Understanding bacterial diversity with
metagenomics 16S sequencing and Ion
Reporter™ Software
Xosé M Fernández
Bioinformatics Consultant – Thermo Fisher Scientific
The world leader in serving science
Overview
• Microbial Field Identification
• Rapid Metagenomics Workflow
• Ion Reporter™ Metagenomics Algorithm
16S rRNA
• Component of 30S small
subunit of prokaryotic
ribosome
• Approximately 1,500bp
• Contains 9 variable regions
flanked by conserved regions
• Used in Tree of Life project
Primer sets with multiple amplicons
Set V2-4-8:
Set V3-6-9:
- Primers to amplify variable
regions 2, 4 and 8 in a single
tube
- Primers to amplify variable
regions 3, 6 and 9 in a single
tube
The library will contain three types
of DNA fragments : 250, 288, 295 bp
107a
520*
323b
V2
357a
V3
757a
V4
538*b
The library will contain three types
of DNA fragments : 215, 260, 209 bp
937b
1060a
V5
808
V67
980a
1294b
V8
1197b
V9
1355a
1503b
Rapid Metagenomics 16S Workflow
PCR Ion 16S
Metagenomics Kit
Library Prep
Template Prep
• DNA dilution Buffer
• AmpliTaq Gold® 360
MM
• Ion PGM™ Template
OT2 400 Kit
• 16S Primer Set V2-4-8
(10X)
• Agencourt®AMPure®
XP Reagent
• Ion PGM™ enrichment
beads
• 16S Primer Set V3-6,79 (10X)
• Agilent High
Sensitivity DNA kit
• Negative Control
water
• Ion Plus Fragment
Library kit
• E. coli DNA control
(30 µg/mL)
• Ion Xpress™ Barcode
Adapters 1-16 kit
Sequencing
• Ion 314™, Ion
316™, or Ion
318™ v2 Chip kit
• Ion PGM™
Sequencing 400
Kit
Analysis
• Torrent Suite™
Software v4.x
• Ion Reporter™ 16S
Metagenomics
Workflow
• Ion Library
Quantitation kit
• Direct integration with the sequencer
• Automated analysis from data generation to species identification
• Simple setup, analysis, interpretation
• Two solutions (local / hosted) optimised to fit your needs
Ion Reporter™ Metagenomics 16S Algorithm
Ion Reporter™ Metagenomics 16S Algorithm
Ion Reporter™ Metagenomics 16S Algorithm
Ion Reporter™ Metagenomics 16S Algorithm
blastn -task megablast -evalue 0.01 -query input01.fasta -db blastDBfile –out result01.txt -max_target_seqs 100 outfmt 6 evalue pident length qstart qend sstart send qseqid stitle
Best-in-class Reference Content Included
1. MicroSEQ® ID
•
•
•
•
Trusted gold-standard
Full length 16S sequences
> 15,000 Organisms
Manually curated for
• Sequence quality
• Length
• Annotation
• Phylogeny
• Taxonomy updates
2. GreenGenes
•
•
•
•
Public 16S data resource
Commonly used in 16S studies
Minimal curation
Over 1.2 million entries
Ion Reporter™ software leverages a two-step mapping process to against manuallycurated and public content
Ion Reporter™ Metagenomics 16S Algorithm
Ion Reporter™ Metagenomics 16S Algorithm
Preprocess
Map / Classify
(Two-step mapping approach)
Produce
FASTA-files for
all unique reads
BAM
-files
Remove and
count all short
reads
Curated MicroSEQ™
BLAST with DB,
results in new
set of result
FASTA-files
Add to unique
read hash table
Good
ID?
Yes
Place read (and
count) in
hierarchy hash
No
Cut with primers
User
settings
Mark read with
level ID for
hierarchy
placement
Check each
result using
CLSI guidelines
If ID level, or
percent ID is too
low, mark read
for second step
mapping, and
BLAST again
CLSI = Clinical and Laboratory Standards Institute
Species ID: 99%
Genus ID: 97%
GreenGenes
How Many Samples Per Chip?
Chip Type
Sample Complexity
Low (10)
Medium (20)*
High (>30)
Ion 314™ Chip
4
2
1
Ion 316™ Chip
20
10
2
Ion 318™ Chip
40
20
4
*Targeting about 350,000 reads per sample; the complexity and
sensitivity will impact the true number of samples
Ion Reporter™ Software
Ion Reporter™ Software
The functionality you need for discovery and translational research
Integration w/ TS
Copy Number Analysis
Annotation Content
• Select Ion Reporter™
workflows directly from
within Torrent Suite™
Software
• Detect CNVs in your Ion
AmpliSeq™ pairedsample workflows
• Rich annotation content
integrated (dbSNP, DrugBank,
ClinVar, and more) or import
custom annotations
Variant Detection
Aneuploidy Workflow
Filter Variants
• Quickly identify somatic or
germline SNP, indels, and
CNVs with one assay and
one workflow
• Detect large chromosomal
abnormalities from low-pass
whole genome sequencing
(0.01x)
• Quickly filter variants to
find those that are
biologically relevant
16S Metagenomics
Broad’s IGV
Data Security
• Taxonomic classification of
your 16S samples
• Interactive taxonomy
visualisation
• One click access to data
visualisation (SNPs, Indels,
CNVs, etc.)
• Customised karyotype
view
• Role-based logins control
access to data
• Audit logs monitor who
does what / when
For Research Use Only. Not for use in diagnostic procedures
Ion Reporter™ Software
Sequence
Import
>
Include Ion Reporter™ Software with
in your metagenomics sequencing
template in Torrent Suite™ Software
Torrent Suite™ software securely
pushes your data into Ion
Reporter™ Software and kicks off
analysis workflow
Ion Reporter™ Software
Simplifying your path to results
Sequence
Import
>
Analyse
>
Select a workflow (if not done
in Torrent Suite™ software),
add samples, and hit go
Identify organisms and
visualize with interactive pie
charts
Ion Reporter™ Software
Simplifying your path to results
Sequence
Import
>
Analyse
>
Visualize and explore your data
via interactive pie-charts
powered by Krona
View population diversity at any
level in the taxonomy and
export images for publication
Ion Reporter™ Software
Simplifying your path to results
Sequence
Import
>
Analyse
>
Identify organisms present in
your sample as genus /
species level
Along with each organism Ion
Reporter™ Software also
provides
• Primer information
• Classification information
• % ID
• Mapping information
Ion Reporter™ Software
Sequence
Import
>
Share data with
anyone just by
entering an e-mail
address and they
get a data download
link. Ion Reporter™
users will also see
the analysis in the
Ion Reporter™
interface
Analyse
>
Share
>
16S Mock Community Validation
• Mock Community DNA samples (BEI)
• 20 known species
www.beiresources.org
• Gold standard in field
Name
Detected
Name
Detected
Acinetobacter baumannii
✓
Listeria monocytogenes
✓
Actinomyces odontolyticus
✓
Neisseria meningitidis
✓
Bacillus cereus
✓
Propionibacterium acnes
✓
Bacteroides vulgatus
✓
Pseudomonas aeruginosa
✓
Clostridium beijerinckii
✓
Rhodobacter sphaeroides
✓
Deinococcus radiodurans
✓
Staphylococcus aureus
✓
Enterococcus faecalis
✓
Staphylococcus epidermidis
✓
Escherichia coli
✓
Streptococcus agalactiae
✓
Helicobacter pylori
✓
Streptococcus mutans
✓
Lactobacillus gasseri
✓
Streptococcus pneumoniae
✓
Combined 16S Mock Community Profile
20. Streptococcus
pneumoniae
19.Streptococcus mutans
3%
7%
18.Streptococcus
agalactiae
6%
1.Acinetobacter
baumannii,
5%
2.Actinomyces
odontolyticus,
2%
3.Bacillus
cereus
6%
17.Staphylococcus
epidermidis
5%
4.Bacteroides
vulgatus
8%
5.Clostridium beijerinckii
5%
16.Staphylococcus aureus
8%
15.Rhodobacter
sphaeroides
2%
14.Pseudomonas
aeruginosa
5%
13.Propionibacterium
acnes
1%
6.Deinococcus radiodurans
3%
7.Enterococcus faecalis
5%
8.Escherichia coli
4%
9.Helicobacter pylori
12.Neisseria meningitidis
5%
4%
11.Listeria monocytogenes 10.Lactobacillus gasseri
8%
7%
16S Mock Community – Consensus Results
16S Mock Community – Consensus Results
All Species Detected by at least One Primer Set
Number of full-length reads found
1.Acinetobacter baumannii,
2.Actinomyces odontolyticus,
3.Bacillus cereus
4.Bacteroides vulgatus
5.Clostridium beijerinckii
6.Deinococcus radiodurans
7.Enterococcus faecalis
8.Escherichia coli
9.Helicobacter pylori
10.Lactobacillus gasseri
11.Listeria monocytogenes
12.Neisseria meningitidis
13.Propionibacterium acnes
14.Pseudomonas aeruginosa
15.Rhodobacter sphaeroides
16.Staphylococcus aureus
17.Staphylococcus epidermidis
18.Streptococcus agalactiae
19.Streptococcus mutans
20. Streptococcus pneumoniae
V2
V4
V8
4052
0
4223
4629
5166
4153
2807
2637
3429
7367
2573
3336
1070
3019
893
2668
3681
2036
3031
1002
2812
739
4827
5454
5232
503
2947
2085
3772
4416
3562
3386
262
2767
1739
2149
451
205
0
865
310
865
1310
144
0
1702
1740
1582
1560
1569
V3
6903
81
7162
8062
7391
0
6489
3474
7348
4458
7463
5104
54
6978
3325
8594
10154 588 9662
5866 3908 5467
5587 3232 9171
2074 1586 2294
V67
V9
234 3370
8243
0
7261
0
13845
0
150
0
4870
0
5564
0
678 4815
5617
0
11253
0
13677
0
0
0
2011
0
4027
0
1793
0
7468
0
6619
0
6323
0
7493
13
4362
0
Total number of reads
19,520
9,514
23,678
31,990
18,804
9,836
18,672
14,999
20,310
27,494
28,977
13,566
4,979
18,351
9,319
29,472
19,962
23,600
28,527
11,318
Shown number of full-length reads. Green – perfect match to the primers; blue – mismatches under the primers; yellow –
perfect match of the primers, low number of full-length reads. Red border shows cases of good detection with partially
matched primers. E.coli has perfect match to all primer sets
16S Mock Community – Results by primer
http://ioncommunity.lifetechnologies.com
>2,000
500
>23,000
Nothing gets better faster – than your publications
Harness the fastest, most scalable and affordable NGS technology
Publications
500
450
400
350
300
250
200
150
100
50
0
Q1
Q2
Q3
2012
Q4
Q1
Q2
Q3
2013
Q4
Q1
2014
Ion Torrent™ publications by application
Microbial Sequencing
Targeted DNA Sequencing
Metagenomics
Mitochondrial
Targeted RNA Sequencing
Gene Expression Profiling
Bacterial Typing
Small RNA
Exome
Transcriptome
Bisulfite Sequencing
Viral Typing
ChIP-seq
Aneuploidy
16S
16SrRNA
rRNAProfiling
profiling
AmpliSeq
Ion AmpliSeq™
Publications
Publications
0
50
100
150
200
Microbial communities of CF sputum samples
PLoS One. 2013; 8(5): e65226
Start sequencing now at
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For Research Use Only. Not for use in diagnostic procedures.
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