Got contigs?

A bacterial genomes finishing tool for structural insights on draft genomes

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CONTIGuator is a Python script for Linux environments whose purpose is to speed-up the bacterial genome finishing process and to obtain a first insight of the genome structure using the well-known artemis comparison tool (ACT), as well as gap-closing PCR primers using primer3

About

Recent developments in sequencing technologies had reduced both labor and cost to completely sequence virtually every bacterial strain. A limiting step is the finishing process, needed to infer the relative position of each contig, in order to reduce the subsequent efforts to close the genome sequence. The availability of a large number of bacterial genomes though, allows geneticists to use complete genomes (usually from the same species) as templates to map the contigs. An additional degree of complexity is given by bacterial species harboring more than one replicon, which are not contemplated by the available programs up to date.

Workflow

The program performs a mapping step against the reference genome using the blastn algorithm; the results are analyzed taking into account the presence of more than one replicon, thus ensuring that no contigs are mapped to more than one replicon.
A mapping algorithm is then used to filter the input contigs and map them to the reference genome, resulting in the generation of one map for each target replicon, with many descriptive features like putative overlaps between mapped contigs and syntenic and non-syntenic regions inside and outside contigs. The maps can be visualized with the artemis comparison tool (ACT).
Once that the maps are generated, primer3 is used to generate the PCR primer pairs in order to design only the significant PCR reactions for efficient gap closure: when the results are ready, the new contigs set can be used for another analysis round, in an iterative fashion that in principle leads to a complete gaps closure.

Requirements

CONTIGuator was developed and tested in a Linux environment and has a few software requirements, listed below:

• Python
• BioPython
• Blast+
• Perl (optional)
• ABACAS (optional)
• MUMmer (optional)
• Primer3 (optional)

To view the results the Artemis comparison tool (ACT) is needed: it can be both downloaded or launched as a Java web-applet. To take advantage of the tblastn search of reference proteins from unmapped regions in the excluded contigs, one or more ptt files should be present in the same directory as the reference genome FASTA file; there should be one ptt file for each reference replicon. If the reference genome FASTA file was downloaded from NCBI, the ptt file name is already in the right format, otherwise the ptt file name should be in the format SEQUENCEID.ptt.

Outputs

The outputs of CONTIGuator are various files and they are divided by folders

"Map_" folders

According to the number of input reference replicons, there will be the same number of directories whose name starts by "Map_", followed by the ID of the reference replicon. Inside each directory there will be a series of files that can be used as input for ACT

• Reference.embl: pseudo-contig ACT file
• PseudoContig.fsa: pseudo-contig fasta file
• PseudoContig.crunch: ACT comparison file
• PseudoContig.embl: pseudo-contig ACT file
• MappedContigs.txt: names (and lenghts) of the contigs mapped to the particular reference molecule
• A shell script to open the ACT map
• A PDF map with publication-quality map

If options -M was selected:

• AlignDetails.tab: tab-delimited file containing details about the mapped hits
• AlignedContigsHits.fsa: mapped hits fasta file (on contigs)
• AlignedReferenceHits.fsa: mapped hits fasta file (on reference)
• UnAlignedContigsHits.fsa: unmapped regions fasta file (on contigs)
• UnAlignedReferenceHits.fsa: unmapped regions fasta file (on reference)

If the primer picking option was selected (-P) the folder will contain other files

• PCRPrimers.tsv: table containing details about the PCR primers generated

"UnMappedContigs" folder

This folder contains those contigs that CONTIGuator was unable to map in fasta format, divided in categories

• Excluded.fsa: all the excluded contigs
• Multi.fsa: contigs mapped to more than one replicon
• Short.fsa: contigs below the lenght treshold (-L)
• NoCoverage.fsa: contigs below the coverage treshold (-C)
• CoverageBorderLine.fsa: contigs near the coverage treshold (-C)
• Discarded.fsa: contigs discarded due to duplicated hits
• UnMappedContigsHits.tab: contains the list of the excluded contigs with the number of tblastn hits
• UnMappedReferenceRegions.tab: contains the reference genome unmapped regions with at least one tblastn hit
• UnMappedContigs.txt: names (and lenghts) of the contigs not mapped to any particular reference molecule

Other files

In addition to these files, a log file (called “CONTIGuator.log”) is present in the source directory: the amount of log output can be modulated using the options -V and -D.

Quick reference

Default run WITHOUT primer picking

Default run WITH primer picking

The -A option can be omitted for manual input of primer3 parameters

Default run with more outputs

Default run showing the maps automatically

The -l option stands for "lazy user" :)

Default run with no N to fill the gaps

Command line options

Inputs

• -c fasta file containing all the contigs
• -r fasta file containing the reference genome; if it contains more than one sequence (i.e. more than one replicon) it can both be in a single file or in more files (just use -r multiple times)
• -f prefix for the output directories

Blast parameters

• -e Blast e-value treshold [Default: 1e-20]
• -b Use the blastn algorithm (for distant genomes)
• -t Number of threads to be used by Blast (useful for large genomes)

Parse Blast mode

• -p parse ready-made Blast output (no Blast runs will be performed) [Default: no]
• -x Blast XML output to be parsed [Default: blast.xml]

Contig profiling parameters

• -L minimal lenght of a contig to be accepted in the analysis [Default: 1000 bp]
• -C minimal coverage of a contig to be accepted in the analysis [Default: 20%]
• -B minimal lenght of a significant Blast hit [Default: 1100 bp]
• -R minimum ratio for best replicon estimation in case of a conflict [Default: 1.5]

Primer picking

• -P do primer picking [Default: no]
• -A use default parameters for primer picking [Default: no]
• -I compute also the inner primers [Default: no]

Output options

• -f prefix for the output directories
• -M prepare even more outputs
• -n Gap size for adjacent contigs
• -N Do not use N to fill the gaps

ACT options

• -a ACT binary location
• -l Open the maps automatically

Logging

• -V verbose
• -D development
• -G debug mode

Changelog

Version 2.7.5

• BUGFIX: be aware of the primer3 version, and treat the output differently

Version 2.7.4

• BUGFIX: return with an error message when a FASTA header contains a tab

Version 2.7.3

• FEATURE: -b option to use thblastn algorithm instead of megablast (for distant genomes)
• FEATURE: -t options to use more than one blast thread (useful for large genomes)
• FEATURE: -n option to regulate the distance between adjacent contigs
• BUGFIX: a cleaner "--version" output

Version 2.7.2

• BUGFIX: correctly import pre-made blast outputs (-p)

Version 2.7.1

• FEATURE: two new files to detail the position of the unaligned regions (-M)

Version 2.7

• FEATURE: new option to modulate the multiple replicons conflicts (-R)
• FEATURE: new option to obtain also the inner primers (-I)
• BUGFIX: multiple replicons collision bugfixes
• BUGFIX: better BioPython version check

Version 2.6.2

• FEATURE: check the input files format

Version 2.6.1

• BUGFIX: Sanitize Blast inputs when files or sequences names contain non-standard chars

Version 2.6

• FEATURE: PDF maps in each "Map" folder (BioPython >= 1.59 is needed)
• FEATURE: -B and -L values can be set below the suggested minimum values
• FEATURE: -N option to generate the maps without the 100 Ns between each contig
• BUGFIX: Coverage option (-C) now working as expected
• BUGFIX: One base misplacement of contigs in ACT maps
• BUGFIX: No overlap check when there is only one contig mapped
• DEPRECATION: -I option removed
• OTHER: Some credits to recent contributors
• OTHER: Changelog file added

Version 2.5.0:

• FEATURE: PCR primers are computed from the last contig to the first one
• FEATURE: Overlaps between contigs are checked using Blast
• FEATURE: Notifications and better errors handling
• BUGFIX: Unused imports clean-up
• BUGFIX: Code header clean-up
• OTHER: Some credits to recent contributors

Version 2.3.3:

• BUGFIX: Older biopython versions support: SeqIO write method may require a list

Version 2.3.2:

• BUGFIX: Correct map directory name for the ACT scripts

Version 2.3.1:

• BUGFIX: Make Blast suport any kind of sequences IDs
• BUGFIX: Clearer messages on Blast failures

Version 2.3:

• FEATURE: Generation of embl files instead of fasta AND tab files (just three files are needed by ACT now)
• FEATURE: An easier way to open the ACT maps is now implemented
  • script are generated in each "Map_" directory
  • the maps can be automatically opened (-l option)
• BUGFIX: Support for Biopython 1.57+
• BUGFIX: EMBL files no more causing crashes of ACT on long contig names
• BUGFIX: PrintStats now gives the correct number of primers
• BUGFIX: Fix two crashes when no contigs are mapped

Version 2.2:

• FEATURE: PCRPrimers.tsv is now produced by default (with -P option)
• FEATURE: Mapped and UnMapped txt files now contain the molecules length
• FEATURE: The script can be launched from any location now (abacas will be found)
• BUGFIX: UnMappedContigs.txt now is correctly created
• DEPRECATION: The abacas generated outputs after primer picking are not used anymore
• DEPRECATION: DNALenghts.tab is no longer produced as an output

Version 2.1:

• FEATURE: UnMappedContigs.txt file
• FEATURE: DNALengths.tab file
• FEATURE: More outputs with -M option:
  • Four fasta files with aligned/unaligned regions (on contigs and reference)
  • PCRPrimers.tsv, containing details about the generated primers
• BUGFIX: Remove those primers generated because of Ns regions INSIDE a contig
• BUGFIX: Fail-safe printing of statistics
• DEPRECATION: Use of legacy-blast now gives a deprecation warning

Version 2.0:

• REFACTOR: Contig profiling and mapping use only Blast
• REFACTOR: Abacas and Mummer are used only for primer picking
• REFACTOR: Removed deprecated functions and added new classes
• FEATURE: New "map" file (MappedContigs.txt), containing the contigs mapped to the reference
• BUGFIX: Minimum contig length and minimum big hit length now have a minimum size
  

Versions 1.x:

• 1.0.1 BUGFIX: removing underscores from contig names
  
• 1.1 FEATURE: added a tblastn from proteins in reference molecules with no homology
• 1.2 BUGFIX: Handling primer3 newer versions // Removed "Linear" option
• 1.3 BUGFIX: Unused splitted contigs correction
• 1.4 FEATURE: added the -A option to automate the primer design parameters input // Various bugfixes
• 1.4.1 BUGFIX: prevention of crashes during PrintStats function
• 1.4.2 FEATURE: added the -G (DEBUG) option // Check for really old BioPython versions
• 1.4.3 FEATURE: add a prefix to the generated directories (-f option)
• 1.4.4 BUGFIX: a little bugfix on prefix option