Recent changes to MutAid_Manual

MutAid_Manual modified by ramvinay

ramvinay — Mon, 27 Jun 2016 14:23:35 -0000

--- v35
+++ v36
@@ -891,12 +891,6 @@

 ##8. Contact Information 

-***PD Dr. Andreas Weinhäusel***
-andreas.weinhaeusel@ait.ac.at
-
-***Dr. Albert Kriegner***
-albert.kriegner@platomics.com
-
 ***Ram Vinay Pandey***
 ramvinay.pandey@gmail.com

MutAid_Manual modified by ramvinay

ramvinay — Fri, 01 Apr 2016 11:34:44 -0000

MutAid_Manual modified by ramvinay

ramvinay — Fri, 01 Apr 2016 11:32:48 -0000

--- v33
+++ v34
@@ -89,7 +89,9 @@

 ######4.1.1 How to use MutAid Virtual Machine?

-**Step1:** Download and install Virtual Box from https://www.virtualbox.org/.
+**Step1.1:** Download and install the *Virtual Box* from http://www.oracle.com/technetwork/server-storage/virtualbox/downloads/index.html#vbox
+
+**Step1.2:** After installation of Virtual Box download and  install the* Virtual Box Extension Pack* from http://www.oracle.com/technetwork/server-storage/virtualbox/downloads/index.html#extpack

 **Step2:** Download MutAid Virtual machine from https://sourceforge.net/p/mutaid/wiki/Virtual_Machine

MutAid_Manual modified by ramvinay

ramvinay — Mon, 21 Dec 2015 20:36:53 -0000

--- v32
+++ v33
@@ -312,7 +312,7 @@

 ### A comma seperated list of chromosomes, in case dont need to use full genome FASTA sequences an GTF annotation. CAN BE EDITED
 ### This is optional and is extremely useful for targeted sequencing.
-#Target_Chromosome = chr11
+Target_Chromosome = chr11

 ### Provide the Hotspot mutation in bed format which will be co-analyzed.  (optional). CAN BE EDITED
 Hotspot_Bed_File = ""
@@ -432,7 +432,7 @@
 UCSC_CpG_Islands_SNPs = ref_input/hg19_annotation/hg19_cpgIslandExt.txt
 UCSC_Coding_DbSnp = ref_input/hg19_annotation/hg19_CodingDbSnp.txt
 UCSC_OrthoPt3Pa2Rm2_SNPs = ref_input/hg19_annotation/hg19_OrthoPt3Pa2Rm2.txt
-UCSC_RefseqGene_Info = ref_input/hg19_gtf/hg19_refseqgenes_dbxref.gtf
+UCSC_RefseqGene_Info = ref_input/hg19_annotation/hg19_refseqgenes_dbxref.gtf

@@ -579,7 +579,7 @@

 ### A comma seperated list of chromosomes, in case dont need to use full genome FASTA sequences an GTF annotation. CAN BE EDITED 
 ### This is optional and is extremely useful for targeted sequencing.
-#Target_Chromosome = chr13,chr17
+Target_Chromosome = chr13,chr17

 ### Provide the Hotspot mutation in bed format which will be co-analyzed.  (optional) CAN BE EDITED
@@ -627,7 +627,7 @@

 ### Mapper name only one mapper at a time allowed. Example Mapper_Name = bwa. CAN BE EDITED

-Mapper_Name = bowtie2
+Mapper_Name = bwa,bowtie2

 ### Provide the Mapper_Parameters it could be any mapper's parameter
@@ -645,7 +645,7 @@
 ##################################################################################################################################

 ### Variant callers name. The default one is samtools. CAN BE EDITED
-Variant_Caller = samtools
+Variant_Caller = varscan,samtools

 ### SNV and INDEL calling parameters
@@ -697,7 +697,7 @@
 UCSC_CpG_Islands_SNPs = ref_input/hg19_annotation/hg19_cpgIslandExt.txt
 UCSC_Coding_DbSnp = ref_input/hg19_annotation/hg19_CodingDbSnp.txt
 UCSC_OrthoPt3Pa2Rm2_SNPs = ref_input/hg19_annotation/hg19_OrthoPt3Pa2Rm2.txt
-UCSC_RefseqGene_Info = ref_input/hg19_gtf/hg19_refseqgenes_dbxref.gtf
+UCSC_RefseqGene_Info = ref_input/hg19_annotation/hg19_refseqgenes_dbxref.gtf

MutAid_Manual modified by ramvinay

ramvinay — Sun, 20 Dec 2015 14:50:19 -0000

--- v31
+++ v32
@@ -1,9 +1,13 @@
 **The user manual of MutAid can be downloaded in PDF format from [here...](https://sourceforge.net/projects/mutaid/files/Manual.pdf)
 **
+# TABLE OF CONTENTS 
 [TOC]

------
+
+
+-----
+

 ##1. What is MutAid?
 1. MutAid is an integrated pipeline for mutation screening in clinical research. It can analyze Sanger sequencing and NGS data from raw reads to list of annotated mutation list with little or no manual work. The important features of MutAid are described below:

MutAid_Manual modified by ramvinay

ramvinay — Sun, 20 Dec 2015 14:49:43 -0000

--- v30
+++ v31
@@ -1,30 +1,6 @@
 **The user manual of MutAid can be downloaded in PDF format from [here...](https://sourceforge.net/projects/mutaid/files/Manual.pdf)
 **
-# TABLE OF CONTENTS 
 [TOC]
-1. What is MutAid?
-
-2. System requirements
-
-3. Softwares/tools required
-
-4. How to obtain MutAid?
-
-    4.1 Download MutAid Virtual Machine
-    4.1.1 How to use MutAid Virtual Machine?
-    4.2 Download MutAid source code
-       4.3 Run MutAid with test inputs
-       
-5. MutAid inputs requirements
-
-6. How to use MutAid?
-   6.1 Prepare reference input files
-    6.2 Sanger data analysis
-    6.3 NGS data analysis
-    
-7. MutAid outputs description
-
-8. Contact information

 -----

MutAid_Manual modified by ramvinay

ramvinay — Sun, 20 Dec 2015 14:48:52 -0000

--- v29
+++ v30
@@ -799,7 +799,7 @@
 ./mutaid --option_file MutAidOptions_NGS
 ~~~~

-###Alternatively MutAid pipline can be run step by step in sequenctial order
+####Alternatively MutAid pipline can be run step by step in sequenctial order

 **Step2: Run MutAid pipeline step-by-step:**
@@ -842,50 +842,50 @@
 ~~~~
 <OUTPUT_FILE>
 <OUTPUT_DIR>
-   |
-- <fastq_files_after_qc>
         |
-        - <patient1_1.fq;patient1_2.fq>
-        - <patient2_1.fq;patient2_2.fq>
-        - <patient3_1.fq;patient3_2.fq>
-        - .
-        - .
-        - .
-        - <patientN_1.fq;patientN_2.fq>
-
-- <QC_report>
-        |
-        - <before_qc_patient1_1.fq_fastqc_qc_report.html>
-        - <after_qc_patient1_1.fq_fastqc_qc_report.html>
-        - <before_qc_patient1_2.fq_fastqc_qc_report.html>
-        - <after_qc_patient1_2.fq_fastqc_qc_report.html>
-        - .
-                - .
-        - .    |
-        - .
-        - <before_qc_patientN_1.fq_fastqc_qc_report.html>
-        - <after_qc_patientN_1.fq_fastqc_qc_report.html>
-        - <before_qc_patientN_2.fq_fastqc_qc_report.html>
-        - <after_qc_patientN_2.fq_fastqc_qc_report.html>
-
-- < bam_files>
-        |
-        - <patient1.bam>
-        - <patient2.bam>
-        - <patient3.bam>
-        - .
-        - .
-        - .
-        - <patientN.bam>
-- <variant_files>
-        |
-        - <patient1.vcf>
-        - <patient2.vcf>
-        - <patient3.vcf>
-        - .
-        - .
-        - .
-        - <patientN.vcf>
+        - <fastq_files_after_qc>
+                |
+                - <patient1_1.fq;patient1_2.fq>
+                - <patient2_1.fq;patient2_2.fq>
+                - <patient3_1.fq;patient3_2.fq>
+                - .
+                - .
+                - .
+                - <patientN_1.fq;patientN_2.fq>
+
+        - <QC_report>
+                |
+                - <before_qc_patient1_1.fq_fastqc_qc_report.html>
+                - <after_qc_patient1_1.fq_fastqc_qc_report.html>
+                - <before_qc_patient1_2.fq_fastqc_qc_report.html>
+                - <after_qc_patient1_2.fq_fastqc_qc_report.html>
+                - .
+                        - .
+                - .    |
+                - .
+                - <before_qc_patientN_1.fq_fastqc_qc_report.html>
+                - <after_qc_patientN_1.fq_fastqc_qc_report.html>
+                - <before_qc_patientN_2.fq_fastqc_qc_report.html>
+                - <after_qc_patientN_2.fq_fastqc_qc_report.html>
+
+        - < bam_files>
+                |
+                - <patient1.bam>
+                - <patient2.bam>
+                - <patient3.bam>
+                - .
+                - .
+                - .
+                - <patientN.bam>
+        - <variant_files>
+                |
+                - <patient1.vcf>
+                - <patient2.vcf>
+                - <patient3.vcf>
+                - .
+                - .
+                - .
+                - <patientN.vcf>
 ~~~~

MutAid_Manual modified by ramvinay

ramvinay — Sun, 20 Dec 2015 14:46:07 -0000

--- v28
+++ v29
@@ -844,48 +844,48 @@
 <OUTPUT_DIR>
    |
 - <fastq_files_after_qc>
-|
-- <patient1_1.fq;patient1_2.fq>
-- <patient2_1.fq;patient2_2.fq>
-- <patient3_1.fq;patient3_2.fq>
-- .
-- .
-- .
-- <patientN_1.fq;patientN_2.fq>
+        |
+        - <patient1_1.fq;patient1_2.fq>
+        - <patient2_1.fq;patient2_2.fq>
+        - <patient3_1.fq;patient3_2.fq>
+        - .
+        - .
+        - .
+        - <patientN_1.fq;patientN_2.fq>

 - <QC_report>
-|
-- <before_qc_patient1_1.fq_fastqc_qc_report.html>
-- <after_qc_patient1_1.fq_fastqc_qc_report.html>
-- <before_qc_patient1_2.fq_fastqc_qc_report.html>
-- <after_qc_patient1_2.fq_fastqc_qc_report.html>
-- .
-       - .
-- .    |
-- .
-- <before_qc_patientN_1.fq_fastqc_qc_report.html>
-- <after_qc_patientN_1.fq_fastqc_qc_report.html>
-- <before_qc_patientN_2.fq_fastqc_qc_report.html>
-- <after_qc_patientN_2.fq_fastqc_qc_report.html>
+        |
+        - <before_qc_patient1_1.fq_fastqc_qc_report.html>
+        - <after_qc_patient1_1.fq_fastqc_qc_report.html>
+        - <before_qc_patient1_2.fq_fastqc_qc_report.html>
+        - <after_qc_patient1_2.fq_fastqc_qc_report.html>
+        - .
+                - .
+        - .    |
+        - .
+        - <before_qc_patientN_1.fq_fastqc_qc_report.html>
+        - <after_qc_patientN_1.fq_fastqc_qc_report.html>
+        - <before_qc_patientN_2.fq_fastqc_qc_report.html>
+        - <after_qc_patientN_2.fq_fastqc_qc_report.html>

 - < bam_files>
-|
-- <patient1.bam>
-- <patient2.bam>
-- <patient3.bam>
-- .
-- .
-- .
-- <patientN.bam>
+        |
+        - <patient1.bam>
+        - <patient2.bam>
+        - <patient3.bam>
+        - .
+        - .
+        - .
+        - <patientN.bam>
 - <variant_files>
-|
-- <patient1.vcf>
-- <patient2.vcf>
-- <patient3.vcf>
-- .
-- .
-- .
-- <patientN.vcf>
+        |
+        - <patient1.vcf>
+        - <patient2.vcf>
+        - <patient3.vcf>
+        - .
+        - .
+        - .
+        - <patientN.vcf>
 ~~~~

@@ -902,13 +902,20 @@
 ***<variant_files>:***
 This output folder contains resulting variants (SNV, Insertion, and Deletion) in Variant Call Format (VCF) for each patient/sample.

+
+
+-----
+
+
 ##8. Contact Information 

-PD Dr. Andreas Weinhäusel 
+***PD Dr. Andreas Weinhäusel***
 andreas.weinhaeusel@ait.ac.at
-Dr. Albert Kriegner
+
+***Dr. Albert Kriegner***
 albert.kriegner@platomics.com
-Ram Vinay Pandey
+
+***Ram Vinay Pandey***
 ramvinay.pandey@gmail.com

MutAid_Manual modified by ramvinay

ramvinay — Sun, 20 Dec 2015 14:43:58 -0000

--- v27
+++ v28
@@ -835,12 +835,83 @@

 -----

-
-
-
-###############################
-
-
-
-##4.3 Run MutAid with test input
-
+##7. MutAid outputs description
+After running MutAid, the results of the MutAid pipeline can be found in the output directory (as specified in the MutAid Options file). Results are provided in the following format.
+
+
+~~~~
+<OUTPUT_FILE>
+<OUTPUT_DIR>
+   |
+- <fastq_files_after_qc>
+|
+- <patient1_1.fq;patient1_2.fq>
+- <patient2_1.fq;patient2_2.fq>
+- <patient3_1.fq;patient3_2.fq>
+- .
+- .
+- .
+- <patientN_1.fq;patientN_2.fq>
+
+- <QC_report>
+|
+- <before_qc_patient1_1.fq_fastqc_qc_report.html>
+- <after_qc_patient1_1.fq_fastqc_qc_report.html>
+- <before_qc_patient1_2.fq_fastqc_qc_report.html>
+- <after_qc_patient1_2.fq_fastqc_qc_report.html>
+- .
+       - .
+- .    |
+- .
+- <before_qc_patientN_1.fq_fastqc_qc_report.html>
+- <after_qc_patientN_1.fq_fastqc_qc_report.html>
+- <before_qc_patientN_2.fq_fastqc_qc_report.html>
+- <after_qc_patientN_2.fq_fastqc_qc_report.html>
+
+- < bam_files>
+|
+- <patient1.bam>
+- <patient2.bam>
+- <patient3.bam>
+- .
+- .
+- .
+- <patientN.bam>
+- <variant_files>
+|
+- <patient1.vcf>
+- <patient2.vcf>
+- <patient3.vcf>
+- .
+- .
+- .
+- <patientN.vcf>
+~~~~
+
+
+
+***<fastq_files_after_qc>:***
+This output folder contains high quality FASTQ file for each patient with Sanger quality encoding. If reads are in paired-end then there will be two files for each patient.
+
+***<QC_report>:***
+This output folder contains Quality control and trimming report in html format generated by FASTQC tool. There are two files for each FASTQ files 1) before quality control and 2) after quality control.
+
+***<bam_files>:***
+This output folder contains resulting BAM file along with BAM index for each patient/sample. These BAM files have been generated after applying all mapping parameters and post-mapping filtering criteria.
+
+***<variant_files>:***
+This output folder contains resulting variants (SNV, Insertion, and Deletion) in Variant Call Format (VCF) for each patient/sample.
+
+##8. Contact Information 
+
+PD Dr. Andreas Weinhäusel 
+andreas.weinhaeusel@ait.ac.at
+Dr. Albert Kriegner
+albert.kriegner@platomics.com
+Ram Vinay Pandey
+ramvinay.pandey@gmail.com
+
+
+
+-----
+

MutAid_Manual modified by ramvinay

ramvinay — Sun, 20 Dec 2015 14:38:01 -0000

--- v26
+++ v27
@@ -265,7 +265,7 @@

 ![MutAid target file](https://a.fsdn.com/con/app/proj/clinqc/screenshots/Screen%20Shot%202015-12-12%20at%2017.28.58.png)

-***Figure 1: Shows Target files (a) Sanger target file, (2) Illumina target file, (3) 454 target file, (4) Ion torrent target***
+***Figure 1: Shows Target files (a) Sanger target file, (b) Illumina target file, (c) 454 target file, (d) Ion torrent target***

 **2. Prepare MutAidOptions_Sanger file**
@@ -515,6 +515,8 @@
 bamToBed = executables/bedtools2/bin/bamToBed
 ~~~~

+
+
 ***Step2: Run MutAid pipeline:***
 After preparing the Target File and MutAidOptions file have been prepared and customized then MutAid pipeline can be run with following command line

@@ -527,6 +529,314 @@

 -----

+###6.3 NGS data analysis
+***Step1: Prepare Input files:***
+**1. Prepare Target file**
+For each analysis, user need to prepare a target file in a predefined format. It is a tab-separated text file, which contains 10 columns. As shown in ***Figure 1a-c*** above, one row for each sequencing file in target files. Target file is a mandatory input, which must be provided. The target file can be given in the MutAidOptions file with the input name ***Target_File=”ngs_target_file.txt”***
+
+**2. Prepare MutAidOptions_NGS file**
+MutAid requires an input configuration file, which can be prepared once by customizing parameters as per the requirement, and the whole pipeline will be run without further user interaction. The MutAid Option file is shown below:
+
+~~~~
+##################################################################################################################################
+## MutAid Options file for NGS data analysis (MutAidOptions_NGS)
+##
+## Note: This configuration file is used for NGS data analysis.
+##
+## This file sets the customizable options for running MutAid-NGS data analysis. The options include the paths to the
+## database cross reference files, paths to executables, annotation files and other parameters of the pipeline
+##
+## This file contains example parameter settings, which can be used to run the test data set
+## provided with MutAid. 
+## For running MutAid with your own data, the example parameter values have to be modified.
+## Please note that only the input value but not the parameter name itself should be changed.
+##
+## Note: 1) Lines starting with # are comment lines. They do not need to be changed.
+##       2) Parameter's name is case sensitive. The parameters can be changed
+##          by replacing the values after the “=” sign.
+##
+##################################################################################################################################
+
+
+
+##################################################################################################################################
+#
+###             Global input parameters
+#
+##################################################################################################################################
+
+
+# Give the path to the fasta file of the genomic reference. The reference fasta should be obtained from UCSC genome browser.
+REFERENCEE_FASTA = "ref_input/hg19_fasta/hg19.fa"
+
+# Give the gene annotation file of your genomic reference in GTF format. The reference GTF should be obtained from UCSC genome browser.
+REFERENCE_GTF_FILE = "ref_input/hg19_annotation/hg19_refseqgenes.gtf"
+
+### Give the number of processors that should be used in MutAid pipeline. CAN BE EDITED
+THREAD = 4
+
+### Reference genome information from UCSC genome browser
+Genome_Assembly = hg19
+Refseq_Genome_Build = GRCh37
+
+
+### dbSNP version from UCSC genome browser
+dbSNP_Version = 142
+
+
+##################################################################################################################################
+#
+#                                       Provide the experiment information files
+#
+##################################################################################################################################
+
+### Target_File contains Sequencing information. (mandetory). CAN BE EDITED
+Target_File = "test_data/illumina/Illumina_target_file.txt"
+#Target_File = "test_data/illumina/BAM_target_file.txt"
+
+### For targeted resequencing or Sanger sequencing provide the assay information file in GTF format (optional) CAN BE EDITED
+Assay_Gtf_File = ""
+
+### A comma seperated list of chromosomes, in case dont need to use full genome FASTA sequences an GTF annotation. CAN BE EDITED 
+### This is optional and is extremely useful for targeted sequencing.
+#Target_Chromosome = chr13,chr17
+
+
+### Provide the Hotspot mutation in bed format which will be co-analyzed.  (optional) CAN BE EDITED
+Hotspot_Bed_File = ""
+
+### Provide the Primer and adapter sequences for trimming. (optional) CAN BE EDITED
+Primer_Adapter_File = ""
+
+
+##################################################################################################################################
+#
+#                                       Output file and directory location
+#
+##################################################################################################################################
+
+### Provide the output directory. CAN BE EDITED
+OUTPUT_DIRECTORY = "test_output/ngs_output_dir"
+
+### Provide the final output file name. CAN BE EDITED
+#OUTPUT_FILE = "test_output/454_output_file"
+
+
+##################################################################################################################################
+#
+###             Step1: Quality control and filtering
+#
+##################################################################################################################################
+
+### Minimum average base quality for trimming the low quality 5' and 3' end of reads
+Minimum_Base_Quality = 20
+
+### Minimum read length to discard the read for further analysis
+Minimum_Read_Length = 50
+
+### Maximum read length to discard the read for further analysis
+Maximum_Read_Length = 1000
+
+
+
+##################################################################################################################################
+#
+###             Step2: Mapping to reference genome
+#
+##################################################################################################################################
+
+### Mapper name only one mapper at a time allowed. Example Mapper_Name = bwa. CAN BE EDITED
+
+Mapper_Name = bowtie2
+
+
+### Provide the Mapper_Parameters it could be any mapper's parameter
+Mapper_Parameters = ""
+BWA_Sampe_Parameters = ""
+
+### Filtering Mapped reads from BAM/SAM file
+Minimum_Mapping_Quality = 20
+
+
+##################################################################################################################################
+#
+###             Step3: Variant detection
+#
+##################################################################################################################################
+
+### Variant callers name. The default one is samtools. CAN BE EDITED
+Variant_Caller = samtools
+
+
+### SNV and INDEL calling parameters
+### Minimum read coverage to call variant
+#Minimum_Coverage = 20
+Minimum_Coverage = 20
+
+### Maximum read coverage to call variant. This is useful to discard the repetative region.
+Maximum_Coverage = 1000
+
+### Minimum Variant allele read count to consider as variant position
+#Minimum_Count = 4
+Minimum_Count = 4
+
+### Minimum allele frequency of Variant allele to consider as variant position
+Minor_Allele_Frequency = 0.01
+
+
+### Provide the Hotspot mutation in bed format which will be co-analyzed.  (optional)
+Hotspot_Bed_File = ""
+
+
+##################################################################################################################################
+#
+###             Step4: Variant effect prediction
+#
+##################################################################################################################################
+
+
+
+
+
+##################################################################################################################################
+#
+###             Step5: Variant functional annotation
+#
+##################################################################################################################################
+
+## Various SNPs information file from UCSC Table browser
+## Provide the full path to all these files. 
+
+UCSC_All_SNPs = ref_input/hg19_annotation/hg19_snpAll.txt
+UCSC_Common_SNPs = ref_input/hg19_annotation/hg19_snpCommon.txt
+UCSC_Flagged_SNPs = ref_input/hg19_annotation/hg19_FlaggedSNPs.txt
+UCSC_HapMap_SNPs = ref_input/hg19_annotation/hg19_HapMapSNPs.txt
+UCSC_Multi_SNPs = ref_input/hg19_annotation/hg19_MultSNPs.txt
+UCSC_COSMIC_SNPs = ref_input/hg19_annotation/hg19_cosmic.txt
+UCSC_GWAS_Catalog_SNPs = ref_input/hg19_annotation/hg19_gwasCatalog.txt
+UCSC_CpG_Islands_SNPs = ref_input/hg19_annotation/hg19_cpgIslandExt.txt
+UCSC_Coding_DbSnp = ref_input/hg19_annotation/hg19_CodingDbSnp.txt
+UCSC_OrthoPt3Pa2Rm2_SNPs = ref_input/hg19_annotation/hg19_OrthoPt3Pa2Rm2.txt
+UCSC_RefseqGene_Info = ref_input/hg19_gtf/hg19_refseqgenes_dbxref.gtf
+
+
+
+
+##################################################################################################################################
+#
+###             Step6: Producing variant summary output
+#
+##################################################################################################################################
+
+### Give the number of based to show in flanking region for each variant in UCSC browser and Ensemble browser
+Flanking_Region = 100
+
+
+
+##################################################################################################################################
+#
+# Third party software/tool executables
+# Provide the full path to the third party executables to run the MutAid pipeline.
+#  
+#
+##################################################################################################################################
+
+### Get samtools to manipulate SAM and BAM files from http://samtools.sourceforge.net/
+# Provide the full path of the executables relative to the <MutAid_1.0>
+###### SAMTOOLS path      
+SAMTOOLS = executables/samtools-0.1.19/samtools
+BCFTOOLS = executables/samtools-0.1.19/bcftools/bcftools
+
+### Define the full path of the Mappers
+BWA = executables/bwa-0.7.9a/bwa
+TMAP = executables/TMAP/tmap
+GSNAP = executables/gmap-2014-12-28/bin/gsnap
+BOWTIE = executables/bowtie-1.1.2/bowtie
+BOWTIE2 = executables/bowtie2-2.2.5/bowtie2
+
+###### FASTQC executables
+FASTQC = executables/FastQC/fastqc
+
+
+###### AlienTrimmer for Adapter and Primer and Homopolymer trimming
+AlienTrimmer = executables/AlienTrimmer_0.4.0/src/AlienTrimmer.jar
+
+###### varient callers path
+#GATK = executables/GenomeAnalysisTK.jar
+GATK_BUNDLE = ref_input/hg19_gatk_bundle
+
+FreeBayes = executables/freebayes/bin/freebayes
+Varscan2 = executables/VarScan.v2.3.7.jar
+
+
+#### PICARD JAR file from http://picard.sourceforge.net/index.shtml
+###### 3 PICARD jar files are required for MutAid pipeline
+PICARD = executables/picard-tools-1.130/picard.jar
+
+
+
+### Get intersectBed executables from a collection of useful utilities called bedtools-
+### from http://code.google.com/p/bedtools/
+###### 3 bedtools
+intersectBed = executables/bedtools2/bin/intersectBed
+bedtools = executables/bedtools2/bin/bedtools
+bamToBed = executables/bedtools2/bin/bamToBed
+
+
+### Filter the variants by minimum number of supported mappers. CAN BE EDITED
+Minimum_Number_of_supported_mapper=2
+
+### Filter the variants by minimum number of supported variant callers. CAN BE EDITED
+Minimum_Number_of_supported_vc=2
+
+
+~~~~
+
+**Step2: Run MutAid whole pipeline:**
+After preparing the Target File and MutAidOptions file have been prepared and customized then MutAid pipeline can be run with following command line
+
+~~~~
+./mutaid --option_file MutAidOptions_NGS
+~~~~
+
+###Alternatively MutAid pipline can be run step by step in sequenctial order
+
+
+**Step2: Run MutAid pipeline step-by-step:**
+Alternatively user can run MutAid pipeline form NGS data analysis in a step-by-step manner. The main advantage of this feature is, user can optimize the parameters for each step independently without spending time to finish whole pipeline. Once all parameters have been optimized then providing the MutAidOptions_NGS file can run whole pipeline.
+Since NGS data are enormous in size and in coverage thus we have facilitated MutAid whole pipeline with many start and stop points and thus user can run 1) Only quality control and filtering step, 2) Only Mapping step and 3) Only variant calling and 4) Only Variant effect prediction, variant annotation and write final variant summary table output.
+
+**Run MutAid only for Quality control and filtering**
+
+~~~~
+./mutaid --option_file MutAidOptions_NGS --qc
+~~~~
+
+**Run MutAid only for Mapping**
+
+~~~~
+./mutaid --option_file MutAidOptions_NGS --map
+~~~~
+
+**Run MutAid only variant detection**
+
+~~~~
+./mutaid --option_file MutAidOptions_NGS --variant_call
+~~~~
+
+**Run MutAid for only writing output**
+With this command MutAid 1) predicts genomic effects like codon change, amino acid change and genomic feature assignment 2) Functional and clinical annotation of all resulting variants and 3) write final output variant summary table.
+
+~~~~
+./mutaid --option_file MutAidOptions_NGS --write_output
+~~~~
+
+
+
+-----
+
+
+

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