Roche LightCycler 480

Research and Testing Laboratory Lubbock, TX 3/1/2013 1

Using Next-Gen Sequencing in a Clinical Setting

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Problem, Purpose, Process, and Potential

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Research and Testing Laboratory LLC

Formed in 2004 with the intent to use molecular diagnostic methods to understand and fight microbial infections.

Biofilms and Chronic wounds. (No case study pictures I promise)

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Problem

Chronic wounds Venous Leg Wounds Decubitus Ulcers Diabetic Foot Ulcers

Recovery Months Years Amputation Death

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Problem

Tissue Oxygen < 30 mm Hg (Hypoxia) Blood Sugars > 350 (Systemic Disease) Albumin less than 2.7 (Malnutrition) Repetitive Trauma (Pressure) Increased Bioburden (Infection)

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Problem

Treatment Plan Debride Culture wound Treat dominant infection Redebride Reculture wound Treat dominant infection Rinse and Repeat

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Problem

Why difficult to treat? Immunocompromised

Biofilms

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Recruitment

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Problem

Polymicrobial

Resistance

Dormancy

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Medical Biofilm US Incidence Annual Cost Diabetic foot ulcers (P) 3 M 50,000 deaths, 30% of

hospital cost for diabetics Venous leg ulcers (P) 2.5 M Decubitus ulcers (P) 3-5 M (P) 27%NH > 50,000 Surgical site infections 500,000 $5-10 B, 5,000 deaths Burn wounds 1.1 M 15,000 deaths Chronic meningitis 1,400-2,800 140-390 deaths Bacterial prostatitis (P) 162,800 All odontogenic infections Chronic tonsillitis 11,000 $121.5 M Gallstones 430,000 $5 B Crohns disease 36,000-60,000 Ulcerative colitis 24,000-40,000 COPD (P) 30 M $37.2 B, 120,000 deaths Bronchiolectasia 110,000

Gen

eral

Inf

ecti

ons

Pneumonia (non-VAP) 1.2 M $14-$25 B, 54,000 deaths Medical Biofilm US Incidence Annual Cost

Vascular graft infection 16,000 $640 M Cardiac pacemakers 4,000-20,000 Peritoneal dialysis peritonitis ~20-25,000 on CPD Ventilator acquired pneumonia 135,000 $1.5 B, 61,000 deaths Endotracheal tubes 100s of thousands* $5 B Urinary catheter cystitis Millions 4,500 deaths

Nos

ocom

ial

Central venous catheters 250,000 $296 M-$2.3 B, 30-62.5 K deaths

Total 20 Million $100 B, >500k deaths

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Disease Incidence Annual Cost Cardiovascular Disease 2.28 M per year $431.8 B, 650,000 deaths Cancer 1.5 M per year $206.3 B, 550,000 deaths Diabetes 1.5 M per year, ages 20+ $132 B, 73,000 deaths Medical Biofilm > 10M per year > $200 B, > 500,000 deaths

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Process

Image Scientific American: Explore the Human Microbiome [Interactive]

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Process

Estimated to be 10x more bacteria cells on and in humans than actual human cells

Microbial constituents help with food digestion, vitamin synthesis, immune system regulation and appetite Microbes contribute more genes responsible for

human survival than human genes

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Members of the same household have similar microbiomes

Community structure is unique to an individual Varies due to age, hormones, pets, stress,

medication, health Implication to biofilms in chronic wounds

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Characterization Culture

Many bacterial species are resistant/difficult to culture

May not reflect the total community structure

90-99%

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16S rRNA Sequencing

Siqueria et. al. (2012)

PresenterPresentation NotesOther barcodesITS Cytochrome SSU LSU Plastids

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Culture v molecular methods

51 Human wound samples were split for culture and 16S deep sequencing. 14 genera were identified by traditional methods, 145 identified by sequencing

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Culture often misses minor constituents

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Characterization Molecular microbiology techniques are

rapidly becoming the gold standard for microbial diversity studies and diagnosis of infection

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Clinical Result

48%

62%

91%

0% 20% 40% 60% 80% 100%

CTRL Group

DNA Dx/Systemic

DNA Dx/Topical

Full Wound Closure % (Healed)

CTRL Group DNA Dx/Systemic DNA Dx/Topical

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The Roche LightCycler 480 II

Can provide sensitive and accurate detection using appropriately designed target primers and probes.

More sensitive in comparison to other high-through put platforms when quantifying specific analytes.

Quick and cost-effective.

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Ion Torrent

Cheap sequencing Fast preparation Small fragments

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Wound Panel ENT Gastroinstestinal Dental Non-biofilms

Lymes Trypanosoma

Virus difficult

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Samples

DNA extraction

DNA pool of PCR products. Purification, quantification, and dilution

emPCR-- PCR in oil-water emulsion, resulting in clonal amplification of a single bead-bound target sequence

Bead recovery

Amplified DNA bound to a single bead is sequenced with the use of pyrosequencing on a GS FLX Titanium System

Phylogenetic assignment, alignment, and clustering of genetic fragments

FLX 454 Flow Diagram

Samples

Samples

Samples

PCR to amplify genetic region and also introduce linkers and tags on the DNA fragments

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Sample Capabilities

To extract Feces, swab, tissue, water, filters, soil, whole

organisms, blood, cerebral fluid, urine, biofilms

Genomic DNA cDNA RNA Pooled regions

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Samples

DNA extraction

DNA pool of PCR products. Purification, quantification, and dilution

emPCR-- PCR in oil-water emulsion, resulting in clonal amplification of a single bead-bound target sequence

Bead recovery

Amplified DNA bound to a single bead is sequenced with the use of pyrosequencing on a GS FLX Titanium System

Phylogenetic assignment, alignment, and clustering of genetic fragments

FLX 454 Flow Diagram

Samples

Samples

Samples

PCR to amplify genetic region and also introduce linkers and tags on the DNA fragments

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Linker and Tag purpose

Linkers A and B Recognition site for DNA binding bead Priming site for enrichment and sequencing

reactions

Tag Unique barcode for pooled samples (8 bp)

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Samples

DNA extraction

DNA pool of PCR products. Purification, quantification, and dilution

emPCR-- PCR in oil-water emulsion, resulting in clonal amplification of a single bead-bound target sequence

Bead recovery

Amplified DNA bound to a single bead is sequenced with the use of pyrosequencing on a GS FLX Titanium System

Phylogenetic assignment, alignment, and clustering of genetic fragments

FLX 454 Flow Diagram

Samples

Samples

Samples

PCR to amplify genetic region and also introduce linkers and tags on the DNA fragments

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Pooling, Purification, Quantification

PCR gel band size and strength Small fragment removal

Ampure beads Diffinity Tips Other techniques

Double stranded quantification Single strand enrichment Single stranded quantification

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Samples

DNA extraction

DNA pool of PCR products. Purification, quantification, and dilution

emPCR-- PCR in oil-water emulsion, resulting in clonal amplification of a single bead-bound target sequence

Bead recovery