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From 2013.igem.org

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Tutorial

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NucleoMod

NucleoMod can modify CDS based on synonymous mutation. It has 5 applications. Firstly, NucleoMod is used to design CRISPR sites on NeoChr so that we can silence the wild type genes. Secondly, it can erase specific enzyme sites according to the users' selection. Thirdly, users can create an enzyme site in selected region of specific genes. Fourthly, it can optimize the codon efficiency to increase the expression level. Finally, it can smash the tandem repeat bases to reduce the synthesis difficulty.

Plugins

This module contains 5 plugins: CRISPR design, erase enzyme site, create enzyme site, codon optimization, repeat smash. All plugins are included in the main program.

2.1 CRISPR design

This plugin is used to design CRISPR site of NeoChr genes so that we can silence the wild type genes. We use blast+ to ensure the uniqueness of CRISPR sites. If you are using more than one plugin at the same time, this plugin will start firstly and deliver the data to next plugin. Otherwise it will generate a new fasta file for sequence and gff file for annotation.

2.1.1 Internal operation

First, this plugin extracts sequence and annotation from the NeoChr FASTA file and GFF3 file, respectively. Regular expression will be applied to find the 23bp basic structure of CRISPR site, with a head of ‘G’ then following 20 facultative bases and finally followed by ‘GG’. All the sequences and locus will be record in an array.
Second, the blast+ will be used to check whether the 12bp sequences (from 9th to 20th) are uniq in the wild type genome. Only uniq sites will be reserved.
Third, synonymous substitution method will be applied to change one base between the 9th to 20th bases of the CRISPR structure. The result will be record in GFF as an element of gene. If –verbose is set, the designed number will be report in STDOUT.
Finally, if this plugin is the last module, the sequence and annotation information will be recreated in FASTA and GFF format.

2.1.2 Example

We have two input forms to execute the plugin:
Run CRISPR design plugin only:
perl NucleoMod.pl -inputfa NeoChr.fa -inputgff NeoChr.gff -outputgff new_annotation.gff -outputfa new_chr.fa -crisprnum 2 -database saccharomyces_cerevisiae_chr.fa

2.1.3 Parameters

Parameter	Description	Default	Selectable range
inputfa	The NeoChr sequence file in FASTA format		string
inputgff	The NeoChr annotation file in GFF3 format		string
outputgff	Output of new chromosome annotation in GFF3 format		string
outputfa	Output of new chromosome sequence in FASTA format		string
verbose	Output the detailed information in STDOUT	none	option
crisprnum	Number of CRISPR site to be design per gene		Int (>0)
database	The sequence of reference genome, used as blast+ database		string
help	Show help information

2.1.4 The format of output file

The output files are standard GFF and FASTA format files.
1. GFF file

2. FASTA file

3. Detailed information in STDOUT

2.2 Erase enzyme site

Given a list of restriction enzyme information, this plugin will erase the restriction sites in every gene. If you are using more than one plugin at the same time, this plugin will start after CRISPR design and deliver the data to next plugin. Otherwise it will generate a new fasta file for sequence and gff file for annotation.

2.2.1 Internal operation

The enzyme information will be extracted. (If the –borbrickstandard parameter is set, it will also remove EcoRI, XbaI, SpeI, PstI and NotI) The recognize site will be reformatted to regular expression and searched in the CDS regions. Once a restriction site is matched, synonymous substitution method will be applied to try to erase the enzyme site. When the substitution is finished, the plugin will restart the next search from 1 base after the last matched position. If this plugin is the last module, the sequence and annotation information will be recreated in FASTA and GFF format.

2.2.2 Example

perl NucleoMod.pl -inputfa NeoChr.fa -inputgff NeoChr.gff -outputgff new_annotation.gff -outputfa new_chr.fa –biobrickstandard [-delenzymelist enzyme.list ]

Format of enzyme.list:
Company enzyme_name enzyme_site …
　　Eg. NEB BamHI G/GATCC

2.2.3 Parameters

Parameter	Description	Default	Selectable range
inputfa	The NeoChr sequence file in FASTA format		string
inputgff	The NeoChr annotation file in GFF3 format		string
outputgff	Output of new chromosome annotation in GFF3 format		string
outputfa	Output of new chromosome sequence in FASTA format		string
verbose	Output the detailed information in STDOUT	none	option
biobrickstandard	Erase the biobrick standard enzyme site	none	option
delenzymelist	The file of enzyme going to delete		string
detail	Show the erased enzyme site in new gff	none	option
help	Show help information

2.2.4 The format of output

The output files are standard GFF and FASTA format.
1. GFF file
2. FASTA file
3. Detailed information in STDOUT

2.3 Create enzyme site

Given a list of restriction enzyme information, this plugin can create a new enzyme site in specific region of selected gene. If you are using more than one plugin at the same time, this plugin will start after erase enzyme site and deliver the data to next plugin. Otherwise it will generate a new fasta file for sequence and gff file for annotation.

2.3.1 Internal operation

First, information of enzyme site will be extracted. According to 3 reading frames, a searching tree will be constructed and converted to regular expression. The plugin will search the selected regions and then change the sequence to enzyme site by synonymous substitution method. If this plugin is the last module, the sequence and annotation information will be recreated in FASTA and GFF format.

2.3.2 Example

perl NucleoMod.pl -inputfa NeoChr.fa -inputgff NeoChr.gff -outputgff new_annotation.gff -outputfa new_chr.fa -addenzymelist enzyme.list -addenzymeconfig gene_id,start_pos,end_pos,enzyme_name

2.3.3 Parameters

Parameter	Description	Default	Selectable range
inputfa	The NeoChr sequence file in FASTA format		string
inputgff	The NeoChr annotation file in GFF3 format		string
outputgff	Output of new chromosome annotation in GFF3 format		string
outputfa	Output of new chromosome sequence in FASTA format		string
verbose	Output the detailed information in STDOUT	none	option
addenzymelist	The file of enzyme to get enzyme site information		string
addenzymeconfig	A array of string to specify enzyme and regions		string,int,int,string
help	Show help information

2.3.4 The format of ouput

The output files are standard GFF and FASTA format.
1. GFF file
2. FASTA file
3. Detailed information in STDOUT

2.4 Codon optimization

Given a codon priority list, this plugin is used to optimize the codon so that we can increase the expression of selected genes. If you are using more than one plugin at the same time, this plugin will start after create enzyme site and deliver the data to next plugin. Otherwise it will generate a new fasta file for sequence and gff file for annotation.

2.4.1 Internal operation

The codon with same amino acid will be separated into 3 ranks, best normal and worst. Every codon of selected gene will be check whether the codon is in best rank. The codon in normal or worst will be change to best rank by synonymous substitution method. If this plugin is the last module, the sequence and annotation information will be recreated in FASTA and GFF format.

2.4.2 Example

perl NucleoMod.pl -inputfa NeoChr.fa -inputgff NeoChr.gff -outputgff new_annotation.gff -outputfa new_chr.fa -codonoptimize CodonPriority.txt -optimizeallgene [-optimizegenelist gene1,gene2,gene3 ]

2.4.3 Parameters

Parameter	Description	Default	Selectable range
inputfa	The NeoChr sequence file in FASTA format		string
inputgff	The NeoChr annotation file in GFF3 format		string
outputgff	Output of new chromosome annotation in GFF3 format		string
outputfa	Output of new chromosome sequence in FASTA format		string
verbose	Output the detailed information in STDOUT	none	option
codonoptimize	Codon priority list to get the ranking information		string
optimizeallgene	Optimize all genes in inputgff		option
optimizegenelist	A list of gene going to optimize, separate by comma		string,string,string,...
detail	Show the optimization sequence in new gff	none	option
help	Show help information

2.4.4 The format of ouput

The output files are standard GFF and FASTA format.
1. GFF file
2 .FASTA file
3. Detailed information in STDOUT

2.5 Repeat smash

This plugin go through the CDS region to find out the tandem repeat bases. Synonymous substitution method will be applied to break long tandem repeat base to reduce the synthesis difficulty. If you are using more than one plugin at the same time, this plugin will start finally and then it will generate a new fasta file for sequence and gff file for annotation.

2.5.1 Internal operation

Regular expression is used to find out the tandem repeat bases longer then specified length (usually longer than 5bp). From the third of the matched sequence, synonymous substitution method will be applied to break the tandem repeat bases. If the substitution is successful and the rest sequence is still longer than the cutoff, then it will move to next 3 bases and do the same thing. The sequence and annotation information will be recreated in FASTA and GFF format.

2.3.2 Example

perl NucleoMod.pl -inputfa NeoChr.fa -inputgff NeoChr.gff -outputgff new_annotation.gff -outputfa new_chr.fa -repeatsmash 5

2.3.3 Parameters

Parameter	Description	Default	Selectable range
inputfa	The NeoChr sequence file in FASTA format		string
inputgff	The NeoChr annotation file in GFF3 format		string
outputgff	Output of new chromosome annotation in GFF3 format		string
outputfa	Output of new chromosome sequence in FASTA format		string
verbose	Output the detailed information in STDOUT	none	option
repeatsmash	The tandem repeat bases longer or equal to this cutoff will be smashed		int
detail	Show the repeat smash result in new gff	none	option
help	Show help information

2.3.4 The format of ouput

The output files are standard GFF and FASTA format.
1. GFF file
2. FASTA file
3. Detailed information in STDOUT