Next Generation Sequencing for Food Authenticity ...Simplest NGS workflow for targeted sequencing...

The world leader in serving science

Erin Dreyling, PhD

Regulatory Leader for Food Protection

Thermo Fisher ScientificMay 31, 2018

Next Generation Sequencing for Food Authenticity, Traceability and Safety

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The World Leader in Serving Science

Mission: We enable our customers to make the

world healthier, cleaner and safer.

Our Customer Focus:

We help accelerate innovation

and enhance productivity for

our customers.

in revenues$20B

employees>65,000

countries50

R&D investment>$900M

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The world leader in serving science

Ion Torrent

Sequencing

Technology

For Research Use Only. Not for use in diagnostic procedures.

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Simplest and Fastest NGS System for Targeted Sequencing


Simplest NGS workflow for targeted sequencing

• <15 minutes to set up a sequencing run

• <45 minutes total hands-on time from DNA to data with Ion

Chef System

Fastest run time

• As little as 3.5 hours from sequence to BAM files

Lowest capital investment

• Single platform for all applications, with flexibility to scale

from 2 to 260 M reads per run*

Lowest DNA/RNA input requirements

• As little as 1 ng using Ion AmpliSeq technology

Easy setup and training

• Single-day installation, plug-and-play cartridge-based

reagents

Ion 520™ Chip5 M reads

Ion 530™ Chip15–20 M reads



Ion 550™ Chip100–130 M

reads

*Requires using two Ion 550 chips per run on Ion GeneStudio S5 Prime System to achieve >130 M reads per run

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• Ion torrent uses CMOS semiconductor technology just like

high end digital cameras

• Instead of light, ion torrent chip detects chemical changes as

the sequencing reaction proceeds

Ion Torrent Technology

High End Interchangeable Lens Camera CMOS Light Sensing Chip Ion Torrent Sequencing Chip

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• Why Targeted Sequencing?

• Focus on the genomic regions of

interest to minimize cost and

maximize speed

• Only 1.5% of the genome

encodes protein

• Many genes have now been

mapped to targets of interest

• Whole genome data creates

storage and analysis challenges

• Ion Ampliseq technology

enables researchers to focus

on the genomic regions they

care about

Targeted Sequencing

Internal Use Only / No external distribution

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~45 Minutes Hands-On Time from DNA to Data | Only 2 Pipetting Steps Per Sample

Automatedlibrary construction

Automated template

preparationSequencing


Pipet

sample and primer

pools into Ion Chef

System for library prep

Load cartridge

into Ion Chef System

Pipet

library into Ion Chef

System for templating

and chip loading

Load cartridge

into Ion Chef System

Transfer chip

to Ion GeneStudio S5

System for sequencing

15 min

hands-on time

15 min

hands-on time

15 min

hands-on time

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Sequence Fast, Faster, and Faster with the Ion GeneStudio S5 Series

Ion GeneStudio S5

System

Ion GeneStudio S5 Plus

System

Ion GeneStudio S5 Prime

System

Speed*19 hrs 10 hrs 6.5 hrs

Output (max/day)**15 Gb 30 Gb 50 Gb

Number of reads (max/day)✝ 80 M 160 M 260 M

Compatible chips Ion 510, 520, 530, or 540 chips Ion 510, 520, 530, 540, and 550 chips

* Sequencing and analysis time based on Ion 540 chip.

** Requires using two Ion 540 chips per run on Ion GeneStudio S5 Plus System ✝Requires using two Ion 550 chips per run on Ion GeneStudio S5 Prime System


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For multiplex samples,

multiple targets

Ion Torrent Applications: Food Integrity

Food Fraud, Authenticity,

Labeling

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Food Authenticity: where fingerprints are strong

We need to confirm whether a

species is present in sample

(e.g. allergies , religious taboos,

lower cost substance)

• Meat

• Halal Foods

• Plants, Spices

• GMO

Adulteration/Food Fraud

Mislabelling of fish, meat, fruit

and vegetable products is

common:

• Wild ↔ farmed

• Origin and feeding diet

• Organic versus inorganic

Mislabeling

We need to confirm the place of

origin of a product, label

declaration.

• Dairy

• Cheeses

• Plants, Spices

Authenticity, Origin

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Food Traceability by Ion Torrent™ sequencing

• Multi Species Identification

Accurate species ID by sequencing of cytochrome subunits and

other genes (“barcodes”) even in mixed samples

Sequencing results show the percentage composition of different

species in a sample

• Geographic Origin Determination of SNP frequencies by GBS of high diversity genes

• Pathogen Detection 16S gene sequencing for identification of bacterial organisms

18S/ITS gene sequencing for identification of fungal organisms

Metagenomics

Whole genome sequencing

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Thermo ScientificTM NGS solutions for Food Authenticity:

•The new Thermo Scientific™ Multi Species ID System is a complete,

automated, next generation sequencing workflow and software database for multi-

species ID to ensure confidence and consumer trust.

The NGS Food Integrity System includes:

Thermo Scientific™

Ion Chef™ System

Ion GeneStudio™

S5 System

Next Generation

Sequencer

Torrent Suite™ and

SGS ™ ALL Species

ID Analyses

Software

SGS™ ALL Species

ID DNA Analyser

Kits

DNA, Library & Template Preparation

34 hours

Sequencing

12 hrs

Data Analysis

1 hr

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NGS FOR FOOD AUTHENTICITY, TRACEABILITY AND SAFETY

In the present study an innovative NGS-based approach was tested for meat,

spices/cereals and fish species identification

In Partnership with:

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Origanum sp. Origanum

Tragelaphus strepsiceros Kudu

Felix catus Cat

Rattus norvegicus Rat

Vulpes vulpes Fox

Alces alces Elk

Coturnix japonica King quail

Bubalus bubalis Buffalo

Camelus dromedarius Camel

Crocodylus niloticus Crocodile

Lophura inornata Pheasant

Oryx leucoryx Oryx gazella

Alcelaphus buselaphus Gnu

Bos grunniens Cattle Yak

Equus asinus Donkey

Meles meles Badger

Tragelaphus scriptus Antilope

Corvus macrorhynchos Daw

Mustela erminea Weasel

Ondatra zibethicus Muskrat

Anas sp Mallard duck

Crocodylus siamensis Crocodile

Phasianus colchicus Pheasant

Alectoris chukar Partridge

Aepyceros melampus Impala


Ovis aries Sheep

Capra hircus Goat

Lepus capensis Hare

Oryctolagus cuniculus Rabbit

Macropus rufus Kangaroo

Capreolus capreolus Roe Deer

Cervus elaphus Red Deer

Rangifer tarandus Reindeer

Antidorcas marsupialis Springbok

Equus hemionus Zebra

Lama glama Lama

Gallus gallus Chicken

Canis familiaris Dog

Bison bison Bison

Cervus dama Fallow Deer

Equus caballus Horse

Sus scrofa Pork

Bos taurus Beef

Meleagris galopavo Turkey

Cairina moscata Duck

Alopochen aegptiacus Goose

Struthio camelus Ostrich

Columba livia Pigeon

Numida meleagris Guinea fowl

Dromaius novaehollandiae Emu

MEAT SPECIES RESULTS

• Number of samples tested: 148 Number of species tested: 49

• Species mix: up to 3 Spiked level: 1%, 10%, 50%

99.3

0.7

Meat Species

positve negative

95.3

4.7

Meat species spiked 1%

positive negative

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Laurus_nobilis Sweet bay

Manihot_esculenta Cassava

Mentha spicata Spearmint

Myristica fragrans Nutmeg

Ocimum basilicum Sweet basil

Oryza_sativa Rice

Panicum miliaceum Millet

Papaver somniferum Opium poppy

Petroselinum crispum Parsley

Pimpinella anisum Anis

Piper nigrum Black pepper

Rosmarinus_officinalis Rosemary

Sesamum indicum Sesame

Sinapis_alba White mustard

Sorghum_bicolor Sorghum

Thymus_vulgaris Garden Thyme

Triticum_aestivum Wheat

Triticum_durum Durum wheat

Zingiber_officinale Garden Ginger

Laurus_nobilis Sweet bay

Manihot_esculenta Cassava

Mentha spicata Spearmint

Myristica fragrans Nutmeg

Ocimum basilicum Sweet basil


Allium schoenoprasum Wild chives

Allium_sativum Garlic

Anethum graveolens Dill

Argemone sp Prickly poppy

Avena sativa Oat

Brassica napus Rape

Capsicum annuum Cayenne

pepper

Carum carvi Caraway

Ceratonia siliqua St John's bread

Conium maculatum Poison henlock

Coriandrum sativum Coriander

Crocus sativus Saffron

Cuminum_cyminum Cumin

Curcuma longa Turmeric

Elettaria cardamomum Cardamom

Foeniculum vulgare Sweet fennel

Glycine max Soybean

Hordeum vulgare Barley

Juniperus_communis Juniper


Allium schoenoprasum Wild chives

Allium_sativum Garlic

Anethum graveolens Dill

Argemone sp Prickly poppy

PLANTS SPECIES (SPICES/CEREALS) RESULTS


• Species mix: up to 5 Spiked level: 1%, 5%, 10%, 20%

98.9

Plant species

positive negative

97.1

2.9

Plant species spiked 1%

positive negative

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Trisopterus luscus Norway pout

Cynoglossus senegalensis Witch flounder

Oncorhynchus

chrysogaster

Pink salmon

Lophius piscatorius Angler

Oncorhynchus nerka Sockeye salmon

Pangasianodon

hypophthalmus

Silver carp

Scomber scombrus Atlantic mackerel

Oncorhynchus gorbuscha Pink salmon

Merluccius hubbsi Argentine hake

Merluccius productus North Pacific hake

Macruronus magellanicus Patagonian grenadier

Merluccius gayi South Pacific hake

Thunnus obesus Bigeye tuna


Salmo salar Atlantic Salmon

Thunnus albacares Yellowfin tuna

Gadus morhua Atlantic cod

Hippoglossus hippoglossus Pacific halibut

Limanda limanda Common dab

Merluccius merluccius European hake

Melanogrammus aeglefinus Haddock

Katsuwonus pelamis Skipjack tuna

Thunnus alalunga Albacore

Pleuronectes platessa European plaice

Molva molva Ling

Sander lucioperca Pike-perch

Pollachius pollachius Pollack

FISH SPECIES RESULTS


• Species mix: up to 2 Spiked level: 1%, 2%, 5%, 10%

94.4

5.6

Fish species >2% spiked

positive negative

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• The NGS workflow identified all of the species tested individually

• Identification of pure material at spiked levels higher than 5% was successfully

obtained for more than 94% of the samples tested

• At the level of 1% spiking, more that 95% of the samples were correctly assigned

to the species name

• When combined, meat, plants and fish species could be analyzed simultaneously

in a single NGS run which significantly reduces the NGS costs

• Extension of the databases is an on-going activity to include additional species

with various collaborators

Conclusions

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• Retailer asked for NGS

spices in Curry mixture

(containing more than 10

spices according to label)

• All the spices except

laurel (scientifica name

Laurus nobilis) could be

detected

• NGS spices was

performed and the

majority of the sample (>

90%) was composed by a

very similar plant of the

laurel family but not

Laurus nobilis (laurel) -

potentially a toxic plant

• The supplier was mixing

in the curry preparation

not Laurel but something

else similar to Laurel but

with less than 10% of true

laurel

Real-Time Response Case Study: Laurel raw material

Rapid ResponseHours to days to sequence sample, and

provide accurate analysis back

Customer SolutionSpecific for Plant Species ID testing in

food

SITUATION RESPONSE VALUE DELIVERED

Increased profitabilityFast and accurate analysis for prevention

of intentional adulteration of food supply

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• Retailer asked for grouper

identification in a fish fillet

that should be a single

grouper species. Therefore

no NGS was as performed

because the sample was a

fillet (single species

product)

• Identification through

Sanger sequencing that is

appropriate for single

species products originated

a mixture of DNA

sequences and no

identification results

• NGS was suggested to

the retailer

• NGS revealed 4 different

fish species and some of

them not included on the

list of authorized fish

species to be

commercialized – one of

the species toxic

• Supplier after knowing the

NGS results confirmed

that in spite of the fillet

format of the product it

was not a true fillet but

processed fish sample

with a fillet format

Real-Time Response Case Study: Grouper fillet

Rapid ResponseHours to days to sequence sample, and

provide accurate analysis back

Customer SolutionSpecific for Fish Species ID testing in

food

Scientific ExpertiseHighly skilled and supportive scientists

dedicated to food safety and integrity

SITUATION RESPONSE VALUE DELIVERED

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Investigate more….

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Appendix

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Ion Torrent Technology

• Ion torrent chips contain

millions of microscopic wells

• DNA is copied onto beads,

then loaded into wells

• DNA on bead is single

stranded, sequential flow of

nucleotides over chip builds

complement strand

• Each extension releases H+,

measured per well and

translated into sequence

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Plug-and-Play Reagents on Ion GeneStudio S5 Series Systems


Large HD

touch

screen

Temperature-controlled

chip clamp

Sequencing reagent

cartridgeWash solution and

waste cartridge(behind W2 solution)

Automated

cleaning

solution

Standard power

Gas

Water

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• Output: DNA sequences

• Each DNA molecule is sequenced (ATCGTGTA…..)

• Millions of DNA molecules are sequenced individually and simultaneously

• Detect all different DNA in a sample

• Provides identification when compared with sequence databases

• Thousands of species are included in the databases

• Identifies content species in food products

• Untargeted approach

DNA Barcodes for Food Authenticity

Next Generation Sequencing for Food Authenticity ...Simplest NGS workflow for targeted sequencing...

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