from sample to result – workflow solutions for genotyping and pathogen detection

Sample to Insight

Pathogen detection with HRM and multiplex real-time qPCR technology:

Challenges, workflow and applications

1

James Qin, Senior Scientist, MDx Applications, QIAGEN

Sample to Insight

2

Legal disclaimer

Pathogen detection with HRM and multiplex RT-qPCR

• QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis,

prevention or treatment of a disease.

• For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user

manual. QIAGEN kit handbooks and user manuals are available

at www.QIAGEN.com or can be requested from QIAGEN

Technical Services or your local distributor.

http://www.qiagen.com/

Sample to Insight

4

Agenda

Pathogen detection with HRM and multiplex RT-qPCR 3

Microbiome utilities and associated diseases

Microbial DNA isolation challenges and solutions

Pathogen detection methods

• Challenges

• Real-time PCR and Multiplex real-time PCR

• Quantitative high-resolution melting (HRM)

Applications

1

2

3

Sample to Insight

4

Agenda





• Challenges



Applications

1

2

3

Sample to Insight

• Well-known microorganisms that

cause GI infections

• Adenovirus

• Campylobacter

• Clostridium difficile

• Cryptosporidium

• Entamoeba histolytica

• Escherichia coli

• Escherichia coli O157:H7

• Giardia

• Helicobacter pylori

• Rotavirus

• Salmonella and Shigella

• Staphylococcus aureus

• Yersinia enterocolitica

• Microbiome ecology: Suggests unique microbial residents are tuned to

the environment of one’s body (genetics, diet, and developmental

history)

• Microbiome diversity: Hints immune and metabolic disorder might be

related to a degraded microbiome and also because antibiotic resistance

is limiting human ability to kill pathogens.

• Microbiome composition: Associated with long-term distortions in the

composition, function, and antibiotic resistance of the intestinal

microbiota

• Microbiome population profiling: Imbalance of the gut microbiota is

linked with gastrointestinal conditions such as IBD, IBS, obesity, type 2

diabetes, and atopy

• Microbiome transplants: New evidence linking changes in microbial

diversity to a variety of diseases, including C. diff colitis; Helping to

rationalize and validate the effectiveness of this controversial treatment

strategy

• Others – Monitoring disease progression and treatment …

Microbiome and human diseases


Sample to Insight

4

Agenda





• Challenges



Applications

1

2

3

Sample to Insight

7

Pathogen detection workflow: From Sample to Insight

Overview of multiplex PCR assay workflow

Sample isolation

Multiplex amplification

Detection and analysis

Extract and purify

microbial DNA

45 minutes, manually

or automated

Multiplex amplification of

targeted pathogens

2 hour

1. Non-specific detection

using DNA binding dyes

2. Specific detection using

target specific probes

Detection: 1 hr

• What pathogens or microorganisms are present?

• What is the relative abundance?

• Are there any specific mutations associated with antimicrobial resistance?

Sample collection & treatment

Pretreat stool specimens

to break down parasitic

30-45 minutes


Sample to Insight

General challenges of pathogen detection & ID workflow

8

Sample collection

DNA or RNA extraction

Detection

Data analysis

Different sample type represents challenges collecting samples: hair bulb, skin, dry blood, ear punch, FFPE, biopsy, saliva/ sputum, urine, stool…

May contain agents that hamper DNA or RNA extractionVariable extraction efficiencyDifficult to determine extraction efficiency

Primer design: High diversity of microbes and majority unknown. It is difficult to design primers that capture all activity.

Nucleases, enzyme inhibitors caninterfere with qRT-PCR; sample abundancy may affect certain type of assays

Normalization, quantitation methods: internal standards, housekeeping genes,


Sample to Insight

9

Some considerations in sample extraction

Microbial DNA extraction

• PCR inhibitors presented in samples is one of big challenges

• High abundance of host DNA can introduce bias of results

Core questions to ask:• What samples are being analyzing?

• Sample type: stool, soil, swabs, urine

• How are samples collected and processed?• Lysis methods strongly depend on sample material (mechanical vs. enzymatic)

• Additional pre-treatments might be necessary (e.g. deparaffinization)

• How do you get rid of all the other stuff?• Most common biomolecules are relatively easy to remove (proteins, lipids, sugars)

• Some metabolites are might be co-purified and inhibit your analysis

• Deplete host DNA and enrich bacterial microbiome DNA

• How much DNA can I possible get from my sample• Species may have low abundance

• Stability of DNA


Sample to Insight

10

QIAGEN Sample to Insight solutions for pathogen and microbial extraction

Detection and analysis

• QIAmp DNA Mini Kit

• QIAmp UCP Pathogen Mini Kit

• QIAmp DNA Stool Fast/ Mini Kit

• QIAamp DNA Microbiome Kit• QIAamp 96 DNA QIAcube HT

Kit• QIAsymphony mericon Bacteria

Kit

MO BIO Laboratories for NGS• PowerSoil DNA Isolation Kit

• PowerFecal DNA Isolation Kit

• …

• QuantiFast Pathogen +IC

Kits

• QuantiTect Virus Kits

• QuantiNova Probe PCR

kits

• Microbial DNA qPCR

Arrays and Assays

• Type-it HRM PCR kit for

genotyping assays

• Rotor-Gene Q

• QIAxcel DNA Kits

• Allprotect

• InhibitEx

• RNAlater


Sample isolation

Sample collection & Stabilization

qPCR or qRT-PCR


Sample to Insight

11

Sample extraction: QIAamp 96 DNA QIAcube HT kit

Automate extraction process: free up you time by automating DNA extraction

Automatable on the QIAcube

Lyse Bind Wash Elute

Fast and reliable 96-well DNA purification

QIAamp silica-membrane technology

Sample: genomic, mitochondrial, and pathogen

DNA from blood, cells, and tissue samples

• Number of samples: 24-96 samples

• Elution volume: 200 ul

• Duration: 24 samples in 45 mins, 96 samples in 96

mins

• Cost/prep: $2.0 per sample


Sample to Insight

• Vortex 10 min• Heat de-activation 10 min• PK/ AL digestion 10 min• Quick spin; and transfer

QIAamp 96 DNA QIAcube HT kit: Experiment data

Move from manual process to fully automated process:

• High yield• Simple and fast


Sample to Insight

4

Agenda

13




• Challenges



Applications

1

2

3


Sample to Insight

14

Current methods in pathogen detection and identification

Each species of pathogens carries a unique DNA or RNA signature

that differentiates it from other organisms• Culture

• Gold standard for bacteria, such as Salmonella and Shigella• Utilized for pathogen identification, not directly characterize virulence factors• Laborious and time-consuming, and expensive

• Rapid antigen• Simple and rapid• Can test a range of viruses and certain bacteria and toxins

• Genomic assay: Singleplex and multiplex• High sensitivity and specificity

• Genomic assay: NGS and Pyrosequencing• Allows quantitative detection and identification of all existing microbial

• Proteomic assays • Rapid and allow function correlation


Sample to Insight

15

Multiplex PCR-based pathogen detection

• PCR based methods are the best for the specimens collected by non-invasive methods such as stool samples• Specific: only detects target sequence• Rapid: easy to set up, and run time within few hours• Sensitive: can detect low copy numbers• Standardized: can be automated and use stable chemical design

• Can be used for the detection of bacteria as well as for characterization of pathogenic genes and specific mutations associated with antimicrobial resistance

• Multiplex allows to focus either a panel of viruses or a panel of bacteria suspected in gastroenteritis with one test

Challenge: The presence of PCR inhibitors in stool samples may cause low sensitivity ； Assay designs.

QIAamp DNA Kit can efficiently remove the inhibitors


Sample to Insight

16

Multiplex real time PCR method

SYBR Green-based detection method

• Detection based on target specific primers only using non-specific DNA binding dyes

• SYBR Green is the most widely used double-strand DNA-specific dye for real time PCR

Target specific probe-based detection method

• Specifically detect real time PCR with oligonucleotide probes labeled with both a reporter

fluorescent dye and a quencher dye.

• Hydrolysis probes such as TaqMan probes

• Molecular Beacons

• FRET Hybridization Probes

• Scorpion Primers

• Quantitative mRNA expression studies of cDNA

• Microbial load or copy number measurements from

microbial genomes

• Allelic discrimination assays or SNP genotyping

• Verification of microarray results


Sample to Insight

17

QIAGEN Sample to Insight solutions for pathogen and microbial detection

• QIAmp DNA Mini Kit

• QIAmp UCP Pathogen Mini Kit

• QIAmp DNA Stool Mini Kit• QIAamp 96 DNA QIAcube HT

Kit• QIAsymphony mericon Bacteria

Kit

MO BIO Laboratories• PowerSoil DNA Isolation Kit

• PowerFecal DNA Isolation Kit

• …

• QuantiNova Probe RT-PCR

Kit• QuantiNova SYBR Green

RT-PCR Kit• QuantiTect Multiplex PCR • QuantiFast Pathogen +IC

Kits• Type-it HRM PCR Kit• Microbial DNA qPCR Arrays

and Assay Kits

• Rotor-Gene Q• QIAxcel DNA Kits

• Allprotect

• InhibitEx



Detection and

analysis

Sample isolation

Sample collection & Stabilization

qPCR or qRT-PCR

Sample to Insight

18

QIAGEN RT-qPCR and multiplex PCR product lines

• QuantiNova Probe RT-PCR Kit and QuantiNova SYBR® Green RT-PCR Kit• Novel QuantiNova two-phase hot-start mechanism

• Reliable: Increased reliability of gene expression results using gDNA reduction• Convenient: Room-temperature reaction setup without compromising • Visual pipetting control: limit pipetting errors• Internal controls: positive in-process verification of successful RT-PCR and monitoring RT-PCR

inhibition• Sensitive: High sensitivity for low-copy RNA targets


Sample to Insight

19


• QuantiTect Multiplex PCR Kit and QuantiTect Probe PCR Kit • Real-time PCR and two-step RT-PCR

• High PCR specificity with integrated hot start• Reliable quantification of low-abundance transcripts• Accurate quantification over several logs of template• Available with or without uracil-N-glycosylase (UNG)• No need to optimize reaction and cycling conditions

Wide dynamic rangeHigh specificity

Fast: Faster results with time savings of up to 50%

Multiplex: Successful multiplex PCR without the need

for optimization

Sensitive: Detect up to 4 targets in 1 tube

Reliable: Quantify low- and high-abundance targets


Sample to Insight

20


• Type-it HRM PCR Kit• Fast and accurate detection of gene mutations and SNPs by High-Resolution

Melting (HRM) analysis

Scan for mutation

Type mutation


Sample to Insight

4

Agenda

21




• Challenges



Applications

• Cannabis contamination detection (Food Safety)

• Bovine respiratory and GI track pathogen detection

• HRM: Microbiome pattern recognition in clinical research and more

1

2

3


Sample to Insight

22

Star: Talaromyces stipitatus; Tree: Aspergillus nidulans Ornaments: Penicillium marneffei; Trunk: Aspergillus terreusHat, Eyes, Mouth, Buttons: Aspergillus niger; Arms: Aspergillus nidulans; Nose: Aspergillus terreus with Penicillium marneffei; Body: Neosartorya fischeri

• Purpose: Develop real-time pathogen detection assays food and agricultural products such as marijuana, organic drinks

• Extraction chemistry: QIAamp DNA kit• Detection Kit: QuantiFast Pathogen + PCR Kit

• Experimental Design and validation strategy

• Result

Development of Cannabis contamination assays in Cannabis quality control with QuantiFast Microbial Kit + IC

Application 1: Cannabis contamination detection (Food Safety)


Sample to Insight

Application 1: Validation strategy

• Assays were tested using synthetic templates

• Microbial qPCR Mastermix, Microbial DNA-Free Water

• Sensitivity• Standard curves from 0 to 1,000,000 copies were prepared• 1000 copy Ct<31 or Ct<34 (low end assays if NTC Ct=40)• Primer efficiency> 80%, R>0.995, LLOQ determined

• Specificity• Human, mouse, rat gDNA• Microbial genomic DNA pools:

• Each pool contains 10 genomic DNA at 2000 genome copy each or complete pool which contains all genomic DNA (110)

• Genomic DNA from same genus put into separate pools• Staph/Strep species made into separate pool• Each specificity test performed in duplicate

• Performance in complex background• Spiked in synthetic template in stool, sputum, sewage• dCt = Ct (background + synthetic template) – Ct (synthetic template) • dCt<3

23Pathogen detection with HRM and multiplex RT-qPCR

Sample to Insight

Application 1: Standard curves using synthetic templates

24

0 1 2 3 4 5 62022242628303234363840

f(x) = − 3.28705357142857 x + 40.0704464285714

Aspergillus niger

Log target copy number

CT

0 1 2 3 4 5 62022242628303234363840

f(x) = − 3.235625 x + 41.0404464285714

Klebsiella pneumoniae


CT

0 1 2 3 4 5 62022242628303234363840

f(x) = − 3.14232142857143 x + 41.64875

Mucor/Rhizopus spp.


CT

0 1 2 3 4 5 62022242628303234363840

f(x) = − 3.16080357142857 x + 41.2038392857143

Pan Aspergillus/Penicillium


CT


Sample to Insight

Pool1 Pool2 Pool3 Pool4

Acinetobacter baumannii Aeromonas hydrophila Alcaligenes faecalis subsp. Faecalis Aspergillus fumigatusBacillus licheniformis Bartonella henselae Bordetella pertussis Brevundimonas diminutaCampylobacter jejuni subsp. Jejuni Candida albicans Candida glabrata Candida parapsilosisCitrobacter freundii Clostridium diffi cile Clostridium perfringens Clostridium thermocellumCorynebacterium glutamicum Enterobacter aerogenes Enterococcus faecalis Enterococcus faeciumFusobacterium nucleatum subsp. Nucleatum Geobacillus stearothermophilus Haemophilus influenzae Helicobacter pyloriLegionella pneumophila subsp. Pneumophila Listeria monocytogenes Mycobacterium tuberculosis Neisseria meningitidisPantoea agglomerans Pediococcus pentosaceus Plesiomonas shigelloides Proteus mirabilis

Rahnella aquatilis Ralstonia solanacearumSalmonella enterica subsp. enterica serovar Paratyphi A Serratia marcescens

Vibrio choleraeYersinia enterocolitica subsp. Enterocolitica Yersinia pestis Stenotrophomonas maltophilia

Pool5 Pool6 Pool7 Pool8

Bacillus cereus-14579D-5Aggregatibacter actinomycetemcomitans Akkermansia muciniphila Anaerococcus prevotii

Burkholderia cenocepacia Bacteroides thetaiotaomicron Bacteroides ureolyticus Bacteroides vulgatusCandida tropicalis Burkholderia cepacia Campylobacter coli Campylobacter concisusCorynebacterium diphtheriae Capnocytophaga gingivalis Cryptobacterium curtum Cryptococcus gattii

Escherichia coli-200928D-5Enterobacter cloacae subsp. Cloacae Gardnerella vaginalis Lactobacillus jensenii

Klebsiella pneumoniae Lactobacillus casei Lactobacillus gasseri Micrococcus luteusOchrobactrum anthropi Methanobrevibacter smithii Mycoplasma pneumoniae Neisseria flavaPseudomonas aeruginosa Mycoplasma orale Porphyromonas gingivalis Prevotella intermediaShigella flexneri Porphyromonas endodontalis Trichomonas vaginalis Ureaplasma parvumYersinia pseudotuberculosis Treponema denticola Staphylococcus haemolyticus Streptococcus mitis

Pool9 Pool10 Pool11 staph/strep

Aspergillus flavus Atopobium rimae Bacillus subtilisStaphylococcus aureus subsp. Aureus-

Bifidobacterium breveBifidobacterium longum subsp. Infantis Bordetella parapertussis

Staphylococcus epidermidis-

Campylobacter gracilis Campylobacter rectus Campylobacter upsaliensisStaphylococcus saprophyticus subsp. Saprophyticus

Cryptococcus neoformans Desulfovibrio desulfuricans Lactobacillus acidophilus Streptococcus gordoniiHaemophilus ducreyi Klebsiella oxytoca Leptotrichia buccalis Streptococcus mutansLactobacillus plantarum Lactobacillus reuteri Mycoplasma hominis Streptococcus pneumoniaeMycobacterium smegmatis Mycoplasma genitalium Peptostreptococcus anaerobius Streptococcus pyogenesNeisseria gonorrhoeae Parabacteroides distasonis Tannerella forsythia Streptococcus sanguinisPrevotella melaninogenica Proteus vulgaris Vibrio parahaemolyticusVeillonella parvula-#1 Vibrio harvey Streptococcus agalactiae

Application 1: Specificity of bacterial fungal assays

Title, Location, Date 25N

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2022242628303234363840

Thermoactinomyces spp. 2Ct

NTC

Tem

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Thermoactinomyces spp. 1Ct

NTC

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Klebsiella pneumoniaeCt

Sample to Insight

Application 1: Testing of assays with genomic DNA

26

0 1 2 3 4 5 62022242628303234363840

f(x) = 2.32053571428571 x + 28.2950025

Aspergillus niger

Log Dilution Factor

CT

0 1 2 3 4 5 62022242628303234363840

f(x) = 3.112405 x + 19.776405

Mucor/Rhizopus spp.

Log Dilution Factor

CT

0 1 2 3 4 5 62022242628303234363840

f(x) = 2.56781080357143 x + 26.307851875

Pan Aspergillus/Penicillium

Log Dilution Factor

CT

0 1 2 3 4 5 62022242628303234363840

f(x) = 2.51111133928571 x + 18.2925016964286

Thermoactinomyces spp. 1

Log Dilution Factor

CT

0 1 2 3 4 5 62022242628303234363840

f(x) = − 0.0137010714285716 x + 39.8811464285714

Thermoactinomyces spp. 2

Log Dilution Factor

CT

Aspergillus brasiliensis gDNA Penicillium chrysogenum gDNA Mucor hiemalis gDNA

Thermoactinomyces vulgaris gDNA Thermoactinomyces vulgaris gDNA


Sample to Insight

Performance and sensitivity (LLOQ)

27

Species slope E R LLOQAspergillus niger -3.53 92% 0.999 60Klebsiella pneumoniae -3.24 104% 0.996 80Mucor/Rhizopus spp. -3.51 93% 1.000 230Pan Aspergillus/Penicillium -3.16 107% 0.995 100Thermo spp. 1 -3.07 112% 0.999 30Thermo spp. 2 -3.01 115% 1.000 60

Aspe

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iger

Kleb

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Ther

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Ther

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-3

-2

-1

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1

2

3

4

dCt (stool-template) dCt (sputum-template) dCt (sludge-template)

dCt (

[met

agen

omic

sam

ple+

synt

hetic

te

mpl

ate]

–

synt

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Assay performance in metagenomic samples

Application 1: Performance


Sample to Insight

• Research purpose• Develop real-time multiplex pathogen detection assays for Veterinary

customers• T. foetus, Calf Diarrhea panel (K99, Sal. Crypto.) assays• Calf Scours 4-plex assay with InType internal control• Bovine Respiratory Disease (BRD) Panel 5-plex assay with InType internal

control

• Current challenges• Long process: Magnetic beads, cRNA, isopropanol, tissue disruption equipment/steps ...

• Lost of sensitivity for calf diarrhea /Johne’s feces because of inhibitors, …

• Cross reactivity and drop of signals: Singleplex assays multiplex assays

• Products used• Extraction: QIAcube HighThroughput (QCHT) automation solution based on QIAamp

DNA 96 kit (96-well plate, silicon columns)• Consistent and comparable performance with QuantiFast Pathogen PCR with IC kit as

a multiplex PCR solution

• Validation strategy and result

Application 2: Bovine respiratory and GI pathogen detection


Sample to Insight

• The enzymes:• HotStarTaq Plus DNA Polymerase• Reactivation within 5 minutes• Unmatched specificity and sensitivity

• The buffer: • Unique combination of K+ and NH4

+ ions

• High specificity• No optimization necessary

• Q-BondTM:• Enables fast cycling by promoting annealing of Taq

and primer/probe to the template

• Synthetic Factor MP• Supports macromolecular crowding• Enables equal amplification of templates that differ in

abundance

Solving problems with fast real-time PCR

High PCR specificity and macromolecular crowding


Sample to Insight

T. Foetus (Fam) Internal Control (Yellow)

TH = 0.05

Tritrichomonas Foetus and InType IC: A clean multiplex assay


Sample to Insight

Negative NTCs; Good IC ranges;Sensitivity <= 10 copies

InType IC

Good correlation & assay efficiency

TH = 0.05

InType Internal Control in Calf Scours 4-plex assay


Sample to Insight

A clean Crypto assay in 4-plex reaction with good dynamic range

Cryptosporidium in 4-plexes assay:Good consistency and sensitivity of Crypto+ in the 4-plex

TH = 0.20

Calf scours panel: E. Coli K99/ Sal./Crypto/ InType 4-Plex assay


Sample to Insight

InType internal control (yellow) 5-plexes

TH = 0.05

Tight range of Internal control, no interference

with the 4 targets vs. IC

Bovine BRD Respiratory Panel

M. haem/H. Somni/ M. bovis/ P. mult InType 5-Plex assay


Sample to Insight

Comparable target Cts. A good specificity on POS vs Negs; and a consistency of M. Haem in singleplex vs. 5-plex assays.

Mannheimia Haemolytica in 5-plexes : Good consistency, specificity, and tighter range of M. Haem+ in the 4-plex

TH = 0.05

Good signal and tight range in IC controls.

InType ICM. Haem+

Bovine BRD Respiratory Panel: M. Haemolytica in 5-plexes


Sample to Insight

Comparable target Cts. Better specificity and great improvement on negative samples in 5-plexes vs. H. Somni singleplex

Histophilus Somni in 5-plexes: Good consistency, specificity, and less cross-reactivity of M. Haem+ in the 4-plex

TH = 0.05

A good signal and tight range in IC controls in 5-plexes.

Bovine BRD Respiratory Panel: H. Somni in 5-plexes


Sample to Insight

Better specificity and good improvement on negative samples in 5-plexes vs. singleplex

Mycoplasm bovis (Green, Fam) in 5-plexes: Good consistency, sensitivity, and less cross-reactivity in M.bovis+ in the 4-plex

TH = 0.05

A good signal and tight range in IC controls in 5-plexes. Overcome primer-probe-target interactions in singleplex in IC.

Bovine BRD Respiratory Panel: Myco. bovis in 5-Plex Assay


Sample to Insight

TH = 0.05

Better specificity and great improvement on negative samples in 5-plexes vs. H. Somni singleplex after optimization

Consistent, good signal and tight range in IC controls.

Pasteurella multocida in 5-plexes: Good consistency, specificity, and tighter range of P. mult + in the 5-plex

Bovine BRD Respiratory Panel: P. mult + Optimization


Sample to Insight

• Purpose • To identify a real-time multiplex kit that is consistent, precise in real-time

PCR, and is fast, easy-to-use and cost-effective

• Extraction chemistry• QIAamp DNA kit• Extraction automation: QIAcube: silica column-based • Detection Kits: QuantiNova real-time probe PCR kit (recommended for

duplex)

• Validation strategy• Use DNA templates contain different copy numbers between target vs.

reference

• Result• Series of template dilutions for ∆∆Ct (relative quantitation) analysis

Application 3: Copy number assay for allele discrimination analysis


Sample to Insight

Comparison study in copy number assay for allele discrimination analysis using QuantiNova and QuantiTect Probe real-time PCR Assays

R2=0.8576

M=-3.361Efficiency=0.98

R2=0.9962

M=-3.275Efficiency=1.02

QuantiNova rt-PCR kit: Comparable result and faster QuantiTect R-PCR Kit: a multiplex alternative (4-plex)

Result is highly comparable.

Application 3: Copy number assay for allele discrimination analysis


Sample to Insight

Purpose & backgroundComplex community microbiome profiling on healthy subjects:

• Qiagen RotorGene Q 5-plex HRM real-time PCR with primers targeting V3 region of 16S rRNA gene (170bp amplicons) on PowerSoil and PowerFecal DNA kits extracted DNA samples from healthy subjects

HRM Profiling for Microbiome Diversity on healthy subjects:

• HRM melt on RGQ 5-plex HRM for possible profiling on the amplified samples above; no PMA-Rx’ed samples

Qiagen kits and automation• Extraction: QIAcube HT with QIAamp DNA kit

• Type-it HRM PCR kit

• QIAgility for liquid handling & assay setup

• Rotor-Gene Q PCR with 100-Disc

Validation strategy• Look at the sample pretreatment options and protocols

• Test samples on QIAcube HT instrument and protocol for an automation and full workflow solution (propose: total 48 – 64 combined fecal + soil samples)

• Reference kit: Data will need to have full range of QC (Qxpert, Nanodrop 8000)

• Will evaluate limited samples with real-time PCR

Results

Application 4: Microbiome profiling by high resolution melting (HRM)


Sample to Insight

Background


Sample to Insight

Primer Set NTC (5ul/20-ul PCR) Run-2

JE/Pro 33.76V3 22.3V5 20.83V6 22.1V7 19.45

Threshold = 0.01Dynamic TubeSlope Correct!

Results: Assay and primer sets testing


Sample to Insight

ºC71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

dF/dT

3.75

3.50

3.25

3.00

2.75

2.50

2.25

2.00

1.75

1.50

1.25

1.00

0.75

0.50

0.25

0.00

JE341/Pro805

V6

V5

V3

V7

RGQ real-time PCR melt curves

Results: RotorGene melt curve analysis on five assays


Sample to Insight

JE/ Pro

V3 V7V5

V6

XX X

Results: Linearity and assay dynamic range check


Sample to Insight

Threshold = 0.18735127 bps

Threshold = 0.15662460 bps

JE341/ Pro805 V6

Ct = 10-12Ct = 22-25

Assay Condition-1 Assay Condition-2

Results: Assay optimization and dynamic range


Sample to Insight

ºC71.5 72.0 72.5 73.0 73.5 74.0 74.5 75.0 75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5

Nor

mal

ised

min

us V

5

55

50

45

40

35

30

25

20

15

10

5

0

-5

-10

JE341/Pro805Normalized to V5:

V6 V5V3

V7

ºC72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

Nor

mal

ised

min

us J

E341

/Pro

805

0

-10

-20

-30

-40

-50

-60

Normalized to JE341/ Pro805:

JE341/Pro805

V6

V3

V5 V7

Results: RotorGene HRM analysis on five assays


Sample to Insight

For many datasets, most of the eigenvalues “lambda” are negligible and can be discarded.

The eigenvalue measures the variationIn the direction e:

Example:

Principal Component Analysis (PCA) in ScreenClust

Finding relationship from a large multi-layer sample set is always a challenge. One way to avoid the dimensionality is by projecting the data onto a lower-dimensional space. It rotates multivariate dataset into a new configuration which is easier to interpret.

Techniques for dimension reduction:Principal Component Analysis (PCA) – most effective!Fisher’s Linear Discriminant Multi-dimensional Scaling. Independent Component Analysis.


Sample to Insight

Application of PCA in genomics

Purposes• Simplify data – PCA is the most commonly used dimension reduction technique.• Look at relationships between variables – PCA is useful for finding new, more informative,

uncorrelated features.• PCR reduces dimensionality by rejecting low variance features.• Look at patterns of units – i.e. new mutations, targets• Analysis of expression data• Analysis of metabolomics data (Ward et al., 2003)

Example RunSophisticated computing and bioinformatics


Sample to Insight

JE341/Pro805

V3

V5

V7

V6

HRM PCR Genotyping Kit/ RGQ: Offers distinct melt curves

HRM analysis with ScreenClust software with RotorGene


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ScreenClust: High predicting power in pattern recognition

Example run report: HRM analysis with ScreenClust software


Sample to Insight

ºC75 80 85 90

dF/d

T2.0

1.5

1.0

0.5

0.0

ºC77 78 79 80 81 82 83 84 85 86 87 88 89

Nor

mal

ised

Flu

ores

cenc

e

100

80

60

40

20

13003

13009

13000

13026

13031 13027

13003DS 13003DP13004

1300013029

HRM is able to pick up individual differences

High Resolution Melt (V6 assay)

Distinct melt curve patterns

HRM and melt curve analysis by ScreenClust on all QCHT extracted Hu. samples


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Result: Clustering Analysis by ScreenClust on All QCHT extracted samples


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• Consumable business that can be forecasted on a monthly basis w/ an annualized value of $50K or greater

• $50K or more on automation for initial purchase or in the form of reagent rental• Combination of the above to meet $50K minimum requirement

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• Set expectations right with limited and achievable key deliverables

• Investment is $10,000 with material/FTE cost for up to four weeks of time. It is free to qualified customers.

If You Are……A small startup that is for-profit and in growth mode, have funding & wants to accelerate their business; …A company that does not have the capacity or the time for development or portfolio expansion but is willing to spend when unique requirements are met. And …

• Companies that have the following challenges:• Liquid cancers • Solid, difficult to extract tissues• Difficult plant, food, drink extraction• Look for assay solutions• Look for one-size-fits-all protocol

• Customer also should have:• Clear, fixed objectives and short timeline• Has an established manual assay but wants to expand or improve• Currently in direct comparison w/ 3rd party offerings w/ a limited timeline to make a decision

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Questions?

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