Protocols for Probing Genome Architecture of Regulatory Networks in Hydrocarbon and Lipid Microorganisms

Abstract

Genome architecture and the regulation of gene expression are expected to be interdependent. Understanding this interdependence is key to successful genome engineering. Evidence for nonrandom arrangement of genes along genomes, defined as the relative positioning of cofunctional or co-regulated genes, stems from two main approaches. Firstly, the analysis of contiguous genome segments across species has highlighted the conservation of gene order (synteny) along chromosome regions. Secondly, the study of long-range regularities along chromosomes of one given species has emphasised periodic positioning of microbial genes that are either co-regulated, evolutionarily correlated, or highly codon biased. Software tools to detect, visualise, systematically analyse and exploit gene position regularities along genomes can facilitate the studies of such nonrandom genome layouts and the inference of transcription factor binding sites and potentially guide rational genome design. Here, a computational protocol is demonstrated for the analysis and exploitation of regular patterns in a set of genomic features of interest (e.g. cofunctional or co-regulated genes, chromatin immunoprecipitation results, etc.). This case study is conducted for genes involved in hydrocarbon metabolism of a marine petroleum-degrading bacterium Alcanivorax borkumensis.

Appendices

Appendix 1: Input File Format for a GREAT:SCAN:patterns Analysis (This Example Contains the Genes from A. borkumensis Involved in Hydrocarbon Degradation)

dhmA :

2734484

alkB1 :

3063242

alkB2 :

130408

aldh :

3066633

alkK :

3110974

alkL :

2198588

alkN :

114433

rubB :

170672

rubA :

171870

GntR :

129582

p450 :

2707944

p450b :

2607794

p450c :

217210

fdx :

216875

alkJ2 :

218643

FAD :

220424

AraC :

215704

alkG :

3064575

alkS :

3060396

Appendix 2: Usage Message of GREAT:SCAN:patterns

usage: patterns.R [-h] -t [<title> [<title> …]]

[-l <genome_in_bp>]

[-a <avgGene_in_bp>]

[-r [<per_bounds> [<per_bounds> …]]]

[-p <pvalue_thres>]

[-s <pvalue_select>]

[-d [<set_coords> [<set_coords> …]]]

[-k [<set_ticks> [<set_ticks> …]]]

[-c <clust_exponent>]

[-z <cluster_size>]

[-m <pvalue_mapping>]

[-i [<a_uniq_ID>]] [-v <path>]

[-o <output_path>]

<file_name>

Systematically analyse, cluster and visualise results from a complete GREAT:SCAN analysis. Full global_spectrum (-DOM and -CIRC analysis) followed by a DBSCAN clustering to identify the in-phase genes and a solenoid_map (sliding window) analysis and visualisation of the spread of all the possible periods.

positional arguments:

<file_name> The input file consisting of two columns of data formatted like this: <entity_ID> <entity_position>

optional arguments:

-h, --help show this help message and exit

-t [<title> [<title> …]], --title [<title> [<title> …]]

A substring to specify a title for the experiment

(default: None)

-l <genome_in_bp>, --chrom_length <genome_in_bp>

The length in bp of the organism chromosome

(default: 4639675)

-a <avgGene_in_bp>, --avg_gene <avgGene_in_bp>

The average gene length of the organism genes

(default: 1000)

-r [<per_bounds> [<per_bounds> …]], --period_range [<per_bounds> [<per_bounds> …]]

The range (min. -- max.) within which periods will be considered for further analysis (default: 5000)

-p <pvalue_thres>, --pvalue_thres <pvalue_thres>

The unweighted p-value threshold for considering a period for further analysis (default: 0.05)

-s <pvalue_select>, --pvalue_select <pvalue_select>

The weighted p-value threshold for selecting which periods will be printed (default: 0.05)

-d [<set_coords> [<set_coords> …]], --plot_coords [<set_coords> [<set_coords> …]]

Specifies a set of genomic coordinates to be printed as significant genome marks in the mapping plot (the E.coli macrodomains are defaults:

[46396, 603158, 1206296, 2180612, 2876552, 3758076])

-k [<set_ticks> [<set_ticks> …]], --plot_ticks [<set_ticks> [<set_ticks> …]]

Specifies a set of axis ticks to be printed as indicators of genome marks in the mapping plot (must be equal size with the coordinates).

(default: ['ori', 'right', 'R/ter', 'ter/L', 'left', 'ori'])

-c <clust_exponent>, --clust_exp <clust_exponent>

The clustering exponent. Assigns the minimum distance d between two points to be members of the same cluster. Specifies the exponent of the ratio between the length of the period and chromosome length (p/L). (default: 0.5)

-z <cluster_size>, --clust_size <cluster_size>

The minimum number of members for a group to be considered as a cluster (DBSCAN parameter)

(default: 2)

-m <pvalue_mapping>, --pvalue_map <pvalue_mapping>

The weighted p-value threshold for selecting which sliding window periods will be plotted (default: 0.001)

-i [<a_uniq_ID>], --uniq_ID [<a_uniq_ID>]

The unique ID for the generation of the results folder. (default: patternAnalysis_ xxxx_xx_xx)

-v <path>, --pv <path>

The path to the 'pv' fit parameters file.

(default: <installation_of_cmdline_programs>)

-o <output_path>, --output_path <output_path>

The absolute path for a directory (existing one including the trailing slash '/') where the output will be kept, or omit for the current working directory. (just the path, the directory name itself is controlled by the -i option).

(default: <current_working_dir>)