Post on 13-Oct-2020
Exact Amplicon Sequence Variants
BIOME 2018
Diversity of Life
Image: Norman Pace
A Molecular CensusMetabarcoding or Marker-gene Sequencing
A Microbial Census
ATTAACGAGATTATAACCAGAGTACGAATACCGAACCACGATTCACAAGGTACCACAAGGTAACATAGCTCCATTAACCCCTTATAACCAGAGTACGAATACCGAACAATTAACGAGATTATAACCAGAGAGAGAATACCGAACCACGATTCTTGTGGTACCACAAGGTAACATAGCTCCCACGATTCACAAGGTACCACAAGGTAACATAGCTCCGGGAACTACAATCTCTAAGGTGAAGTCTCAGTCTATATTAACGAGATTATAACCAGAGTACGAATACCGAACCACGATTCACAAGGTACCACAAGGTAACATAGCTCCATTAACGAGATTATAACCAGAGTACGAATACCGAAC
Metabarcoding or Marker-gene Sequencing
A Microbial Census
ATTAACGAGATTATAACCAGAGTACGAATACCGAACCACGATTCACAAGGTACCACAAGGTAACATAGCTCCATTAACCCCTTATAACCAGAGTACGAATACCGAACAATTAACGAGATTATAACCAGAGAGAGAATACCGAACCACGATTCTTGTGGTACCACAAGGTAACATAGCTCCCACGATTCACAAGGTACCACAAGGTAACATAGCTCCGGGAACTACAATCTCTAAGGTGAAGTCTCAGTCTATATTAACGAGATTATAACCAGAGTACGAATACCGAACCACGATTCACAAGGTACCACAAGGTAACATAGCTCCATTAACGAGATTATAACCAGAGTACGAATACCGAAC
Lactobacillus crispatus 1300 5 0 882 596
Ureaplasma urealytica 15 0 220 0 0
Gardnerella vaginalis 22 0 1 0 412
Prevotella intermedia 0 0 8 12 0
... ... ... ... ... ...
Metabarcoding or Marker-gene Sequencing
A Microbial Census
ATTAACGAGATTATAACCAGAGTACGAATACCGAACCACGATTCACAAGGTACCACAAGGTAACATAGCTCCATTAACCCCTTATAACCAGAGTACGAATACCGAACAATTAACGAGATTATAACCAGAGAGAGAATACCGAACCACGATTCTTGTGGTACCACAAGGTAACATAGCTCCCACGATTCACAAGGTACCACAAGGTAACATAGCTCCGGGAACTACAATCTCTAAGGTGAAGTCTCAGTCTATATTAACGAGATTATAACCAGAGTACGAATACCGAACCACGATTCACAAGGTACCACAAGGTAACATAGCTCCATTAACGAGATTATAACCAGAGTACGAATACCGAAC
Lactobacillus crispatus 1300 5 0 882 596
Ureaplasma urealytica 15 0 220 0 0
Gardnerella vaginalis 22 0 1 0 412
Prevotella intermedia 0 0 8 12 0
... ... ... ... ... ...
Inference
ExplorationVisualization
Metabarcoding or Marker-gene Sequencing
The MGS Microscope
The MGS Microscope
• sample collection • sample storage • DNA extraction • library preparation • PCR • sequencing instrument • bioinformatics
Marker-gene Sequencing2,000,000 bp300 bp
2,000,000 bp300 bp
1
5
10
15
12,000,005
12,000,010
Marker-gene Sequencing
2,000,000 bp300 bp
Thousands to Billions of “Barcodes”
1
5
10
15
12,000,005
12,000,010
Marker-gene Sequencing
Callahan, et al. Nature Methods, 2016.
PCR/sequencing
SampleSequences
AmpliconReads
Callahan, et al. Nature Methods, 2016.
PCR/sequencing
SampleSequences
AmpliconReads
Pick OTUs
OperationalTaxonomic Units
Callahan, et al. Nature Methods, 2016.
PCR/sequencing
SampleSequences
AmpliconReads
PCR/sequencing
SampleSequences
AmpliconReads
Callahan, et al. Nature Methods, 2016.
PCR/sequencing
SampleSequences
AmpliconReads
Callahan, et al. Nature Methods, 2016.
Pick OTUs
Uh oh!
Different OperationalTaxonomic Units
OTU85 is not a consistent label
Exact Sequence Variants…
OTU85 is predictive of a disease? Not in future data!
OTU85 is associated w/ X and Y? Can’t be tested!
OTU85 is in this community? OTUs don’t exist in nature!
McLaren & Callahan, mBio, 2018.
Exact Sequence Variants…
Different OperationalTaxonomic Units
PCR/sequencing
SampleSequences
AmpliconReads
Callahan, et al. Nature Methods, 2016.
Pick OTUs
Different OperationalTaxonomic Units
PCR/sequencing
SampleSequences
AmpliconReads
Callahan, et al. Nature Methods, 2016.
Pick OTUs
Infer Sample Sequences
Exact Sequence Variants…
OTU85 is not a consistent label, but…
ATTAACGAGATTATAACCAGAGTACGAATA…
is consistent!
Sequence Tables
Study 1
Study 2
Study 3
Cross-study comparisonmerge
Eliminates need for joint reprocessing of raw data.
Callahan, et al. ISMEJ, 2017.
…the sequence is the label
Sequence Tables
Study 1
Study 2
Study 3
Cross-study comparisonmerge
Eliminates need for joint reprocessing of raw data.Continuous data integration. Unlimited dataset size.
Callahan, et al. ISMEJ, 2017.
…the sequence is the label
Sequence Tables
Study 1
Study 2
Study 3
Cross-study comparisonmerge
Eliminates need for joint reprocessing of raw data.Continuous data integration. Unlimited dataset size.You in 2020 can work directly with you today.
Callahan, et al. ISMEJ, 2017.
…the sequence is the label
…the sequence is the labelD
ata
Your dataset
Biology
In re
fere
nce
data
base
De Novo Closed-Reference Exact SVs
Valid
Invalid
Callahan, et al. ISMEJ, 2017.
“Replacing OTUs with ASVs makes marker-gene sequencing more precise, reusable, reproducible and comprehensive.”
A rose by another name…
Exact Sequence Variants (ESVs) - Callahan et al. 2017 (by accident)
Amplicon Sequence Variants (ASVs) - Needham et al. 2017 - Callahan et al. 2017
sub-OTUs (sOTUs) - Amir et al. 2017
Zero radius OTUs (zOTUs) - Edgar 2017
Haplotypes, oligotypes, …
A rose by another name…
Exact Sequence Variants (ESVs) - Callahan et al. 2017 (by accident)
Amplicon Sequence Variants (ASVs) - Needham et al. 2017 - Callahan et al. 2017
sub-OTUs (sOTUs) - Amir et al. 2017
Zero radius OTUs (zOTUs) - Edgar 2017
All the same thing! All the same (qualitative) benefits!
Haplotypes, oligotypes, …
Exact Sequence Variants…
PreciseTractableReproducibleComprehensiveCallahan, et al. ISMEJ, 2017.
Exact Sequence Variants…
PreciseTractableReproducibleComprehensiveCallahan, et al. ISMEJ, 2017.
… are replacing OTUs
PreciseTractableReproducibleComprehensive
“the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale…for many sample types, especially plant-associated and free-living communities, one-third of reads or more could not be mapped to [closed-reference OTUs]…Because exact sequences are stable identifiers, unlike OTUs, they can be compared to any 16S rRNA or genomic database now and in the future, thereby promoting reusability [22]… An advantage of using exact sequences is that they enable us to observe and analyse microbial distribution patterns at finer resolution than is possible with traditional OTUs…”
Callahan, et al. ISMEJ, 2017.
Preterm birth is the leading cause of child mortality in the US and worldwide.
Intrauterine infection is the most common mechanism, estimated to occur in 25-40% of cases. This may be an underestimate.
Bacterial vaginosis is a known risk factor (2-4x). Perhaps periodontitis as well.
Potential routes of intrauterine infection
Ascendingroute, nativemicrobiota
Resolution: Preterm Birth
Goldberg, et al. Lancet, 2006.
Resolution: Preterm Birth
Gardnerella
0.01
1.00
0.01 1.00
P−Value (Stanford)
P−V
alu
e (
UA
B)
Resolution
Genus/OTU
Sequence Variant
Frequency
0.1%
1%
10%
Callahan et al. PNAS, 2017.
Resolution: Preterm Birth
G1
G2
G3
Gardnerella
0.01
1.00
0.01 1.00
P−Value (Stanford)
P−V
alu
e (
UA
B)
Resolution
Genus/OTU
Sequence Variant
Frequency
0.1%
1%
10%
Callahan et al. PNAS, 2017.
Resolution: Preterm Birth
00703C2mash
00703Bmash
5515241V 1400E
HMP9231
0288E
14019
75712
315−A
101
00703Dmash6119V5409−055−1
AMD
6420B
G1
G2
G3
Tree from whole genomes
Callahan et al. PNAS, 2017.
=? Reproducible Measurements