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From 2013.igem.org

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Revision as of 05:13, 28 October 2013

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Tutorial

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SegmMan

This module will cut chromosome into pieces with different sizes with Gibson, Goldengate, Homologous adaptors to them so that they are able to be assembled into whole experimentally.

Plugin Scripts

3-1. 01.whole2mega.pl

This utility can split the whole chromosome ( at least 90kbp long ) into about 30k segments and add homologous overlap and adaptors, so that these fragments can be integrated into whole experimentally.

Internal operation

First, this utility searches for the location of centromere and ARSs (autonomously replicating site). The minimal distance between centromere and ARS should NOT be larger than a defined megachunk which is about 30k long.
Second, this utility cuts out the first 30k sequence window containing the centromere and its adjacent ARS, and then adds this megachunk with two original markers and left, right telomeres.
Thirdly, this utility continues to cut more megachunks from the original one to both ends. But these megachunks are not independent, they all have about 1kbp overlaps. Moreover, these new splited window can be given only one marker alternately and only left or right telomere.
The output file will be dealed with 02.globalREmarkup.pl
For more information about segmentation design, please refer to the page ASSEMBLY DESIGN PRINCIPLE .

Example (command line)

perl 01.whole2mega.pl –gff sce_chrI.gff -fa sce_chr01.fa -ol 1000 -ck 30000 -m1 LEU2 -m2 URA3 -m3 HIS3 -m4 TRP1 -ot sce_chrI.mega

Parameters

		default	Option
gff	The gff file of the chromosome being restriction enzyme sites parsing
fa	The fasta file of the chromosome being restriction enzyme sites parsing (The length of the chromosome is larger than 90k)
ol	The length of overlap between megachunks	1000bp
ck	The length of megachunks	30kbp
m1	The first marker for selection alternately	LEU2 (1797bp)	LEU2/URA3HIS3/TRP1
m2	The second marker for selection alternately	URA3 (1112bp)	LEU2/URA3/HIS3/TRP1
m3	The first marker orinally residing in first 30k segmentation	HIS3 (1774bp)	LEU2/URA3/HIS3/TRP1
m4	The second marker orinally residing in first 30k segmentation	TRP1 (1467bp)	LEU2/URA3/HIS3/TRP1
ot	The output file	Prefix(fa filename)+ suffix(.mega)

The format of output:

The output file is stored in /the path where you install GENOVO/Result/ 01.whole2mega.
Besides, there is screen output about the process state and result.
1. Screen output
2. 01.state
 Store the segmentation information

Megachunk_ID	Corresponding location in the designed chromosome
Part ID	Location in the segmentation

3 *.mega
 Store the fasta information of the 30k segments

3-2. 02.globalREmarkup.pl

This utility will parse the exited restriction enzyme sites residing in the chromosome.

Internal operation

This utility searches the exited restriction enzyme sites along the chromosome both plus strand and minus strand, after users define the list of enzymes.
Besides, we tried to find out all the potential restriction enzyme sites, so that maybe some unusual restriction enzyme sites can be created and let segmentation go. But because it had low efficiency, we’re still working on that.
The output file will be dealed with 03.mega2chunk2mini.pl
For more information about segmentation design, please refer to the page ASSEMBLY DESIGN PRINCIPLE .

Example (command line)

perl 02.globalREmarkup.pl -sg 01.whole2mega/sce_chrI.mega -re standard_and_IIB -ct Standard.ct –ot sce_chrI.mega.parse

Parameters

		default	Option
sg	The fasta file of the 30k segmentation, the output of 01.wh2mega.pl
ps	The markup file of the 30k segmentation, the output of 02.globalREmarkup.pl
re	The restriction enzyme sites list. It is devided by different standards, type (IIP, IIA, IIB), cost (standard, nonexpensive) and etc.	Standard_and_IIB	IIP/IIA/IIB/Standard/ Nonexpensive/ Standard_IIB Nonexpensive_IIB
a2	2k to 10k assembly strategy (Gibson or Goldengate)	Gibson	Gibson/ Goldengate
a10	10k to 30k assembly strategy (Gibson or Goldengate)	Goldengate	Gibson/ Goldengate
ckmax2	The maximum length of minichunks	2200 bp
ckmin2	The minimum length of minichunks	1800 bp
cknum	The number of minichunks in a chunk	5

Codon table list:
1 The Standard Code
2 The Vertebrate Mitochondrial Code
3 The Yeast Mitochondrial Code
4 The Mold, Protozoan, and Coelenterate Mitochondrial Code and the Mycoplasma/Spiroplasma Code
5The Invertebrate Mitochondrial Code
6 The Ciliate, Dasycladacean and Hexamita Nuclear Code
7 The Echinoderm and Flatworm Mitochondrial Code
8 The Euplotid Nuclear Code
9 The Bacterial, Archaeal and Plant Plastid Code
10 The Alternative Yeast Nuclear Code
11 The Ascidian Mitochondrial Code
12 The Alternative Flatworm Mitochondrial Code
13 Blepharisma Nuclear Code
14 Chlorophycean Mitochondrial Code
15 Trematode Mitochondrial Code
16 Scenedesmus Obliquus Mitochondrial Code
17 Thraustochytrium Mitochondrial Code
18 Pterobranchia Mitochondrial Code
19 Candidate Division SR1 and Gracilibacteria Code

The format of utput

The output file is stored in /the path where you install GENOVO/Result/. 02.globalREmarkup.
Besides, there is screen output about the process state and result.
1. Screen output
2. *.parse
Store the exited enzyme recognition site in the megachunks
Enzyme ID Start End Recognition site Real site

3-3. 03.chunk_30k_10k_2k.pl

This utility can produce 2k minichunks with Gibson adaptors and 10k chunks with goldengate adaptors.

Internal operation

This utility will segmentate the megachunk produced by 03.mega2chunk2mini.pl into 2k minichunks with Gibson assembly adaptors, so that they can be put together into 10k chunks.
First, this bin will search the inexistent restriction enzyme sites locally, and then decide the size of the minichunks according to the requirements from users, and add two same Gibson adaptors to each sides of minichunks. Secondly, the second part of this bin will define the start and end point of the chunks as users asked and design goldengate assembly adaptors for the chunks.
The output file can be sent in gene synthesis company after human attention and double check.
For more information about segmentation design, please refer to the page ASSEMBLY DESIGN PRINCIPLE .

Example (command line)

perl 03.mega2chunk2mini.pl -re standard_and_IIB -sg 01.whole2mega/sce_chr01_0.mega -ps 02.globalREmarkup/sce_chr01_0.parse -ot 03.mega2chunk2mini

Parameters

		default	Option
sg	The fasta file of the 30k segmentation, the output of 01.wh2mega.pl
ps	The markup file of the 30k segmentation, the output of 02.globalREmarkup.pl
re	The restriction enzyme sites list. It is devided by different standards, type (IIP, IIA, IIB), cost (standard, nonexpensive) and etc.	Standard_and_IIB	IIP/IIA/IIB/Standard/ Nonexpensive/ Standard_IIB Nonexpensive_IIB
a2	2k to 10k assembly strategy (Gibson or Goldengate)	Gibson	Gibson/ Goldengate
a10	10k to 30k assembly strategy (Gibson or Goldengate)	Goldengate	Gibson/ Goldengate
ckmax2	The maximum length of minichunks	2200 bp
ckmin2	The minimum length of minichunks	1800 bp
cknum	The number of minichunks in a chunk	5

If parameter a2 is Gibson, then there are additional parameters:

ol2	The length of overlap	40 bp
tmax2	The maximum melting temperature of the overlap of minichunks	60℃
tmin2	The minimum melting temperature of the overlap of minichunks	56℃
fe2	The minimum free energy of the overlap of minichunks	-3
ex2	The type of exonuclease used for minichunks	T5	T5/T3
lo2	The minimum distance between minichunks overlap and loxpsym	40 bp
en2	The type of enzyme flanking minichunks	IIP
et2
ep2	The maximum unit price of enzyme used in minichunks digestion	0.5 $/unit

If parameter a10 is Goldengate, then there are additional parameters:

en10	The type of enzyme flanking chunks	IIB	IIA/IIB
et10	The temperature of enzyme used in chunks digestion	37℃

The format of ouput

The output file is stored in /the path where you install GENOVO/Result/. 03.mega2chunk2mini.
Besides, there is screen output about the process state and result.
1. Screen output
2. *.2kstate
Store the minichunks states.

Left IIP enzyme site	Right IIP enzyme site	Start	End	Size of minichunks	Melting temperature of overlap

3. *.10kstate
Store the chunks states
Size of chunks

Left IIB enzyme site	Right IIB enzyme site	Start	End

4. *.mini
Store the fasta of designed minichunks.