Microbial communities contribute to most organisms in our environment and have greater capacity to transform the world around. However, a large number of them remain uncultivable. Identification and functional validation of the uncultured microbial communities in environmental samples remains a challenging task. Metagenomics has become one of the indispensable tools for studying the diversity and function of these uncultured microbes. Our sample has been collected from a public toilet of an undeveloped area in North Coimbatore District, Tamil Nadu, INDIA. Waste effluents from this public toilet were used to generate electricity for the area using Microbial Fuel Cell (MFC) which involves bio-film formation which helps in electron transfer. Our study aims in identification of the diverse microbial community present in urine sample and to functionally annotate the gene responsible for biofilm formation aiding in electron transfer process. As metagenomic studies are becoming widespread, a large number of computational tools and a wide range of bioinformatics pipelines have been developed for metagenomic assembly, taxonomic and functional annotation. Choosing an appropriate tool is crucial and requires validation. In this present study, a range of bioinformatics tools were used quantify the sequence reads, taxonomic classification of the microbial population in the urine sample, assembly of the mixed sequence reads of multiple species and to attain functional annotation of the hypothetical protein.
REFERENCES
1.
Sato
, K.
, & Sakakibara
, Y.
(2015
). MetaVelvet-SL: an extension of the Velvet assembler to a de novo metagenomic assembler utilizing supervised learning
. DNA research
, 22
(1
), 69
–77
.2.
Alshalchi
, S. A.
, & Anderson
, G. G.
(2015
). Expression of the lipopolysaccharide biosynthesis gene lpxD affects biofilm formation of Pseudomonas aeruginosa
. Archives of microbiology
, 197
(2
), 135
–145
.3.
Altschul
, S. F.
, Gish
, W.
, Miller
, W.
, Myers
, E. W.
, & Lipman
, D. J.
(1990
). Basic local alignment search tool
. Journal of molecular biology
, 215
(3
), 403
–410
.4.
Wingett
, S. W.
, & Andrews
, S.
(2018
). FastQ Screen: A tool for multi-genome mapping and quality control
. F1000Research
, 7
.5.
Andrews
, S.
(2010
). FastQC: a quality control tool for high throughput sequence data.
6.
Bateman
, A.
, Birney
, E.
, Cerruti
, L.
, Durbin
, R.
, Etwiller
, L.
, Eddy
, S. R.
, … & Sonnhammer
, E. L.
(2002
). The Pfam protein families database
. Nucleic acids research
, 30
(1
), 276
–280
.7.
Cai
, C. Z.
, Han
, L. Y.
, Ji
, Z. L.
, Chen
, X.
, & Chen
, Y. Z.
(2003
). SVM-Prot: web-based support vector machine software for functional classification of a protein from its primary sequence
. Nucleic acids research
, 31
(13
), 3692
–3697
.8.
Caméléna
, F.
, Pilmis
, B.
, Mollo
, B.
, Hadj
, A.
, Le Monnier
, A.
, & Mizrahi
, A.
(2016
). Infections caused by Tissierella praeacuta: a report of two cases and literature review
. Anaerobe
, 40
, 15
–17
.9.
Cayol
, J. L.
, Ducerf
, S.
, Patel
, B. K.
, Garcia
, J. L.
, Thomas
, P.
, & Ollivier
, B.
(2000
). Thermohalobacter berrensis gen. nov., sp. nov., a thermophilic, strictly halophilic bacterium from a solar saltern
. International journal of systematic and evolutionary microbiology
, 50
(2
), 559
–564
.10.
Charuvaka
, A.
, & Rangwala
, H.
(2011
, December). Evaluation of short read metagenomic assembly
. In BMC genomics
(Vol. 12
, No. 2
, pp. 1
–13
). BioMed Central.11.
National Research Council
. (2007
). The new science of metagenomics: revealing the secrets of our microbial planet
. National Academies Press
.12.
De Filippo
, C.
, Ramazzotti
, M.
, Fontana
, P.
, & Cavalieri
, D.
(2012
). Bioinformatic approaches for functional annotation and pathway inference in metagenomics data
. Briefings in bioinformatics
, 13
(6
), 696
–710
.13.
Fakruddin
, M.
, & Mannan
, K.
(2013
). Methods for analyzing diversity of microbial communities in natural environments
. Ceylon Journal of Science (Biological Sciences)
, 42
(1
).14.
Finn
, R. D.
, Bateman
, A.
, Clements
, J.
, Coggill
, P.
, Eberhardt
, R. Y.
, Eddy
, S. R.
, … & Punta
, M.
(2014
). Pfam: the protein families database
. Nucleic acids research
, 42
(D1
), D222
–D230
.15.
Gasteiger
, E.
, Gattiker
, A.
, Hoogland
, C.
, Ivanyi
, I.
, Appel
, R. D.
, & Bairoch
, A.
(2003
). ExPASy: the proteomics server for in-depth protein knowledge and analysis
. Nucleic acids research
, 31
(13
), 3784
–3788
.16.
Gasteiger
, E.
, Hoogland
, C.
, Gattiker
, A.
, Wilkins
, M. R.
, Appel
, R. D.
, & Bairoch
, A.
(2005
). Protein identification and analysis tools on the ExPASy server
. The proteomics protocols handbook
, 571
–607
.17.
Haas
, B. J.
, Gevers
, D.
, Earl
, A. M.
, Feldgarden
, M.
, Ward
, D. V.
, Giannoukos
, G.
, … & Human Microbiome Consortium
. (2011
). Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons
. Genome research
, 21
(3
), 494
–504
.18.
Handelsman
, J.
(2004
). Metagenomics: application of genomics to uncultured microorganisms
. Microbiology and molecular biology reviews
, 68
(4
), 669
.19.
Hasan
, N. A.
, Chowdhury
, W. B.
, Rahim
, N.
, Sultana
, M.
, Shabnam
, S. A.
, Mai
, V.
, … & Alam
, M.
(2010
). Metagenomic 16S rDNA targeted PCR-DGGE in determining bacterial diversity in aquatic ecosystem
. Bangladesh Journal of Microbiology
, 27
(2
), 46
–50
.20.
Huelsenbeck
, J. P.
, & Ronquist
, F.
(2001
). MRBAYES: Bayesian inference of phylogenetic trees
. Bioinformatics
, 17
(8
), 754
–755
.21.
Huerta-Cepas
, J.
, Szklarczyk
, D.
, Forslund
, K.
, Cook
, H.
, Heller
, D.
, Walter
, M. C.
, … & Bork
, P.
(2016
). eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences
. Nucleic acids research
, 44
(D1
), D286
–D293
.22.
Inbakandan
, D.
, Sriyutha Murthy
, P.
, Venkatesan
, R.
, & Ajmal Khan
, S.
(2010
). 16S rDNA sequence analysis of culturable marine biofilm forming bacteria from a ship’s hull
. Biofouling
, 26
(8
), 893
–899
.23.
Koonin
, E. V.
(2002
). The Clusters of Orthologous Groups (COGs) Database: phylogenetic classification of proteins from complete genomes
. The NCBI Handbook.
24.
Lai
, B.
, Ding
, R.
, Li
, Y.
, Duan
, L.
, & Zhu
, H.
(2012
). A de novo metagenomic assembly program for shotgun DNA reads
. Bioinformatics
, 28
(11
), 1455
–1462
.25.
Lavigne
, R.
, Seto
, D.
, Mahadevan
, P.
, Ackermann
, H. W.
, & Kropinski
, A. M.
(2008
). Unifying classical and molecular taxonomic classification: analysis of the Podoviridae using BLASTP-based tools
. Research in microbiology
, 159
(5
), 406
–414
.26.
Lee
, K. J.
, Lee
, M. A.
, Hwang
, W.
, Park
, H.
, & Lee
, K. H.
(2016
). Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria
. Biofouling
, 32
(7
), 711
–723
.27.
Letunic
, I.
, Goodstadt
, L.
, Dickens
, N. J.
, Doerks
, T.
, Schultz
, J.
, Mott
, R.
, … & Bork
, P.
(2002
). Recent improvements to the SMART domain-based sequence annotation resource
. Nucleic acids research
, 30
(1
), 242
–244
.28.
Li
, Y. H.
, Xu
, J. Y.
, Tao
, L.
, Li
, X. F.
, Li
, S.
, Zeng
, X.
, … & Chen
, Y. Z.
(2016
). SVM-Prot 2016: a webserver for machine learning prediction of protein functional families from sequence irrespective of similarity
. PloS one
, 11
(8
), e0155290
.29.
Liu
, H.
, Ramnarayanan
, R.
, & Logan
, B. E.
(2004
). Production of electricity during wastewater treatment using a single chamber microbial fuel cell
. Environmental science & technology
, 38
(7
), 2281
–2285
.30.
Marchler-Bauer
, A.
, Lu
, S.
, Anderson
, J. B.
, Chitsaz
, F.
, Derbyshire
, M. K.
, DeWeese-Scott
, C.
, … & Bryant
, S. H.
(2010
). CDD: a Conserved Domain Database for the functional annotation of proteins
. Nucleic acids research
, 39
(suppl_1
), D225
–D229
.31.
McGuffin
, L. J.
, Bryson
, K.
, & Jones
, D. T.
(2000
). The PSIPRED protein structure prediction server
. Bioinformatics
, 16
(4
), 404
–405
.32.
Qiu
, Y. Q.
, Tian
, X.
, & Zhang
, S.
(2015
). Infer metagenomic abundance and reveal homologous genomes based on the structure of taxonomy tree
. IEEE/ACM transactions on computational biology and bioinformatics
, 12
(5
), 1112
–1122
.33.
Murzin
, A. G.
, Brenner
, S. E.
, Hubbard
, T.
, & Chothia
, C.
(1995
). SCOP: a structural classification of proteins database for the investigation of sequences and structures
. Journal of molecular biology
, 247
(4
), 536
–540
.34.
Muyzer
, G.
, De Waal
, E. C.
, & Uitterlinden
, A. G.
(1993
). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA
. Applied and environmental microbiology
, 59
(3
), 695
.35.
Muyzer
, G.
, & Smalla
, K.
(1998
). Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology
. Antonie van Leeuwenhoek
, 73
(1
), 127
–141
.36.
Nakao
, R.
, Ramstedt
, M.
, Wai
, S. N.
, & Uhlin
, B. E.
(2012
). Enhanced biofilm formation by Escherichia coli LPS mutants defective in Hep biosynthesis
. PloS one
, 7
(12
), e51241
.37.
Namiki
, T.
, Hachiya
, T.
, Tanaka
, H.
, & Sakakibara
, Y.
(2012
). MetaVelvet: an extension of Velvet assembler to de novo metagenome assembly from short sequence reads
. Nucleic acids research
, 40
(20
), e155
–e155
.38.
Narasingarao
, P.
, Podell
, S.
, Ugalde
, J. A.
, Brochier-Armanet
, C.
, Emerson
, J. B.
, Brocks
, J. J.
, … & Allen
, E. E.
(2012
). De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hyper-saline microbial communities
. The ISME journal
, 6
(1
), 81
–93
.39.
Oulas
, A.
, Pavloudi
, C.
, Polymenakou
, P.
, Pavlopoulos
, G. A.
, Papanikolaou
, N.
, Kotoulas
, G.
, … & Iliopoulos
, L.
(2015
). Metagenomics: tools and insights for analyzing next-generation sequencing data derived from biodiversity studies
. Bioinformatics and biology insights
, 9
, BBI
–S12462
.40.
Read
, S. T.
, Dutta
, P.
, Bond
, P. L.
, Keller
, J.
, & Rabaey
, K.
(2010
). Initial development and structure of biofilms on microbial fuel cell anodes
. BMC microbiology
, 10
(1
), 1
–10
.41.
Salamov
, A. A.
, & Solovyev
, V. V.
(2000
). Ab initio gene finding in Drosophila genomic DNA
. Genome research
, 10
(4
), 516
–522
.42.
Sanli
, K.
, Bengtsson-Palme
, J.
, Nilsson
, R. H.
, Kristiansson
, E.
, Alm Rosenblad
, M.
, Blanck
, H.
, & Eriksson
, K. M.
(2015
). Metagenomic sequencing of marine periphyton: taxonomic and functional insights into biofilm communities
. Frontiers in microbiology
, 6
, 1192
.43.
Sauer
, K.
(2003
). The genomics and proteomics of biofilm formation
. Genome biology
, 4
(6
), 1
–5
.44.
Schmieder
, R.
, & Edwards
, R.
(2011
). Quality control and preprocessing of metagenomic datasets
. Bioinformatics
, 27
(6
), 863
–864
.45.
Schultz
, J.
, Milpetz
, F.
, Bork
, P.
, & Ponting
, C. P.
(1998
). SMART, a simple modular architecture research tool: identification of signaling domains
. Proceedings of the National Academy of Sciences
, 95
(11
), 5857
–5864
.46.
Söding
, J.
, Biegert
, A.
, & Lupas
, A. N.
(2005
). The HHpred interactive server for protein homology detection and structure prediction
. Nucleic acids research
, 33
(suppl_2
), W244
–W248
.47.
Sussman
, J. L.
, Lin
, D.
, Jiang
, J.
, Manning
, N. O.
, Prilusky
, J.
, Ritter
, O.
, & Abola
, E. E.
(1998
). Protein Data Bank (PDB): database of three-dimensional structural information of biological macromolecules
. Acta Crystallographica Section D: Biological Crystallography
, 54
(6
), 1078
–1084
.48.
Tatusov
, R. L.
, Galperin
, M. Y.
, Natale
, D. A.
, & Koonin
, E. V.
(2000
). The COG database: a tool for genome-scale analysis of protein functions and evolution
. Nucleic acids research
, 28
(1
), 33
–36
.49.
Tatusov
, R. L.
, Natale
, D. A.
, Garkavtsev
, I. V.
, Tatusova
, T. A.
, Shankavaram
, U. T.
, Rao
, B. S.
, … & Koonin
, E. V.
(2001
). The COG database: new developments in phylogenetic classification of proteins from complete genomes
. Nucleic acids research
, 29
(1
), 22
–28
.50.
Tennant
, R. K.
, Sambles
, C. M.
, Diffey
, G. E.
, Moore
, K. A.
, & Love
, J.
(2017
). Metagenomic analysis of silage
. Journal of visualized experiments: JoVE
, (119
).51.
Dunlop
, A. L.
, Mulle
, J. G.
, Ferranti
, E. P.
, Edwards
, S.
, Dunn
, A. B.
, & Corwin
, E. J.
(2015
). The maternal microbiome and pregnancy outcomes that impact infant health: a review
. Advances in neonatal care: official journal of the National Association of Neonatal Nurses
, 15
(6
), 377
.52.
Wang
, Q.
, Garrity
, G. M.
, Tiedje
, J. M.
, & Cole
, J. R.
(2007
). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy
. Applied and environmental microbiology
, 73
(16
), 5261
.53.
Xu
, J.
, Li
, M.
, Kim
, D.
, & Xu
, Y.
(2003
). RAPTOR: optimal protein threading by linear programming
. Journal of bioinformatics and computational biology
, 1
(01
), 95
–117
.54.
Yadav
, P. K.
, & Rana
, J.
(2011
). Computer aided epitope prediction for glycoprotein-B in human cytomegalovirus
. Elixir BioPhy
, 39
, 5021
–5025
.55.
Rho
, M.
, Tang
, H.
, & Ye
, Y.
(2010
). FragGeneScan: predicting genes in short and error-prone reads
. Nucleic acids research
, 38
(20
), e191
–e191
.56.
Zerbino
, D. R.
, & Birney
, E.
(2008
). Velvet: algorithms for de novo short read assembly using de Bruijn graphs
. Genome research
, 18
(5
), 821
–829
.
This content is only available via PDF.
© 2022 Author(s).
2022
Author(s)
You do not currently have access to this content.
