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Bioinformatics Approaches in Studying Microbial Diversity

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Management of Microbial Resources in the Environment

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Abstract

Proper understanding of molecular sequences, identification and phylogenetics of microorganisms are very important in many branches of biological science. Generation of genomic DNA sequence data from different organisms including microbes requires the application of bioinformatics tools for their analysis. Bioinformatics tools including BLAST, multiple sequence alignment tools etc. are used to analyze nucleic acid and amino acid sequences for phylogenetic affiliation. The emerging fields of comparative genomics and phylogenomics require the substantial knowledge and understanding of computational methods to handle the large scale data involved. The introduction of comparative rRNA sequence analysis represents a major milestone in the history of microbiology. Also single gene based phylogenetic inference and alternative global markers including elongation and initiation factors, RNA polymerase subunits, DNA gyrases, heat shock and RecA proteins are of immense importance. The analysis of the sequence data involves four general steps including: (i) selection of a suitable molecule or molecules, (ii) acquisition of molecular sequences, (iii) multiple sequence alignment and (iv) phylogenetic evaluation. This chapter explains in detail how raw data of molecular sequences from any microbe may be used for the detection and identification of microorganisms using computer based bioinformatics tools.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, Uttar Pradesh, 202002, India

    Mohammad Tabish, Shafquat Azim, Mohammad Aamir Hussain, Sayeed Ur Rehman, Tarique Sarwar & Hassan Mubarak Ishqi

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  1. Mohammad Tabish
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  2. Shafquat Azim
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Correspondence to Mohammad Tabish .

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Editors and Affiliations

  1. Department of Agricultural Microbiology Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India

    Abdul Malik (Visiting Professor/Scientist) (Visiting Professor/Scientist)

  2. Division of Infectious Diseases, University Medical Center Freiburg, Freiburg, Germany

    Abdul Malik (Visiting Professor/Scientist) & Elisabeth Grohmann (Visiting Professor/Scientist) & 

  3. Center of Biological Engineering, University of Minho, Braga, Portugal

    Madalena Alves

Glossary

Homology Searches: BLAST & FASTA

Background Information: The three BLAST programs that one will commonly use are BLASTN, BLASTP and BLASTX. BLASTN will compare your DNA sequence with all the DNA sequences in the nonredundant database (nr). BLASTP will compare your protein sequence with all the protein sequences in nr. In BLASTX nucleotide sequence of interest will be translated in all six reading frames and the products compared with the nr protein database. A tutorial is also available at NCBI.

BLAST Homepage – (NCBI)

Found at http://blast.ncbi.nlm.nih.gov/Blast.cgi. It is widely used for homology searches. BLAST stands for Basic Local Alignment Search Tool and it displays a number of organism and query specific blast.

Nucleotide BLAST (BLASTN)

N.B. the default database is “human genomic and transcript” not “nucleotide collection (nt/nr)”

Protein BLAST (BLASTP)

This program is also coupled with a motif search. If you suspect that your protein may only show weak sequence similarity to other proteins, I would suggest clicking on the PSI-BLAST (Position-Specific Iterated BLAST) feature. NCBI also provides a PSI-BLAST tutorial. PSI-BLAST searches to yield better delineation of true and false positives.

Translated BLAST (BLASTX)

TBLASTX searches translated nucleotide databases using a translated nucleotide query; while TBLASTN searches translated nucleotide databases using a protein query. These are useful resources if you are interested in homologs in unfinished genomes. Undeter “Databases” select “genomic survey sequences”, “High throughput genomic sequences” or “whole-genome shotgun reads”

Blast with Microbial Genomes (BLASTN, TBLASTN, TBLASTX etc.).

It permits us to compare a nucleic acid or protein sequence against finished archaeal and bacterial genomes. Depending upon the time of day your results may appear almost immediately or the search may be delayed or not accepted at all. For PSI-BLAST, and other searches it is recommended to frequently enter information in the “Entrez Query” section e.g. Escherichia coli [organism] or Viruses [organism] to see “hits” specifically to E. coli or viruses/bacteriophages. It is advisable to always select “Show results in a new window”

EMB BLAST-(European Molecular Biology network).

Very convenient since it permits one to specifically search databases such as prokaryote, bacteriophage, fungal, & 16S rRNA using BLASTN, and specific bacterial genomes or SwissProt using BLASTX or BLASTN.

ParAlign

It employs a heuristic method for sequence alignment. In essence, ParAlign is about as sensitive as Smith-Waterman but runs at the speed of BLAST.

GTOP

Sequence Homology Search (Laboratory for Gene-Product Informatics, National Institute of Genetics, Japan) – offers BLASTP search capability against individual archaea, bacteria, eukaryota, and viruses.

T4-like Phage NCBI MegaBLAST (Tulane Univ., New Orleans, U.S.A. & CNRS, Toulouse, France)

This includes a growing list of T4-like completed phage sequences as well as those in the draft and contig stages of completion.

WU-BLAST (Washington University BLAST)

The emphasis of this tool is to find regions of sequence similarity quickly, with minimum loss of sensitivity. This will yield functional and evolutionary clues about the structure and function of the novel sequence.

Batch BLAST (Greengene web server; University of Massachusetts, Lowell, U.S.A.)

was developed by Michael V. Graves for DNA or protein BLAST sequence analysis against the NCBI databases. It allows one to submit a file that contains multiple sequences and then will organize the results by each individual sequence contained in the file.

HHPred Homology detection & structure prediction

is a method for database searching and structure prediction that is as easy to use as BLAST but is much more sensitive in finding remote homologs. HHpred is the first server that is based on the pairwise comparison of profile hidden Markov models (HMMs). Whereas most conventional sequence search methods search sequence databases such as UniProt or the NR, HHpred searches alignment databases, like Pfam or SMART. This greatly simplifies the list of hits to a number of sequence families instead of a clutter of single sequences. HHpred accepts a single query sequence or a multiple alignment as input.

PSI-BLAST or PHI-BLAST search

Position-Specific Iterative BLAST creates a profile after the initial search.

BLAST 2

BLAST two sequences against one another. This utilizes BLASTN, P, X as well as TBLASTN and TBLASTX.

Gene Context Tool

It is an incredible tool for visualizing the genome context of a gene or group of genes.

TC-BLAST

It scans the transport protein database (TC-DB) producing alignments and phylogenetic trees. The TC-DB details a comprehensive classification system for membrane transport proteins known as the Transport Commission (TC) system.

MEROPS BLAST

This permits one to screen protein sequences against an extensive database of characterized peptidases.

SEARCHGTr

It is web-based software for the analysis of glycosyltransferases involved in the biosynthesis of a variety of pharmaceutically important compounds like adriamycin, erythromycin, vancomycin etc. This software has been developed based on a comprehensive analysis of sequence/structural features of 102 GTrs of known specificity from 52 natural product biosynthetic gene clusters.

PipeAlign

It offers an integrated approach to protein family analysis through a cascade of different sequence analysis programs (BALLAST, DbClustal multiple alignment program, Rascal alignment analysis) removal of any sequences that do not belong to the protein family are performed by the NorMD, and clustered into potential functional subfamilies using Secator or DPC.

MPsrch (EMBL-EBI)

This sequence sequence comparison tool implements the true Smith and Waterman algorithm identifying hits in cases where Blast and Fasta fail and also reports fewer false-positives. This software provides information on Match %; % Query Match (% of the query sequence matched); Conservative changes; Mismatches; Indels; and Gaps.

GOAnno

This web tool automatically annotates proteins according to the Gene Ontology using hierarchised multiple alignments. Positioning the query protein in its aligned functional subfamily represents a key step to obtain highly reliable predicted GO annotation based on the GOAnno algorithm.

COMPASS

This is a profile-based method for the detection of remote sequence similarity and the prediction of protein structure. The server features three major developments: (i) improved statistical accuracy; (ii) increased speed from parallel implementation; and (iii) new functional features facilitating structure prediction. These features include visualization tools that allow the user to quickly and effectively analyze specific local structural region predictions suggested by COMPASS alignments.

MineBlast

It performs BLASTP searches in UniProt to identify names and synonyms based on homologous proteins and subsequently queries PubMed, using combined search terms in order to find and present relevant literature.

Comparison of homology between two small genomes:

SCAN2 (Softberry.com) It provides one with a colour-coded graphical alignment of genome length DNAs in Java. In the top panel regions of high sequence identity are presented in red. By highlighting the grey yellow, green, black boxes one can select specific regions for examination of the sequence alignment.

Advanced PipMaker

It aligns two DNA sequences and returns a percent identity plot of that alignment, together with a traditional textual form of the alignment. We may need to download it for viewing and manipulating the output from pairwise alignment programs such as PipMaker representations of the alignments.

JDotter: A Java Dot Plot Viewer

(Viral Bioinformatics Resource Center, University of Victoria, Canada) – a dot matrix plotter for Java. It produces similar diagrams to the above mentioned programs, but with better control on output.

multi-zPicture: multiple sequence alignment tool

provides nice dotplot graphs and dynamic visualizations. If simple gene locations are provided in the form (e.g. >2,000–5,000 RNA_polymerase; indicates that the RNA polymerase gene is found on the plus strand between bases 2,000 and 5,000) this data will be added to the dynamic visualization. zPicture alignments can be automatically submitted to rVista to identify conserved transcription factor binding sites.

GeneOrder 3.0

This is ideal for comparing small GenBank genomes (up to 2 Mb). Each gene from the Query sequence is compared to all of the genes from the Reference sequence using BLASTP. There are two display formats: graphical and tabular.

CoreGenes

This programme is designed to analyze two to five genomes simultaneously, generating a table of related genes – orthologs and putative orthologs. These entries are linked to their GenBank data. It has a limit of 0.35 Mb, while the newer version CoreGenes 2.0 extends the limit to approx. 2.0 Mb. If data is not present in GenBank, using this site will be very helpful.

CoreGenes 3.0

This is the latest member in the CoreGenes family of tools. It determines unique genes contained in a pair of proteomes. This has proved extremely useful in determining unique genes in comparisons between large Myoviridae.

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Tabish, M., Azim, S., Hussain, M.A., Rehman, S.U., Sarwar, T., Ishqi, H.M. (2013). Bioinformatics Approaches in Studying Microbial Diversity. In: Malik, A., Grohmann, E., Alves, M. (eds) Management of Microbial Resources in the Environment. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-5931-2_6

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