Ion Torrent infectious diseases NGS Solution Titelstijl van ......Ion PGM and Ridom SeqSphere+ Software CompeteComplete: A complete solution for bacterial typing research Fast: less

Titelstijl van model bewerken Ion Torrent infectious diseases NGS Solution

Mathieu Boimard

September 28th 2016

Titelstijl van model bewerken

Pathogens impact Human Health

Women’s

Health

Antibiotic

Resistance Gastric Ulcers

Respiratory Tuberculosis Outbreaks


Genomic Research Applications

Microbes and

Viruses


Ion Torrent Infectious Disease Research Menu

Whole genome

sequencing

Ion Xpress™ Plus

Fragment Library Kit

Thermo Scientific™

MuSeek Library

Preparation Kit

Applied Biosystems™

Library Builder™

System

16S rRNA sequencing

metagenomics

Ion 16S™ Metagenomics Kit

Ion Reporter

Software

Metagenomics

Workflow

Ion AmpliSeq™

Panels

TB Research Panel

Tuberculosis

Ebola Research Panel

Learn more at thermofisher.com/microbialgenomics

Custom Panel


Whole genome sequencing


Ion Torrent Microbial Sequencing Workflow

Construct

library

Prepare

template

Run

sequence Analyze data

Ion Xpress Plus

Fragment Library

Kit

Ion Chef System or

Ion OneTouch 2

System

Ion S5 or S5XL

Systems

Torrent Suite

Software

Microbial Sequencing De novo assembly

and secondary

analysis

Ion Xpress Plus

3 hours

Ion OneTouch 2

5 hours

Run time

4 hours

Analysis time

8/17.5 hours Ion Chef

11 hours

Titelstijl van model bewerken Ion Xpress Plus Fragment

Ligate adapters and nick translate

P B

P B

P B

P B

P B

P B

A


Diagenode

Bioruptor™

Device

Library Options

Fragment

Library

Methods

Barcodes

Size Selection

Ion Xpress Plus

Fragment

Library Kit

Ion Plus

Fragment

Library Kit

Library

Builder

System

13 libraries in 2.5 hours

Ion Xpress™ DNA Barcode

Adapters 1–96

Ion Xpress™ RNA-Seq Barcode 1–16 Kit

Sage Science Pippin

Prep™ System

Covaris™ M220

MuSeek

Library

Preparation

Kit

Invitrogen™ E-Gel™ SizeSelect™ Gels


Ion S5

sequencing Data mapping

De novo

assembly Typing

Whole genome

(de novo) Basecalling

Create contigs and

assemblies

Gene by gene

comparison

Ion Xpress Plus

Fragment Kit /

MuSeek Library

Preparation Kit / Ion

520 or Ion 530™

Chips

Torrent Suite

Software SPAdes

Ridom™

SeqSphere+

Software

12 hours 8 or 17.5 hours

(S5) A few hours 2–30 minutes

Bacterial Typing Research Workflow


Ion PGM and Ridom SeqSphere+ Software

Compete Complete:

A complete solution for bacterial typing

research

Fast:

less than a day

Accurate:

For needs to perform more

laborious research applications

User friendly:

No scripting or bioinformatician skills

needed


Typing and detection of antimicrobial resistance directly from urine using Whole-genome-sequencing

• Epidemiological genotyping and antimicrobial resistance (AMR) markers were determined directly from urine for Neisseria gonorrhoeae

• Urine was enriched for microbial DNA

• After sequencing, human reads were filtered, sequence was de novo

assembled and analyzed with Ridom SeqSphere software

For research use only. Not for use in diagnostic procedures.


Ion AmpliSeq Panels for Infectious Disease Research


Simple Workflows with Ion AmpliSeq Technology

Ion AmpliSeq technology

Amplify Digest Ligate

Exomes Gene

Panels

RNA

Expression Fusions Community

Panels CNV Infectious

disease

research panels


Ion AmpliSeq technology Workflow

PB PB


Complete Workflow for Targeted Resequencing

• Automated workflow

• Minimum hands-on time

• You can also customize a panel with Ion AmpliSeq Designer

Construct library Prepare template Run sequence Analyze data

Total time: 5.5 hours Total time: 11 hours Total time: 2.5 hours Total time: 5 hours

Ion AmpliSeq

Infectious Disease

Research Panels

Ion Chef System

Ion S5 System

Ion PGM System

Torrent Suite Software


Ion AmpliSeq Research Panels Available

Panel Ion AmpliSeq TB

Research Panel

Ion AmpliSeq Ebola

Research Panel

Organism Mycobacterium tuberculosis Ebola virus (EBOV)

Amplicons 109 150

Pools 2 2

Genes targeted

Antibiotic-resistant genes: embB,

eis, gyrA, inhA, katG, pncA, rpoB,

rpsL

99.49% of the Ebola genome

Input required 10–100 ng DNA 10 ng RNA

Recommended

multiplex

70 per Ion 520 Chip

(1000X coverage)

26 per Ion 520 Chip

(2000X coverage)


Ion AmpliSeq TB Research Panel

Gene Function Major Drug Resistance

embB Membrane indolylacetylinositol

arabinosyltransferase B

Ethambutol

eis Aminoglycoside acetyltransferase Kanamycin

gyrA DNA gyrase subunit A Fluoroquinolone

inhA NADH-dependent enoyl-[acyl-carrier-protein]

reductase

Isoniazid

katG Catalase-peroxidase-peroxynitritase T Isoniazid

pncA Pyrazinamidase/nicotinamidase Pyrazinamide

rpoB DNA-directed RNA polymerase β-chain Rifampicin

rpsL 30S ribosomal protein S12 Streptomycin


Ebola Virus Research

• Whole-genome sequencing on

the virus was done using the

Ion AmpliSeq Ebola

Research Panel

• Data shows genetic similarity

across the archived samples

from 2014

• Ion PGM System and Ion Chef

System installed in a research

laboratory (tent) in Makeni,

Sierra Leone For research use only. Not for use in diagnostic procedures.

Titelstijl van model bewerken • Ion AmpliSeq Designer™ be used for custom primer design

• Microorganism genome can be uploaded as custom reference

• BED file with masked region should be provided (SNP)

• Target regions provided in custom reference coordinates

Ion AmpliSeq™ Designer : Custom Genome


Design results

Titelstijl van model bewerken Species Application

HPV all genotypes Epidemiology, Cancer research

HPV-16 Epidemiology, Cancer research

Hep-A virus Whole genome to detect food poisoning

Cytomegalovirus Drug resistance

PPRV Epidemiology, vaccine development

H. Pylori Submucosal drug sensitivity

Pan-Pathogen design Identification of pathogens

Antimicrobial resistance genes Detection of ABR genes

Custom Ion AmpliSeq

These AmpliSeq panels are all custom made (no launched products)


Pan-bacterial detection using Ion AmpliSeq

• Proof of concept to detect

microbes in a background of

complex DNA

• 467 primer pairs for species-

level detection of Bacillus

anthracis, Yersinia

pestis, Francisella tularensis,

and Burkholderia pseudomallei

• Effective identification of

microbial genomic material

present in as little as 10 copies

per ml



Metagenomics Research


Metagenomics Research

Identification of mixed populations in

environmental samples

Research of the human microbiome

Research on correlation of

microbiome and human health


Complementary Research Solutions: Microbial ID

Ion 16S Metagenomics Kit

on Ion S5/S5XL Systems

Rapid ID Species ID

• Population analysis

• Sample pooling

• Relative enumeration

• Genus to species ID

• Can combine with Fungal/Viral ID

• Ideal for limited samples

• Medium to high throughput

• Verified reference databases

• Long read lengths for species level ID

• Complete solution for QC

microbiology labs

Applied Biosystems™ MicroSEQ™ ID

on capillary electrophoresis


Ion 16S Metagenomics Kit

Primer Sets

• Set V2-4-8

• Set V3-6, 7-9

Master Mix

• Environmental

Master Mix

v2.0

Buffers

• DNA Dilution

Buffer

• Negative

Control Water

Control DNA

• E. coli

control DNA


Sequence

Ion S5 and S5XL

Systems

Rapid Metagenomics Ion 16S Workflow

Sample Analyze

• Automated analysis from data generation to species identification

• Simple setup, analysis, interpretation

• Two solutions (local/hosted) depending on your need

Ion 16S

Metagenomics

Kit

Tube 1: V2, V4, V8

Tube 2: V3, V6-7, V9

Ion Reporter

Software

Your lab

Titelstijl van model bewerken Ion Reporter Software leverages a two-step mapping

process against manually-curated and public content

High Quality Reference Content Included

1. Curated MicroSEQ ID 2. Curated GreenGenes

• Curation

• Full-length 16S

sequences

• >9,000 organisms

• Manually curated for

• Sequence quality

• Length

• Annotation

• Phylogeny

• Taxonomy updates

• Public 16S data resource

• 1.2 million sequences

• Minimal curation

• Over 400,000 organisms

after Ion curation

process


Research Study of NGS-based 16S rRNA Profiling Identified More Bacteria Compared with Culture Method


• Detection of polymicrobial infections in retrospective samples from

bacterial brain abscesses

• Sequencing was done directly on samples (no culture)

• 16S rRNA profiling results were compared to culture and Sanger

sequencing-based detection

• Triple the number of bacteria identified compared to Sanger/culture


© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are

the property of Thermo Fisher Scientific and its subsidiaries unless otherwise

specified. Covaris™ M220 is a trademark of Covaris Inc.

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

Documents

Ion Torrent infectious diseases NGS Solution Titelstijl van ......Ion PGM and Ridom SeqSphere+ Software CompeteComplete: A complete solution for bacterial typing research Fast: less