OMICS, Comics and Metaproteomics - Galaxy-Pgalaxyp.org/wp-content/uploads/2019/01/OmicComics_Pune_PJagta… · For example, Protein Database Downloader downloads ... Sci Rep. (2018)

OMICS, Comics and Metaproteomics:

Analyzing Microbiomes using proteomics

Pratik Jagtap

@ Saturday Meet Programme

DEPARTMENT OF MICROBIOLOGY

SAVITRIBAI PHULE PUNE UNIVERSITY

• Omics & Multiomics: Benefits and Challenges

• Functional microbiome analyses

• Metaproteomics, Galaxy and Projects

• Extending tools for metaproteomics

• Microbiology and Microbiome



https://wildlifesnpits.wordpress.com/2015/11/18/transcriptomics-for-conservation/

Mias and Snyder, Quant Biol. (2013) 1(1): 71–90. doi: [10.1007/s40484-013-0005-3]

https://wildlifesnpits.wordpress.com/2015/11/18/transcriptomics-for-conservation/

Mass Spectrometry and Proteomics

Multiomics / trans-omics

https://doi.org/10.1007/978-1-4939-7717-8_7

Multiomics / trans-omics

https://doi.org/10.1007/978-1-4939-7717-8_7

https://doi.org/10.1007/978-1-4939-7717-8_7

multiomics data: Analytical challenges

• Omics & Multiomics: Benefits and ChallengesMultiomics Research is emerging and will offer a more complete biological perspective. Multiomicsdata analysis presents challenges and opportunities.














http://draxe.com/microbiome/

MICROBIOME RESEARCH

http://draxe.com/microbiome/

Microbiome: Microbial genetic potential and response

Metagenomics: Identifies

species present within complex community (16S rRNA and Whole Genome Sequencing).

DNA from samples. 16S rRNA (economical) or

Shotgun sequencing (expensive).

Multiple studies that correlatetaxonomy with observed phenotype.

Multiple studies have shown correlation of microbial composition with physiological conditions. Also used to study interaction with environment.

tinyurl.com/medgadgetcom

Morgan, Xochitl & Huttenhower, Curtis. (2012). Chapter 12: Human Microbiome Analysis. PLoS computational biology. 8. e1002808. 10.1371/journal.pcbi.1002808.

MICROBIOME RESEARCH

Human Microbiome Project Consortium. Nature. 2012 Jun 13;486(7402):215-21. doi: 10.1038/nature11209.

microbial taxa vary while metabolic pathways

remain stable within a healthy population


• Functional microbiome analysesMultiomics approach in microbiome research has the potential to offer mechanistic insights.













www.revitin.com

Microbiome: Microbial genetic potential and response

Multiple studies have shown correlation of microbial composition with physiological conditions.

Metagenomics: DNA Sequencing identifies species present within complex community (16S rRNA and Whole Genome Sequencing).

Metatranscriptomics: RNA Sequencing identifies species present and possible functions within complex communities (RNASeq).

Metaproteomics: The large-scale characterization of the entire protein complement of environmental microbiota at a given point in time. Potential to unravel the mechanistic details of microbial interactions with host / environment by analyzing the functional dynamics of the microbiome.

Metaproteomics approach goes beyond studies of compositional dynamics and sheds light on mechanistic details of microbial interactions with host / environment.

Bond and Wilmes 2015

“Through the application of metaproteomics to different microbial consortia over the past decade, we have learnt much about key functional traits in the various environmental settings where they occur.” Proteomics. doi:10.1002/pmic.201500183.

Metaproteomics

Bond and Wilmes 2004

“The large-scale characterization of the entire protein complement of environmental microbiota at a given point in time” Environ. Microbiol. 6, 911–920.

Metaproteomic Challenges

SINGLE-ORGANISM

PROTEOMICS

METAPROTEOMICS

SINGLE

SMALL TO MEDIUM SIZE (10 K TO

100K SEQUENCES)

SINGLE + CONTAMINANTS

SEARCH DATABASE- SIZE

- COMPLEXITY

LARGE (1 MILLION AND ABOVE)

MULTI-ORGANISM DATABASE WITH HOMOLOGOUS PROTEINS

• SEARCH ALGORITHMS BEING DEVELOPED TO ADDRESS LARGE AND COMPLEX

DATABASE SEARCHES

• PROTEIN GROUPING AT MULTI-ORGANISM LEVEL

• IDENTIFICATION STATISTICS AFFECTED BY LARGE DATABASES

• TAXONOMY BASED ON UNIQUE PEPTIDE IDENTIFICATIONS

• FUNCTIONAL ANALYSIS BASED ON PROTEINS IDENTIFIED

Disparate tools andmultiple processing

steps.

Solution: Galaxy Platform

Goecks J et al Genome Biol. 2010;11(8):R86.

• A web-based bioinformatics data analysis platform.• Software accessibility and usability. • Share-ability of tools, workflows and histories. • Reproducibility and ability to test and compare results after using multiple parameters.• Ability to assimilate disparate software into integrated workflows.


For example, Protein Database Downloader downloads

UniProt protein FASTA databases of various organisms.

Software tools can be used in a sequential manner to generate analytical workflows that can be reused, shared and creatively modified.

DATABASE

SEARCH

&

STRATEGIES

•

•

DATABASE

GENERATION

FASTQ

Protein / Peptide FASTA

TAXONOMY

ANALYSIS

Unique Peptides

FUNCTIONAL

ANALYSIS

Proteins

Known Function

Peptides

Search Algorithm

Spectra

Hypothetical Function

Unknown Function

Shared TaxonomyUnassigned Taxonomy

Metaproteomics Workflow


Metaproteomic analysis using the Galaxy framework.

Proteomics. (2015) doi:10.1002/pmic.201500074.

• Plaque microcosms were grown from carious lesions in 12 children

• Extensive inter-individual variation in dental plaque composition

Metaproteomics Projects

Sucrose-induced dysbiosis in dental caries.

Rudney et al., BMC Microbiome DOI: 10.1186/s40168-015-0136-z

NS: Absence of sucrose WS: Presence of sucroseP: Plaque samples

• Paired design – microcosms grown in presence/absence of sucrose• Sucrose induces acid production in dental caries (dysbiosis)• 16S rRNA sucrose microcosms retained inter-individual taxonomic variation

RAW data conversionDatabase generation

Database Search against HumanOral Microbiome Database (HOMD)

Convert microbial peptide lists to FASTA format

BLAST-P Analysis (30K to 60K peptides)

MEGAN Analysis

1 1

2

2

3

4

5

6

3

4

5

6




SEED Functional Analysis



• 2-D LC-MS/MS Metaproteome was analyzed for taxonomy and function.


MEGAN5 Taxonomic Analysis

NS: Absence of sucroseWS: Presence of sucrose

• Taxonomically-diverse sucrose-pulsed microcosms clustered more closely by function. Function-based changes may be better indicators of dysbiosis


Clinical Projects

• MICROBIOME OF HUMAN CERVICAL-VAGINAL FLUID was analyzed

after collecting samples using residual Pap test fixatives from a pool of 40

normal samples and 5 individual women with normal cytology.

• Understanding the nature of the microorganisms and the functional role of

the expressed proteins is an important step to develop diagnostic tools for

gynecological conditions and malignancies.

MEGAN Analysis

1190 Microbial Peptides

30 genera. 42 species

Function: 939 peptides assigned to 137

InterPro2GO proteins

Skubitz Lab

MEGAN Analysis

776 Microbial Peptides

53 genera. 71 species

Function: 232 peptides assigned to 107

InterPro2GO proteins

• BRONCHOALVEOLAR LAVAGE FLUID (BALF)

MICROBIOME: Microbiome of fluid proximate to the site of lung

injury – acquired by using a bronchoscopy procedure – was analyzed.

• BALF from subjects with Acute Respiratory Disease (ARDS) patients

and lung injury after Hematopoietic Stem Cell Transplant (HSCT) were

analyzed.

Bhargava Lab

Jagtap P et al BAL Fluid Metaproteome in Acute Respiratory Failure. Am J Respir Cell Mol Biol. (2018) 59(5):648-652. doi: 10.1165/rcmb.2018-0068LE.

Afiuni-Zadeh S et al Evaluating the potential of residual Pap test fluid as a resource for the metaproteomic analysis of the cervical-vaginal microbiome. Sci Rep. (2018) 8(1):10868. doi: 10.1038/s41598-018-29092-4.

SATELLITE Workshop @ ABRF Annual Meeting

March 25 2017San Diego, CA

• https://galaxyproteomics.github.io/abrf2017/

Noble Lab,University of WASeattle, WA

Workshop @ ASMS Annual

MeetingJune 7 2017

Indianapolis, IN

Workshop @ GCC Annual Meeting

June 28 2017Montpellier, France

• https://galaxyproteomics.github.io/asms2017/

• https://galaxyproteomics.github.io/gcc2017/

Metaproteomics Gateway

Gateway on JetStream with documentation, tools & workflowsused at the ABRF workshop

z.umn.edu/metaproteomicsgateway



• Metaproteomics, Galaxy and ProjectsMetaproteomics workflows are available within Galaxy platform and are utilized workflows in research studies. The workflows can be accessed via public instance metaproteomics gateway.












DATABASE

SEARCH

&

STRATEGIES

•

•

DATABASE

GENERATION

FASTQ

Protein / Peptide FASTA

TAXONOMY

ANALYSIS

Unique Peptides

FUNCTIONAL

ANALYSIS

Proteins

Known Function

Peptides

Search Algorithm

Spectra

QUANTITATIVE

ANALYSIS

Spectral counts OR

Intensity data

Hypothetical Function

Unknown Function

Shared TaxonomyUnassigned Taxonomy

Metaproteomics Workflow

metaQuantome: An integrated, quantitative metaproteomics approach reveals

connections between taxonomy and protein function in complex microbiomes

metaQuantome: An integrated, quantitative metaproteomics approach reveals

connections between taxonomy and protein function in complex microbiomes

• Under the high-sucrose conditions, streptococcal sucrose

metabolism results in production of lactate, a stronger acid.

• Fusobacteria lack acid tolerance mechanisms, so they do not

survive well under a regimen of sucrose pulsing.

• Moreover streptococci can then compete more effectively with

fusobacteria for mucin oligosaccharides, which they are also

capable of metabolizing.

• Fusobacterium preferentially metabolizes amino acids.

• When mucin oligosaccharides are present, fusobacteria also can metabolize their

constituent sugars, such as galactose and mannose.

• However, most strains are unable to metabolize sucrose.

• Under those circumstances, the end products of carbohydrate fermentation in NS

conditions are weak acids.

Integrated multi-omics of the human gut microbiome in a case study of familial type 1 diabetes.Heintz-Buschart et al NATURE MICROBIOLOGY 2, 16180 (2016) | DOI: 10.1038/nmicrobiol.2016.180

Microbiome Multi-omic Analysis

FUNCTIONAL QUANTITATION

TAXONOMY-FUNCTION QUANTITATION

MetaPhlAn2

GraphPhlAn KRONA

Taxonomic abundance

Visualization of community structureGene Family

AbundancePathway

Abundance

GO Slim TermAbundance

HUMAnN2

Assembly Graph

Metabolic assignment

Predicted ORFs

Graph2Pep

Peptide Sequences

Protein Sequences

Peptides supported by MS/MS

Search

Graph2Pro

Protein DatabaseFor Metaproteomics

Search

Metatrancriptomics Data

Preprocessing(QC, trimming, dereplication, sorting)

Reads (FASTA)

FastQC, TrimGalore!, VSearch

SortmeRNA

Non-ribosomal RNA/DNA Reads

MEGAHITMetaSPAdes

Contigs

Taxonomy datafor UniPept

Mass spectrometry data

MAGs MaxBin

FragGeneScan

A workflow for integrated metatranscriptomic-metaproteomic informaticsASaiM

Graph2Pro

TAXONOMY QUANTITATION

INTEGRATED MULTI-OMICS

ANALYSIS

Taxonomic Correlation Functional Correlation

Quantitative Correlation Visualization WP3

METATRANSCRIPTOMICS DATA

Quantitative Data

Taxonomic Abundance

Functional Abundance

Protein FASTA

METAPROTEOMICS DATA

Peptides

Taxonomy Analysis

Functional Analysis

Taxonomic Abundance

Functional Abundance

Binning Data

METAGENOMICS DATA

WP1 WP2

Quantitative Data Analysis

Protein FASTABinning Data

Multi‐omic informatics for characterizing microbiomes

and their role in health, disease and environment.

Norwegian Centennial Chair Program




• Extending tools for metaproteomicsMetaproteomics workflows are getting extended to allow quantitative and multiomics analysis.







• Extending tools for metaproteomicsMetaproteomics workflows are getting extended to allow quantitative and multiomics analysis.




Microbiology and Microbiome

http://www.geograph.org.uk/photo/2815842

http://www.microbiome.at/sites/ami/files/images/Microbiome_1_CharisTsevis.jpeg

http://www.geograph.org.uk/photo/2815842

http://www.microbiome.at/sites/ami/files/images/Microbiome_1_CharisTsevis.jpeg

Conclusions

• Multiomics Research is emerging and will offer a more complete biological perspective. Multiomics data analysis presents challenges and opportunities.

• Multiomics approach in microbiome research has the potential to offer mechanistic insights

• Metaproteomics workflows are available within Galaxy platform and are utilized workflows in research studies. The workflows can be accessed via public instance metaproteomics gateway.

• Metaproteomics workflows are getting extended to allow quantitative and multiomics analysis.

Acknowledgements

Funding

galaxyp.org Follow us on: twitter.com/usegalaxyp

The Galaxy-P Team at University of Minnesota

http://twitter.com/usegalaxyp

Minnesota Supercomputing InstituteJames JohnsonThomas McGowanMichael Milligan

Ira CookeMelbourne , Australia

University of Minnesota

Timothy GriffinPraveen KumarCandace GuerreroSubina MehtaAdrian Hegeman (Co-I)Art EschenlauerShane HublerRay SajulgaCaleb EasterlyAndrew Rajczewski

Biologists / collaboratorsLaurie ParkerJoel RudneyManeesh BhargavaAmy SkubitzChris WendtBrian CrookerSteven FriedenbergKevin VikenKristin BoylanMarnie PetersonSomiah AfiuniBrian SandriAlexa PragmanWanda WeberAmy Treeful

Harald Barsnes Marc Vaudel University of Bergen, Norway

University of Freiburg,Freiburg, Germany

VIB, UGhent, Belgium

Judson HerveyNaval Research InstituteWashington, D.C.

Matt ChambersNashville, TN

Alessandro TancaPorto Conte Ricerche, Italy

Carolin KolmederUniversity of Helsinki, Finland

Thilo MuthBernhard RenardRobert Koch Institut

Thomas DoakJeremy Fisher Indiana University

Josh EliasStanford University

Brook NunnU of Washington

Lennart Martens (Co-I)Bart MesuereRobbert G Singh

Bjoern GrueningBérénice Batut

Lloyd Smith (Co-I)Michael ShortreedUW-Madison

Karen ReddyMo HeydarianJohns Hopkins University

Anamika KrishanpalPriyabrata PanigrahiPersistent Systems Limited

Stephan KangIntero Life Sciences

galaxyp.org

FundingACKNOWLEDGMENTS

Magnus Øverlie Arntzen (Co-I)Francesco DeloguNMBU,Oslo, Norway

Documents

OMICS, Comics and Metaproteomics - Galaxy-Pgalaxyp.org/wp-content/uploads/2019/01/OmicComics_Pune_PJagta… · For example, Protein Database Downloader downloads ... Sci Rep. (2018)