study of organism and functional diversityInvestigate the complex interactions between bacterial populations (e.g. oral and cardiovascular disease)Link between the human oral disease and cardiovascular disease.Link between gut microbiome and common age-related diseases such as cancer and potentially cardiovascular disease through modification of classical risk factors such as obesity, insulin resistance and plasma lipids.Microbes have symbiotically evolved in the gut to perform functions not evolved in humans, including the extraction of calories from indigestible components of our diet, synthesis to essential vitamins and amino acids.Hypothesised that an imbalance in microbial health can cause obesity.Allows direct study of organisms in natural environmentMicroorganisms may play an even more important role in maintaining human health than in generating disease, makes metagenomics one of the most relevant areas of future research.

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Molecular quantification of microbes Used clinically for prognostic monitoring

Important for defining accurate limit of detection for microbial identification

Therefore for traceable molecular measurement (quantitative and ID) there is a need to investigate:Quantitative methods Control material development and requirements

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HIV viral load testing is an integral component of the management of HIV infection. Used to monitor success of anti-retroviral therapy and to detect the emergence of viral resistance, evidenced by a rise in the viral load despite ongoing therapy.HIV viral loads predict progression of disease and give prognostic information.Plasma/serum SARS-CoV quantification represents a potential useful early diagnostic and prognostic tool for SARS.12

The ComplicationsHigh level of complexity of information offered by microbial molecular profiling methodsPoses a considerable challenge during analytical standardisationDisparate methods could cause problemsSo how can control materials help?13

Advances in DNA sequencing enable the analysis of the genetic content of microbial populations (the metagenome)Associated studies commonly measure the composition of the component organisms in the environment but what about quantification?Early evidence suggest that sample processing and preparation can have a strong influence on the measured abundance of the microbes13

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A Control Material for Microbial Molecular Profiling

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Previous Work

We have previously described the application of a control material to compare different library preparation methods

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The Metagenomic Control Material (MCM) A Prototype Reference Material16

Mix of DNA from 10 bacterial species prepared

~ 200 units prepared in May 20122 X material @ 1 ng/LGram PositivesStaphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Streptococcus agalactiae Enterococcus faecalis

Gram NegativesPseudomonas aeruginosa Klebsiella pneumoniae Acinetobacter baumannii Escherichia coli Neisseria meningitidis

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Microbial Metagenomics

Proportions of the MCM bacterial gDNA mixtures presented as a pie chart based on the estimation of mass and analysed using pre-PCR and post-PCR protocols with assays 23 and 456. Klebsiella pnuemoniae and Streptococcus pneumoniae were not measured as a predominant species when pre-PCR protocols are performed using assays 23 and 456 respectively. The data above just shows the results for assay 23. The effect was less prominent when post-PCR protocols were employed.

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StudyAim: to perform a comprehensive investigation into the inherent error in profiling microbial communitiesUse the control material (metagenomic control material MCM) for community analysis which was already preparedCharacterise control material using two independent methods dPCR and fluorescenceCan use the MCM to investigate errors when performing different sequencing strategies (amplicon v shotgun)Compare the relative abundances of the component organisms of the MCM as determined by dPCR, fluorescence and next generation sequencing methods

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Any measurement can be judged by the following meta-measurement criteria values: level of measurement (which includes magnitude), dimensions (units), and uncertainty. They enable comparisons to be done between different measurements and reduce confusion. Even in cases of clear qualitative similarity or difference, increased precision through quantitative measurement is often preferred in order to aid in replication. Science of measurement is metrology.Measurements are most commonly made in the SI system, which contains seven fundamental units: kilogram, metre, candela, second, ampere, kelvin, and mole. Six of these units are artefact-free (defined without reference to a particular physical object which serves as a standard); the definition of one remaining unit, the kilogram is still embodied in an artefact which rests at the BIPM outside Paris. Eventually, it is hoped that new SI definitions will be uniformly artefact-free.

describing the sources of bias during sample preparationreporting the accuracy of NGS platforms over a broad dynamic range establishing the capabilities for measuring minority targets defining the requirements of a reference material when conducting next generation sequencing

Relative data comparabilityCross-laboratory comparisonsStandardisationPropagation of uncertainty

Biases can be investigated using control materials to interrogate the different software packages, the impact of sample preparation, 16S primer choice and amplicon preparation, direct sampling and library preparation. In addition to this, the performance characteristics of the different sequencing platforms can be investigated. Amplicon sequencing can also be used for measuring the microbiome and uses PCR to amplify conserved homologues. The most popular target, the 16S ribosomal rRNA gene, has been used in taxonomic studies for classifying bacteria at the molecular level for decades. It is a key genetic marker in phylogenetic studies and consequently is a popular alternative to WGS for microbiome analysis 18

Relative copy number, expressed as % of MCM

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Good agreement with dPCR.Sequencing was able to identify the species in the MCM and also able to quantify them.

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Relative copy number, expressed as % of MCM

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Good agreement with dPCR.Sequencing was able to identify the species in the MCM and also able to quantify them.

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ConclusionsdPCR offers a simple method for providing confidence when measuring control materialsAll sequencing methods performed comparablyGood agreement between sequencing methods and dPCR (WGS demonstrating very high reproducibility)Any discrepancy between abundances were in general < 3 foldCare should be taken when comparing small differences; need to apply control materialProvides a foundation for future work looking at microbial abundances in a communityFuture work looking at impact of extraction methods

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One of our key findings is that due to the high precision afforded by most of the methods, significant differences between methods are frequently observed. However this is seldom greater than three fold suggesting that researchers applying these methods should treat significant differences that are less than threefold with caution/ apply control materials etc21

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Profiling Food Spoilage Microorganisms

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Microorganisms in Food SpoilageMicroorganisms, and their by-products can be harmful in food Traditional methods can be slow, laborious and often inconsistentMicrobial profiling methods could be used to understandthe complex communities in foodstuffs and which microorganisms are involvedhow changes in the microbiota as whole could indicate food spoilage conditionshow the shelf life could be improved through rapid ID of spoilage bacteria

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Traditionally, bacterial species have been identified using techniques involving culturing processes combined with morphological, physiological, and biochemical characterization. Primary identification involves a few simple assays such as analysing colony morphology, growth conditions and Gram staining. For a better approximation of bacterial identification, other techniques such as microbial observation and resistance to different substrates are employed.In addition to these tests being slow, laborious and often inconsistent, they can also only be used with the limited number of microorganisms that are compatible with in vitro cultivation.Molecular methods of microbial profilingTraditional methods of bacterial profiling have now been largely replaced with molecular techniques including antibody-based assays, such as ELISAs, and DNA-based methods such as real-time PCR and DNA sequencing. These molecular techniques are much faster, more accurate and sensitive.The development of DNA sequencing technologies has enabled the genomic analysis of whole microbial communities (metagenomics) as opposed to the analysis of specific species or partial community analysis. This allows the capturing of microdiversity, or variations in strains of bacteria between different samples. Such differences can be very subtle and the ability to detect such changes is crucial to detecting shifts in composition towards spoilage. High-throughput DNA sequencing of 16S rDNA gene amplicons has revolutionized the capacity and depth of microbial community profiling by providing a culture-independent, rapid method of profiling whole micriobial communities.

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Test CasesGammon samples stored at +4oC and -20oC for several weeksFreeze-dried chicken samples (LGC Standards)Perform 16S rDNA sequencing on the Roche GS Junior

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Gammon samples have been analysed by standard microbial culture techniques.Freeze-dried chicken samples which are used for PT schemes, spiked with food spoilage bacterial species Lactobacillus alimentarius and Pseudomonas aeruginosa.24

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Figure 2 shows the microbial profiles for the two gammon samples stored at different temperatures. Data is presented as a taxonomic tree with each sequence assigned to the lowest level of identification possible. For example, at the far left of the chart for the gammon stored at 40C one sequence could only be identified as bacteria, 5534 sequences could be identified as Firmicutes (phylum level), 490 sequences identified as Leuconostoc (Genus level) and 265 sequences could be identified down to the species level of Leuconostoc carnosum (a key food spoilage agent).For both storage conditions large numbers of sequences could only be identified to the genus level or higher. However, differences in profiles could be seen between the two storage conditions. The main species identified by culture at CBRI were lactobacilli.

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Figure 3. shows the chicken microbial profile. The data is plotted according to 16S rDNA amplicon (456c or 12 piecharts), but for the purposes of this study it is only the species identified that are relevant. As for the gammon, large numbers of sequences could only be assigned to a genus level identification. The two bacteria that were spiked in (Lactobacillus Alimentarius and Pseudomonas Aeruginosa) could again be identified at the genus level (marked by arrows)

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Conclusions from Test CasesPerformed microbial profiling of food samples to identify food spoilage agentsRequire more sequencing depth to determine rarer and under-represented species16S rDNA strategy was not able to identify down to species levelDifferent storage conditions resulted in differences in profilesNeed to develop strategy for the identification of moulds and yeasts27

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Further Impact of Molecular Methods on Microbial Quantification

28To investigate how targeting different bacterial chromosomal positions effect the measurement.

Molecular vs CultureP Q01.1 Report on the first pilot study- Establishing Comparability for Microbial Culture Quantitation (NMIA)Dr. Dean Clarke, NMIA

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Morpeth et al ISPPD 2012

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gDNA materialsAvailable from IRMM for E. coli O157, (strain EDL 933)L. monocytogenes (strain 4B, NCTC 11994) Available from NIM China forE.coli O157 (Strain EDL 931, ATCC 35150)L. monocytogenes (Strain Li 23,ATCC 19114)

Positive control for PCR reactions for diagnostics PCRs

Provided lyophilised (Nominal g value and uncertainty provided)

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Considerations for molecular quantification Chromosomal sizeE. coli = ~5 MbL. monocytogenes = ~2.9 Mb

Gene dosageReplication Multicopy genes (16S rDNA)

EpigeneticsMethylation

Optical methods will be effected by chromosomal size, however dPCR will not.There is extensive size variation between species.

Bacteria can have more than one chromosome.Gene dosage is the number of copies of a gene present in a cell or nucleus. An increase in gene dosage can cause higher levels of gene product if the gene is not subject to regulation from elsewhere in the body.

If DNA is methylated has a different molecular weight so this needs to be factored in when calculating DNA concentration using a32

Bacterial replication

www.pasteur.fr33

Figure shows the elements associated with the organisation of the bacterial chromosome.Green and red distinguish between the leading and lagging strands.The bacterial chromosome is circular.Replication begins on the bacterial chromosome at a specific sequence of nucleotides called the origin (ori) and finished at the terminus of replication, encircled arrows indicate the direction of replication fork progression.Replication proceeds by the bidirectional progression of the replication forks along the chromosome.In each replication fork, a complex with two DNA polymerases replicates the two DNA strands. The pace of the replication fork varies significantly between bacteria.Genes near the origin are >8 times more abundant in the cell than are genes near the terminus.

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ABCABC

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Short sequence reads can be used to generate useful assemblies of both prokaryotic and eukaryotic genome sequence, albeit containing many gaps. However new algorithms can help in read assembly.Biases in real short read sequence data. A set of Illumina 36 nt genomic sequence reads from P. Syringae was aligned against the previously published. Figure illustrates the depth of coverage by aligned reads over the 6 Mb circular chromosome. Coverage is shallower around the 3 Mb region that it is far away from the origin of replication position.Observe a systematic over-representation of sequences close to the origin of replication.34

Chromosomal TargetsE. coli has large genomeTarget four regions Ori, sequence closer to the origin of replicationdif , sequence close to the termination siteuidA, diagnostic sequence used by LGC16S rDNA, multi copy sequence (7 in the strains under investigation)

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As already mentioned about 5 Mb.35

E. coli CFT073http://www.ncbi.nlm.nih.gov/nucleotide/26111730?report=genbank&log$=nuclalign&blast_rank=74&RID=KW9A1N24016http://www.ecogene.org/old/genemap/map.php?search_topic=132

4680kb0kb4160kb3640kb3120kb2600kb2080kb1560kb1040kb520kbEscherichia coli CFT0735231428 bprrsDrrsGrrsArrsBrrsErrsCrrsHoriCdifuidAClockwise = Strand +Anticlockwise = Strand -E. coli gDNA 36

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Questions?How does chromosomal location effect result (qPCR, dPCR)

Is this similar between StrainsPreparations/Laboratories 37

qPCR is not as affected by chromosomal location as you normalise your results to the standard curve.dPCR is affected by chromosomal position as it is an absolute method and is not related to a standard.37

E. coli gDNA Three different E. coli strains O157:H7, (strain EDL 933). Supplied by IRMMO157:H7, (strain EDL 931), Supplied by NIMCCFT073, Supplied by ATCCPrepared by 3 different laboratories (China, US, Belgium)Re-suspended at LGCQuantified by fluorescence Same amount of each extract measured by dPCR 38

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E. coli gDNA 39

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E. coli gDNA 40

Has the 16S rRNA result been normalised to the number of 16S copies in the genome?40

E. coli gDNA 41

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Absolute QuantificationConcordance between assays is very impressivedPCR has the potential to improve microbial molecular measurementDo we need to assign a value to gDNA extracts?Other things to considerDifferent cultures (strains/growth stages etc.)MatricesOrganismsAssay specificity42

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How much precision is necessary?What are we aiming for with regards to precision

Viral load testing decisions rarely made on differences that are < 1 log

What about limit of detection?

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Other considerationsHow do we standardiseqPCR/amplicon sequencing/shotgun?Different laboratories?

If we produce a reference material what do we trace it to:Colony forming units?DNA gram weight?Genome equivalents?

Just what does 100 bacterial genomes mean to:Microbe viability ?Microbe infectivity?Microbe toxicity?

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AcknowledgementsLGCJim Huggett, Carole Foy, Tan Temisak, Nicholas RedshawUniversity of ExeterDavid Studholme, Thomas LaverSt. Georges University of LondonPhilip Butcher, Ken LaingGreat Ormond Street HospitalKathryn HarrisNational Measurement Institute ChinaLianhua Dong

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46Thanks for listeningdenise.osullivan@lgcgroup.com

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