The Ocean Sampling Day's Metagenome Analysis: Standards, Pipelines and First Results

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The Ocean Sampling Day's Metagenome Analysis:

Standards, Pipelines and First Results

Microbial Genomics and Bioinformatics Research GroupRenzo Kottmann

[email protected], 2015-11-18

MAX PLANCK INSTITUTEFOR MARINE MICROBIOLOGY

Investigation of the diversity,structure and distribution ofmicrobial populations through theapproaches based onnucleic acid analyses

Junior Group of Molecular EcologyDr. Rudolf Amann


Common Bioinformatics View on Metagenomics:

Give me a big metagenomic sequence data,I assign gene functions


Data Centric View on Metagenomics

Sketch from Martin Fowler: http://martinfowler.com/articles/bigData/


OSD Sampling

Scientists

Masame/GustaME


A global mega-sequencing campaign

June Solstice 2014/15



MyOSD Citizen Science Project

www.my-osd.org



Key facts > 200 sites participated in OSD

• 4 OSD Pilot Events (Jun/Dec 2012 + Jun/Dec 2013) • 2 OSD Main Events (June 2014 and June 2015)

Participating OSD sites ranged from: • subtropical waters in Hawaii to extreme

environments such as the Fram Strait in the Arctic Ocean

Each main year:• ~150 Metagenomes• ~200 Amplicon (16S/18S) samples

2 Citizen Science Campaigns• MyOSD 2014 and 2015 • ~190 Amplicon (16S/18S) samples


Standards & Data Harvesting

Scientists

Masame/GustaME


Standard Sampling Protocols: OSD Handbook

http://www.microb3.eu/sites/default/files/osd/OSD_Handbook_v2.0.pdf P ten Hoopen et al.

http://www.microb3.eu/sites/default/files/osd/OSD_Handbook_v2.0.pdf

http://www.microb3.eu/sites/default/files/osd/OSD_Handbook_v2.0.pdf


Data Standards: M2B3

Core:• Absolute minimum for Micro-

B3 data sets Time and place Event/sample ID Temperature Salinity Molecular sampling protocol

Recommended:• Remainder of mandatory fields

for existing standards and others

Optional• Useful fields from existing

standards

P ten Hoopen et al. Marine microbial biodiversity, bioinformatics and biotechnology (M2B3) data reporting and service standards

https://scholar.google.de/citations?view_op=view_citation&hl=en&user=uHcm-xEAAAAJ&sortby=pubdate&citation_for_view=uHcm-xEAAAAJ:BmWJbWwHJAwC




Environmental Data

• Air temperature• Water temperature• Salinity• Phosphate• Secchi depth• …


Information System

Curation

Contextual Data


MicroB3 Summer School, May-June 2014

Logsheets


OSD Citizen App

https://itunes.apple.com/us/app/osd-citizen/id834353532?mt=8

https://play.google.com/store/apps/details?id=com.iw.esa

Early, consistent, digital acquisition of environmental data

https://itunes.apple.com/us/app/osd-citizen/id834353532?mt=8

https://play.google.com/store/apps/details?id=com.iw.esa


Sample Registration


Logistics


More than 1000 filters (OSD2014)

High-throughput Visualization of Big Diversity


SAMPLESWith BARCODES

METADATA

SAMPLING

CONTEXTUALDATA

SequencingCentre +

SEQUENCINGDATA

CONTEXTUALDATA & METADATA

SEQUENCINGDATA & METADATA

Ocean Sampling Day: Data Flow Overview

http://www.oceansamplingday.org

http://www.oceansamplingday.org/



Bioinformatics Pipelines

Scientists

Masame/GustaME





Pre-Processing


Sequence Data Pre-Processing Definition: Filter original raw sequence data

• based on well defined sequence quality criteria Goal:

• provide all OSD participants (and the whole scientific community) with a single, quality-controlled dataset

• ensure comparability and repeatability of analysis results.

Scope: • covers both amplicon (i.e. 16/18S rDNA) and

shotgun (i.e. metagenome) data sequenced with Documentation:

http://tinyurl.com/osd-pre-processing





Raw reads

Quality evaluation

Pre-processing

Read-level analysis

Assembly-level analysis

Pipeline View



MetagenomicsAmplicons 16S/18S



Scientists

Masame/GustaME


Open Collaboration All data publically available beFORe analysis Open Analysis Group

• Working on > 40 scientific questions/topics Analysis grouped into 3 topic areas:

1. Diversity(Using OTU-based metrics and alternatives such as MED, UniPept etc.)

2. Insights metabolic functions (with focus on human impact) and their role in the ecosystems from Metagenomes

3. Towards an understanding of broad-scale ecological patterns


Some preliminary Results

By Antonio Fernandez Guerra(MPI Bremen, Oxford University)


MalaspinaGOS TARA OSD

OSD in Context of other mega-sequencing campaigns


Sunagawa, et al., 2015

Most complete view of the Ocean Microbiome

First sampling 2013

243 sites

7.2Tbp


Ocean Microbial-Reference Gene Catalog (OM-RGC) Assembled reads Annotated Clustered to generate

a non-redundant set of reference genes.

Sunagawa et al., http://ocean-microbiome.embl.de/companion.html


OM-RGCIncremental clustering

(cd-hit-2d):~ 7 M

OSD genes present in OM-RGC

After Clustering at 95%:

~3.6 M

OSD genes NOT present in OM-RGC

~4.5 M

Adding OSD to the Ocean Microbiome Reference Gene Catalogue


MalaspinaGOS TARA OSDOSD is 75% Coastal Sampling Day

~50% of OSD genes NOT present in OM-RGC Under-sampling of coasts

• Where 30-40% of human population lives


The Ocean Resistome

(slides left-out, please contact for further Information)


The dark side of the metagenomes

Text

Unravelling the unknown in the metagenomic protein universe


unknownknown

Knowns and unknowns in the metagenomic protein universe

sequences with significant similarity to known protein domains (PFAM)

all the sequences without an assigned function


unknownknownenvironmentalunknown

genomicunknown

Knowns and unknowns in the metagenomic protein universe

sequences with significant similarity to known protein domains (PFAM)

all the sequences without an assigned function


Metagenomic Network Analysis

Cluster1800572 environmental unknown

SAR11_0487 Tryptophan synthase

SAR11_1266 hypothetical protein

SAR11_0686 hypothetical protein

SAR11_1277 aspartate racemase

Discover the unknowns (Global Ocean Survey)



Scientists

Masame/GustaME


Ecological Analysis Tools: Gustame

http://mb3is.megx.net/gustame




Buttigieg & Ramette, 2014


Multivariate AnalysiS Applications for Microbial Ecology (MASAME)

http://mb3is.megx.net/masame/




Summary Ocean Sampling Day

• A great open collaboration, open science community• Sharing data is pre-requisite!

Build on existing infrastructure(s)• Only added missing components• Cover all aspects of studying marine microbiomes

Data gathering Bioinformatics Analysis Archiving/Dissemination Ecological Analysis

Comparative metagenomics which takes into account whole data lifecycle• From sampling to ecological and biotechnological analysis



Thanks for your attention

1st Marine Board Forum: Marine data Challenges: from Observation to Information

http://twitter.com/Micro_B3http://www.oceansamplingday.org

http://twitter.com/Micro_B3



Ecological Traits of Metagenomes

Established Database of calculated traits • Available at http://mb3is.megx.net/mg-traits

PCA of Codon Usage (see http://mb3is.megx.net/mg-traits/traits-summary)

http://mb3is.megx.net/mg-traits

http://mb3is.megx.net/mg-traits

http://mb3is.megx.net/mg-traits/traits-summary

http://mb3is.megx.net/mg-traits/traits-summary
