MEGASAT - Revision history

Luyaozhan: /* Installing MEGASAT */

2015-10-06T15:12:34Z

Installing MEGASAT

Luyaozhan: /* Running “MEGASAT_Genotype.pl” */

2015-10-06T15:11:00Z

Running “MEGASAT_Genotype.pl”

Luyaozhan: /* Mplot.R */

2015-10-05T23:51:58Z

Mplot.R

Luyaozhan: /* MEGASAT_Genotype.pl */

2015-10-05T23:47:19Z

MEGASAT_Genotype.pl

Luyaozhan: /* Input sequence file */

2015-10-05T23:38:22Z

Input sequence file

Luyaozhan: /* MEGASAT_Genotype.pl */

2015-10-05T23:37:29Z

MEGASAT_Genotype.pl

Luyaozhan: /* MEGASAT_Genotype.pl */

2015-07-09T17:46:30Z

MEGASAT_Genotype.pl

Luyaozhan: /* MEGASAT_Genotype.pl */

2015-07-07T14:09:36Z

MEGASAT_Genotype.pl

Luyaozhan: /* MEGASAT_Genotype.pl */

2015-07-07T14:05:20Z

MEGASAT_Genotype.pl

Luyaozhan: /* Primer file */

2015-07-06T19:27:22Z

Primer file

← Older revision		Revision as of 15:12, 6 October 2015
Line 12:		Line 12:

	== Installing MEGASAT ==		== Installing MEGASAT ==
		+
		+	MEGASAT is free available at https://github.com/beiko-lab/MEGASAT.

	=== Windows ===		=== Windows ===

@@ Line 80: / Line 80: @@
 === Running “MEGASAT_Genotype.pl” ===
-If you don’t want to use command line to invoke scripts, a simple GUI is provided for Windows, Mac users. Double click the “MEGASAT_GUI” will display a pop-up page. In the tab page "main GUI", you can click the first “Open” button to open your input primers file. The text field under the “Open” button will display the directory of your primers file. The second small text field is for typing the number of mismatches that gives the error tolerance to forward primers and reverse primers. For five prime flank and three prime flank, the number of mismatches is set based on their lengths.  The second “Open” button is to open the data set folder that contains the input sequence read files. The “Choose” button is to choose the directory to save your output folder. Two radio buttons in this page offer two options- compress the output folder or not compress the output folder. After all these parameters are filled, click the “Run the program” to run the Perl scripts.
+If you don’t want to use command line to invoke scripts, a simple GUI is provided for Windows, Mac users. Double click the “MEGASAT_GUI” will display a pop-up page. In the tab page "main GUI", you can click the first “Open” button to open your input primers file. The text field under the “Open” button will display the directory of your primers file. The second small text field is for typing the number of mismatches that gives the error tolerance to forward primers and reverse primers. For five prime flank and three prime flank, the number of mismatches is set based on their lengths. The next small text field is for typing the minimum depth threshold (we set it to 20 in our experiment). The second “Open” button is to open the data set folder that contains the input sequence read files. The “Choose” button is to choose the directory to save your output folder. Two radio buttons in this page offer two options- compress the output folder or not compress the output folder. After all these parameters are filled, click the “Run the program” to run the Perl scripts.
-If you want to run the scripts from command line, for Windows users, make sure you already have Perl installed in your system. We assume that “MEGASAT_Genotype.pl” and your primers txt file “primers.txt” are saved in the directory “C:\Users\Andy\Downloads”. And the data set folder “dataset” is also saved in the directory “C:\Users\Andy\Downloads”. In order to run the Perl script, first step is go back to the command prompt and type “perl C:\Users\Andy\Downloads\ MEGASAT_Genotype.pl C:\Users\Andy\Downloads\primers.txt 2 C:\Users\Andy\Downloads\dataset C:\Users\Andy\Desktop”.
+If you want to run the scripts from command line, for Windows users, make sure you already have Perl installed in your system. We assume that “MEGASAT_Genotype.pl” and your primers txt file “primers.txt” are saved in the directory “C:\Users\Andy\Downloads”. And the data set folder “dataset” is also saved in the directory “C:\Users\Andy\Downloads”. In order to run the Perl script, first step is go back to the command prompt and type “perl C:\Users\Andy\Downloads\ MEGASAT_Genotype.pl C:\Users\Andy\Downloads\primers.txt 2  20 C:\Users\Andy\Downloads\dataset C:\Users\Andy\Desktop”.
-The first command-line argument is the directory of primers txt file. The second command-line argument is the number of mismatches (2 is a good choice to set). Next argument is the directory of data set folder that contains input sequence read files. The last command-line argument specifies the directory where you want to save your output. After this script is completed, an output folder called “Output_dataset” will be in the saving directory you type in the command line.
+The first command-line argument is the directory of primers txt file. The second command-line argument is the number of mismatches (2 is a good choice to set). The third command-line argument is the minimum depth threshold. Next argument is the directory of data set folder that contains input sequence read files. The last command-line argument specifies the directory where you want to save your output. After this script is completed, an output folder called “Output_dataset” will be in the saving directory you type in the command line.
 === Running “MEGASAT_Update.pl” ===

← Older revision		Revision as of 23:51, 5 October 2015
Line 113:		Line 113:

	=== Mplot.R ===		=== Mplot.R ===
−	The output folder of "Mplot.R" contains many pdfs and each pdf contains many ~~bar plots that represent the~~ length ~~distribution of each individual and~~ each locus. One pdf shows the length ~~distribution~~ of all individuals for one locus.	+	The output folder of "Mplot.R" contains many pdfs and each pdf contains many histograms of sequence length variations for each single locus genotype. One pdf shows the sequence-length frequency distributions of all individuals for one locus.

@@ Line 103: / Line 103: @@
 == Output folder ==
 === MEGASAT_Genotype.pl===
-The output folder generated by “MEGASAT_Genotype.pl” has three types of files. In this folder, “Genotype.txt” is a tab-separated txt file that gives all the genotype information for all the individuals and loci. In this Genotype.txt file, “X X” means that this locus doesn’t occur in this individual. “0 0” means that the depth of alleles is too small to score. “Unscorable Unscorable” means that there are three possible real alleles, which makes the genotype difficult to be determined. "Number_Discarded.txt" is a tab-separated txt file that counts the number of discarded sequences for all the individuals and loci. Those discarded sequences are sequences that have 5' microsatellite primers but have no flank, repeat_unit_sequence and reverse-complement of 3' microsatellite primers. In "Number_Discarded.txt", "X" means that there is no discarded sequences for this individual at this locus. You can use Microsoft Excel to open these tab-separated txt files, which makes these files more easier to read.
+The output folder generated by “MEGASAT_Genotype.pl” has three types of files. In this folder, “Genotype.txt” is a tab-separated txt file that gives all the genotype information for all the individuals and loci. In this Genotype.txt file, “X X” means that this locus doesn’t occur in this individual. “0 0” means that the depth of alleles is too small to score. “Unscored Unscored” means that there are three possible real alleles, which makes the genotype difficult to be determined. "Number_Discarded.txt" is a tab-separated txt file that counts the number of discarded sequences for all the individuals and loci. Those discarded sequences are sequences that have 5' microsatellite primers but have no flank, repeat_unit_sequence and reverse-complement of 3' microsatellite primers. In "Number_Discarded.txt", "X" means that there is no discarded sequences for this individual at this locus. You can use Microsoft Excel to open these tab-separated txt files, which makes these files more easier to read.
-Those txt files whose names start with “Genotype” and follow by the individual names show the length distribution of each microsatellite locus. In those txt files, the first row illustrates the different length for all the loci in one individual. Each row under the first row shows the number of the occurrences of different lengths for each locus. The last column is the genotype information for all loci in one individual. The txt file whose name start with "Ratios" and follow by the data set name shows the ratios group for each microsatellite locus.
+Those txt files whose names start with “Genotype” and follow by the individual names show the sequence length distribution for each microsatellite locus. In those txt files, the first row illustrates the different sequence length for all the loci in one individual. Each row under the first row shows the count of length variants for each locus. The last column is the genotype information for all loci in one individual. The txt file whose name start with "Ratios" and follow by the data set name shows the ratios group for each microsatellite locus.
 And those split files whose names start with “Sorted” and follow by the individual names and loci names contain all the non-trimmed(just trim off 5' microsatellite primers) sequences for one individual & one locus. Obviously, those split files whose names start with “Trimmed” have all the trimmed sequences. All the other split files whose names start with "Discarded" have all the discarded sequences(5' microsatellite primers are trimmed off but cannot find 3' flank,5' flank, repeat_unit_sequence or reverse-complement of 3' microsatellite primers).

@@ Line 59: / Line 59: @@
 ==== Input sequence file ====
-Input sequence read files must be in standard FASTQ format.
+Input sequence read files could be in standard FASTQ format or FASTA format.
 Here is an [[Media:MEGASAT Example.fastq|example of a short FASTQ file that will work with the primer file above.]]

@@ Line 36: / Line 36: @@
 == Input file formats ==
 === MEGASAT_Genotype.pl===
-“MEGASAT_Genotype.pl” requires an input file with information about PCR primers, and a set of .fastq files representing reads from each sampled locus.
+“MEGASAT_Genotype.pl” requires an input file with information about PCR primers, and a set of .fastq or .fasta files representing reads from each sampled locus.
 ==== Primer file ====

@@ Line 50: / Line 50: @@
 In this text file, a header line is required to specify the column name. If one locus doesn’t have 3’ flank and 5' flank, a character “X” needs to be written in the 3’ flank column and 5' flank column in that text file. Column 7 has six ratios that are separated by comma. Here explains how to use these six ratios. We assume A1 is the largest allele peak, A2 is the second largest allele peak, R1&R2&R3&R4&R5&R6 represent six ratios respectively.
-When A1 is smaller than A2, first calculate the quotient of depth(A2) and depth(A1) to see if it is larger than R1. If not, it will be homozygous, which means that A1 A1 will be scored as real alleles. If so, check If there is a stutter peak A3 or A4 that is one repeated unit larger than A2 and depth(A3) or depth(A4)/depth(A2)> = R3, A1 A3 or A1 A4 will be real genotype. If depth(A3 or A4)/depth(A2) is in the range(R2,R3), it will be scored as "unscorable unscorable" since it may have three alleles. If depth(A3 or A4)/depth(A2) is smaller than R2, A1 A2 will be real genotype.
+When A1 is smaller than A2, first calculate the quotient of depth(A2) and depth(A1) to see if it is larger than or equal to R1. If not, it will be homozygous, which means that A1 A1 will be scored as real alleles. If so, check If there is a stutter peak A3 or A4 that is larger than A2 and depth(A3 or A4) >=R2. If so, check if A3 or A4 is one repeated unit larger than A2 and depth(A3) or depth(A4)/depth(A2)> = R3. If this condition is met, A1 A3 or A1 A4 will be real genotype. But if not, it will be scored as "unscorable unscorable" since it may have three alleles. If depth(A3 or A4)/depth(A2) is smaller than R2, A1 A2 will be real genotype.
-When A1 is larger than A2, we need to check if A1-A2>=3 and depth(A2)/depth(A1)>=R4  or A1-A2<=2 and depth(A2)/depth(A1)>=R5 (requirement 1). If so, check if there is a stutter peak A3 > A1 and depth(A3)/depth(A1) > R6. If this requirement is met, it will be scored as "unscorable unscorable". If not, A2 A1 will be real genotype. If requirement 1 is not met,we need to check if A3>A1 and depth(A3)/depth(A1) > R6(requirement 2). If requirement 2 is met, check if there is a stutter peak A4 that is one repeated unit larger than A3 and depth(A4)/depth(A3)> = R3. If so, A1 A4 will be real genotype. If not, A1 A3 will be real genotype. If requirement 1 and requirement 2 are both not met, it will be homozygous, which means that A1 A1 will be scored as real alleles.
+When A1 is larger than A2, we need to check if A1-A2>=3 and depth(A2)/depth(A1)>=R4  or A1-A2<=2 and depth(A2)/depth(A1)>=R5 (requirement 1). If so, check if there is a stutter peak A3 > A1 and depth(A3)/depth(A1) > R6. If this requirement is met, it will be scored as "unscorable unscorable". If not, A2 A1 will be real genotype. If requirement 1 is not met,we need to check if A3>A1 and depth(A3)/depth(A1) >= R6(requirement 2). If requirement 2 is met, check if there is a stutter peak A4 that is one repeated unit larger than A3 and depth(A4)/depth(A3)> = R3. If so, A1 A4 will be real genotype. If not, A1 A3 will be real genotype. If requirement 1 and requirement 2 are both not met, it will be homozygous, which means that A1 A1 will be scored as real alleles.
 If you want to use all the default ratios which is (0.15,0.4,0.7,0.6,0.8,0.2) to predict genotypes, you don't need to write this column in the primer file. But if you want to change part of these six ratios, you can write your own ratios in the corresponding positions in column7. For other ratios you don't want to change, a space can be used in the corresponding position. For example, if the user just want to change the first ratio to 0.3, the column7 format will be (0.3, , , , , ). In the column7, you don't need to write brackets.