CSBB - Computational Suite For Bioinformaticians and Biologists -> -> -> version3.0
Developer : Praneet Chaturvedi, Senior Analyst Bioinformatics @ Cincinnati Children's Hospital and Medical Center
CSBB provides 18 modules focused on analytical tasks like performing upper-quantile normalization, interactive visualization and next generation sequencing pipelines. CSBB now also has capability to process public data. Providing User with End to End pipeline experience. Detailed instructions on how to execute each module is give below. Also refer to white paper in the package for detailed explanation of each module!!!
Step 1: Open the terminal/Command prompt in MAC OS, LINUX & Windows respectively.
Step 2: Browse to the directory where you have saved the package.
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cd —> change directory
ls —> list directory
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For example: let’s say you have downloaded the package in Downloads.
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MACOS :
cd /Users/xxx/Downloads/CSBB-v3.0
Linux :
cd /home/xxx/Downloads/CSBB-v3.0
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Requirements For CSBB-v3.0::
1) Perl -> -> -> [MACOS and Linux are preloaded with perl, but if perl is missing on your system then please intall perl using : https://www.perl.org/get.html]
2) Python2.7+ -> -> [MACOS and Linux are preloaded with python, but if python is missing on your system then please intall pyhton using : https://www.python.org/downloads/]
3) R -> -> -> -> -> [Please install R on your machine from : https://www.r-project.org/]
We recommend using R-3.3.3 or R-3.4.4 [CSBB-v3.0 is throughly tested with these R versions]
CSBB-v3.0 installs all required R packages automatically and if required packages are not installed then running time of the module which require packages will increase [Only one time process] [CSBB-v3.0 automatically checks for latest updates for R packages]
Note:
1) When you run CSBB-v3.0 first time, then please do run install module to automatically download files and install dependencies [tools/softwares]
2) CSBB-v3.0 can auto install samtools/1.8 but if auto install fails then please install samtools/1.8 using : http://www.htslib.org/doc/samtools.html
3) CSBB-v3.0 now downloads all the required files and indexes for running RNA-Seq and ChIP/ATAC-Seq pipelines automatically from https://sourceforge.net/projects/csbb-repository/files/?source=navbar.
If CSBB is taking a long time to download these files then please directly download files from : https://sourceforge.net/projects/csbb-repository/files/?source=navbar and unzip the files
and place all the files and folders inside the Modules folder
4) CSBB-v3.0 can auto install bowtie2 but if auto install fails then please install bowtie2 using : http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
5) CSBB-v3.0 now creates a log folder CSBB-v3.0_Logs inside the package folder and write logs for all modules [Log, Error and Exit Status]
6) Current reference assemblies : Human : hg19 , Mouse : mm10 , Xenopus-Laevis : Laevis_9.2 , Xenopus-Trop : Trop_9.1 and Zebrafish : Zv10
7) If you wish to use your own reference indexes of Bowtie2 and RSEM then please match the current name and file format of the files in the index folders and place them in the Modules folder
8) Now CSBB supports processing of public data from SRA [https://www.ncbi.nlm.nih.gov/sra]
9) If running perl CSBB-v3.0_MacOS.pl or perl CSBB-v3.0_Linux.pl gives an error then follow steps mentioned below:
if MacOS then run commands mentioned below from command line:
*) sudo perl -MCPAN -e 'install Bundle::CPAN'
*) sudo perl -MCPAN -e 'install Capture::Tiny'
if Linux then run commands mentioned below from command line:
if Ubuntu
*) sudo apt-get install build-essential
*) sudo apt-get install libbundle-cpan-perl
*) sudo apt-get install libcapture-tiny-perl
if Redhat
*) sudo yum install build-essential
*) sudo yum install perl-Bundle-CPAN
*) sudo yum install perl-Capture-Tiny
10) CSBB-v3.0 requires complete path to file and folder in use : for example : /Users/xxxx/Desktop/Data.txt or /Users/xxxx/Desktop/CSBB-files
11) Please note that users can simply drag and drop the file/folder Finder Window (MAC) and LINUX respectively when path to a folder or a file is required
12) Please remove space and quotes when files and folders are dragged from finder
13) LINUX users please remove quotes on the file and folder path when using drag and drop
14) If you do not have administrative privileges on your machine where CSBB-v3.0 is installed then please run CSBB with sudo command : for example : sudo perl CSBB-v3.0_MacOS.pl install or sudo perl CSBB-v3.0_MacOS.pl Process-ChIP-ATAC_SingleEnd
15) FetchGEO and FetchSRA modules have been removed from CSBB-v3.0 and three new modules have been added : ProcessPublicData, Process-ChIP-ATAC_SingleEnd and Process-ChIP-ATAC_PairedEnd
16) CSBB-v3.0 uses bash scripts to process RNA-Seq and ChIP/ATAC-Seq data inhouse or public [User can change parameters of tools that CSBB-v3.0 uses in bash scripts. By default, CSBB-v3.0 uses all default parameters. If any change has been done to Bash scripts then please on command line browse to Modules folder and do chmod +x BashScriptName.sh
How to Create SRA-DATA_Table.txt ?
##### Please see all this information can be gathered from https://www.ncbi.nlm.nih.gov/sra
##### SRA PUBLIC DATA INFO FILE
##### SRA ID : For example - SRR2924628
##### SPECIES : human or mouse or xenopus-leavis or xenopus-trop or zebrafish
##### ASSAY TYPE : RNA-SEQ or ATAC or ChIP-TF or ChIP-Epigenetic
##### READ TYPE : SINGLE-END or PAIRED-END
##### SAMPLE NAME : WHOLE-Genome
SRRid Species Assay ReadType SampleName
SRR2924628 xenopus-leavis RNA-SEQ SINGLE-END Sample1
Please Find SRA_DATA_TABLE.txt in the package to test ProcessPublicData Module
~~~Running each module
All modules can be run in two ways:
1) Giving all arguments from command line in one line [Batch Mode Run if you have more than one file as input].
2) Providing arguments when prompted by the application.
Let’s see how to use —help option in the application.
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MAC USERS ::
perl CSBB-v3.0_MacOS.pl —help
LINUX USERS ::
perl CSBB-v3.0_Linux.pl —help
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Below is the sample output from —help option
Operating System is darwin
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CSBB - Computational Suite For Bioinformaticians and Biologists
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Version_3.0
Developed and maintained by Praneet Chaturvedi!!
Please report issues and bugs on https://github.com/csbbcompbio/CSBB-v3.0
CSBB-v3.0 Last run : Wed May 9 17:35:16 2018
Current Run: Wed May 9 17:36:29 2018
/Users/pra7mx/Desktop/Development_CSBB_v3.0/CSBB-v3.0_Logs directory is already present, CSBB will use the same to write module logs
Please See README and White-Paper for getting detailed instructions on running CSBB-v3.0
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Please See : Please run install module if using CSBB-v3.0 for first time [just one time process]
Please use options below to run CSBB - Computational Suite For Bioinformaticians and Biologists
Options::
Citation ---> for citing CSBB please use url : https://github.com/csbbcompbio/CSBB-v3.0
install ---> for installing all the required dependencies for Pipelines ::only one time process
UpperQuantile ---> for performing upper quantile normalization
BasicStats ---> for obtaining stats like mean, median, standard deviation, variance, Sum, min and max for each Gene Expression profile
ExpressionToZscore ---> for obtaining z-scores for Gene Expression in samples
ExtractGeneInfo ---> for obtaining info/expression of list genes from a huge matrix gene info/expression
ExpressionPlot ---> for generating line plot visualizing expression/information of genes/entities across samples/objects
InteractiveHeatmap ---> for generating interactive heatmaps for expression data. User has three options on clustering type and four choices on color theme. Please read README for descriptions and run command.
CorrelationProfiles ---> for obtaining genes correlation profile termed as positively and negatively correlated based on User threshold. One can obtain profile for all genes or just genes of interest (For genes of interest user needs to provide the path to gene list file). Heatmap will only be displayed for genes of interest
Biogrid-Gene-Protein-Search ---> for obtaining gene-protein interactions for Human and Mouse for genes of interest
DifferentialExpression ---> for obtaining DE genes in RNA-SEQ expriments. Uses RUVSeq package in R
PCA ---> for performing Principal Component Analysis
NMF ---> for performing Nonnegative Matrix Factorization on Samples in Expression dataset
ProcessPublicData ---> for processing public data [Includes downloading .sra files from SRA, mapping to reference genome and running processing pipelines based on Assay Type for human, mouse, frog and zebrafish]
InteractiveScatterPlot ---> for generating Interactive Scatter plot based on user preference. User needs to provide path to the file, Column number for x-axis values, Column number for y-axis values and Column number which user needs for color factorization (If users provides No color factorization will not be done) Please see README for extensive explanation
Process-RNASeq_SingleEnd ---> for processing Single End RNASeq data using RSEM for human, mouse, frog and zebrafish
Process-RNASeq_PairedEnd ---> for processing Paired End RNASeq data using RSEM for human, mouse, frog and zebrafish
Generate-TPM-Counts-Matrix ---> for generating TPM and Counts Matrix for Both Isoforms and Genes using RSEM result directory and species of interest
Process-ChIP-ATAC_SingleEnd ---> for processing Single End ChIP and ATAC Seq data for human, mouse and frog
Process-ChIP-ATAC_PairedEnd ---> for processing Paired End ChIP and ATAC Seq data for human, mouse and frog
I) Running install:
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perl CSBB-v3.0_MacOS.pl install
perl CSBB-v3.0_Linux.pl install
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II) Running UpperQuantile:
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perl CSBB-v3.0_MacOS.pl UpperQuantile Path_to_file
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Example Mac Users: perl CSBB-v3.0_MacOS.pl UpperQuantile /Users/xx/Desktop/SAMPLE.txt
Example Linux Users: perl CSBB-v3.0_Linux.pl UpperQuantile /Users/xx/Desktop/SAMPLE.txt
Now if you did not provide the path to file as an argument
Example: perl CSBB-v3.0_MacOS.pl UpperQuantile
— > Application will prompt to provide user input
III) Running BasicStats:
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perl CSBB-v3.0_MacOS.pl BasicStats Path_to_file
perl CSBB-v3.0_Linux.pl BasicStats Path_to_file
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Example Mac Users: perl CSBB-v3.0_MacOS.pl BasicStats /Users/xx/Desktop/FIND.txt
Example Linux Users: perl CSBB-v3.0_Linux.pl BasicStats /Users/xx/Desktop/FIND.txt
IV) Running ExpressionToZscore:
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perl CSBB-v3.0_MacOS.pl ExpressionToZscore Path_to_file
perl CSBB-v3.0_Linux.pl ExpressionToZscore Path_to_file
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Example Mac Users: perl CSBB-v3.0_MacOS.pl ExpressionToZscore /Users/xx/Desktop/FIND.txt
Example Linux Users: perl CSBB-v3.0_Linux.pl ExpressionToZscore /Users/xx/Desktop/FIND.txt
V) Running ExtractGeneInfo:
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perl CSBB-v3.0_MacOS.pl ExtractGeneInfo Path_to_Expression/Info_file Path_to_Gene_List_File
perl CSBB-v3.0_Linux.pl ExtractGeneInfo Path_to_Expression/Info_file Path_to_Gene_List_File
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VI) Running InteractiveHeatmap:
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perl CSBB-v3.0_MacOS.pl InteractiveHeatmap Path_to_File Clustering_Option [Row_Clust, Col_Clust or Row_Col_Clust] Color_theme [YellowGreenOrange, BlueWhiteRed, YellowBlackBlue or GreenWhitePurple]
perl CSBB-v3.0_Linux.pl InteractiveHeatmap Path_to_File Clustering_Option [Row_Clust, Col_Clust or Row_Col_Clust] Color_theme [YellowGreenOrange, BlueWhiteRed, YellowBlackBlue or GreenWhitePurple]
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Example : perl CSBB-v3.0_MacOS.pl InteractiveHeatmap /Users/xxxx/Desktop/Heatmap.txt Row_Col_Clust BlueWhiteRed
In color theme : First color represents low expression and last color represents high expression
VII) Running CorrelationProfiles:
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perl CSBB-v3.0_MacOS.pl CorrelationProfiles Path_to_File Correlation_Threshold [-1 to 1] all/Path to Gene list Correlation_Type[pearson or spearman or kendall]
perl CSBB-v3.0_Linux.pl CorrelationProfiles Path_to_File Correlation_Threshold [-1 to 1] all/Path to Gene list Correlation_Type[pearson or spearman or kendall]
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Example : perl CSBB-v3.0_MacOS.pl CorrelationProfiles /Users/xxxx/Desktop/File.txt 0.75 all pearson
Example : perl CSBB-v3.0_MacOS.pl CorrelationProfiles /Users/xxxx/Desktop/File.txt 0.75 Users/xxx/Desktop/GeneList.txt pearson
**use all to calculate correlation profiles all the genes/entities in the matrix.
**using path to gene list file to calculate correlation profile for a specified set of genes.
**Please specify which correlation method you want CSBB to use [use Pearson for linear dependency and Spearman when interested in ranked correlation]
VIII) Running Biogrid-Gene-Protein-Search:
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perl CSBB-v3.0_MacOS.pl Biogrid-Gene-Protein-Search Human/Mouse Path_to_gene_list
perl CSBB-v3.0_Linux.pl Biogrid-Gene-Protein-Search Human/Mouse Path_to_gene_list
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IX) Running DifferentialExpression
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perl CSBB-v3.0_MacOS.pl DifferentialExpression Path_to_Counts_File Number_of_Controls Number_of_Treatments Counts_Threshold_for_filtering Number_of_Samples_for_Filtering_per_Gene Type_of_Normalization [UpperQuantile or UpperQuantile+Empirical]
perl CSBB-v3.0_Linux.pl DifferentialExpression Path_to_Counts_File Number_of_Controls Number_of_Treatments Counts_Threshold_for_filtering Number_of_Samples_for_Filtering_per_Gene Type_of_Normalization [UpperQuantile or UpperQuantile+Empirical]
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Example: perl CSBB-v3.0_MacOS.pl DifferentialExpression Path_to_Counts_File 10 10 5 8 UpperQuantile
*** Please see that place your controls/control reps first and then treatments/treatment reps
*** Please read https://bioconductor.org/packages/release/bioc/vignettes/RUVSeq/inst/doc/RUVSeq.pdf for understanding types of Normalization
*** Generally if sequencing Quality is good then UpperQuantile normalization works best
*** CSBB advise using both Normalization Types separately and see which gives you robust/best results
X) Running PCA
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perl CSBB-v3.0_MacOS.pl PCA Path_to_File
perl CSBB-v3.0_Linux.pl PCA Path_to_File
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XI) Running NMF
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perl CSBB-v3.0_MacOS.pl NMF Path_to_File
perl CSBB-v3.0_Linux.pl NMF Path_to_File
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XII) Running ProcessPublicData
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perl CSBB-v3.0_MacOS.pl ProcessPublicData Path_to_SRA-DATA_Table Path_to_Folder_to_write_results
perl CSBB-v3.0_Linux.pl ProcessPublicData Path_to_SRA-DATA_Table Path_to_Folder_to_write_results
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*** Please see for SRA-DATA_table example in the CSBB-v3.0 folder
*** All the information has been provided in the example table file
XIII) Running InteractiveScatterPlot
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perl CSBB-v3.0_MacOS.pl InteractiveScatterPlot Path_to_File Column_x_axis_values Column_y_axis_values Column_for_Color_Factorization/No
perl CSBB-v3.0_Linux.pl InteractiveScatterPlot Path_to_File Column_x_axis_values Column_y_axis_values Column_for_Color_Factorization/No
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Example : perl CSBB-v3.0_MacOS.pl InteractiveScatterPlot /Users/xxx/Desktop/DE.txt 3 2 7 -> When you are using column 7 for color factorization
Example : perl CSBB-v3.0_MacOS.pl InteractiveScatterPlot /Users/xxx/Desktop/DE.txt 3 2 No -> When no color factorization is needed
XIV) Running Process-RNASeq_SingleEnd
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perl CSBB-v3.0_MacOS.pl Process-RNASeq_SingleEnd Path_to_Fastq_File Species [human or mouse or xenopus-leavis or xenopus-trop or zebrafish ] Output_Folder_path Phred_Quality_encoding [phred33 or phred64 or solexa]
perl CSBB-v3.0_Linux.pl Process-RNASeq_SingleEnd Path_to_Fastq_File Species [human or mouse or xenopus-leavis or xenopus-trop or zebrafish] Output_Folder_path Phred_Quality_encoding [phred33 or phred64 or solexa]
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Example :
perl CSBB-v3.0_MacOS.pl Process-RNASeq_SingleEnd /Users/xx/Desktop/my_fastq.fastq human /Users/xx/Desktop phred33
perl CSBB-v3.0_Linux.pl Process-RNASeq_SingleEnd /Users/xx/Desktop/my_fastq.fastq human /Users/xx/Desktop phred33
*** More information about phred quality encoding can be gathered from : https://en.wikipedia.org/wiki/FASTQ_format - Encoding
*** From 2011 Illumina [1.8+] version of pipelines use phred33 encoding for reads
XV) Running Process-RNASeq_PairedEnd
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perl CSBB-v3.0_MacOS.pl Process-RNASeq_PairedEnd Path_to_Fastq_File_pair1 Path_to_Fastq_File_pair2 Species [human or mouse or xenopus-leavis or xenopus-trop or zebrafish] Output_Folder_path Phred_Quality_encoding [phred33 or phred64 or solexa]
perl CSBB-v3.0_Linux.pl Process-RNASeq_PairedEnd Path_to_Fastq_File_pair1 Path_to_Fastq_File_pair2 Species [human or mouse or xenopus-leavis or xenopus-trop or zebrafish] Output_Folder_path Phred_Quality_encoding [phred33 or phred64 or solexa]
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Example :
perl CSBB-v3.0_MacOS.pl Process-RNASeq_PairedEnd /Users/xx/Desktop/my_fastq_pair1.fastq /Users/xx/Desktop/my_fastq_pair2.fastq human /Users/xx/Desktop phred33
perl CSBB-v3.0_Linux.pl Process-RNASeq_PairedEnd /Users/xx/Desktop/my_fastq_pair1.fastq /Users/xx/Desktop/my_fastq_pair2.fastq human /Users/xx/Desktop phred33
*** More information about phred quality encoding can be gathered from: https://en.wikipedia.org/wiki/FASTQ_format - Encoding
*** From 2011 Illumina [1.8+] version of pipelines use phred33 encoding for reads
XVI) Running Generate-TPM-Counts-Matrix
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perl CSBB-v3.0_MacOS.pl Generate-TPM-Counts-Matrix Path_to_RNA-SEQ_Result_Directory Species [human or mouse or xenopus-leavis or xenopus-trop or zebrafish] Path_to_Output_Directory
perl CSBB-v3.0_Linux.pl Generate-TPM-Counts-Matrix Path_to_RNA-SEQ_Result_Directory Species [human or mouse or xenopus-leavis or xenopus-trop or zebrafish] Path_to_Output_Directory
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Example :
perl CSBB-v3.0_MacOS.pl Generate-TPM-Counts-Matrix /Users/xx/Desktop/CSBB_RNA-SEQ human /Users/xx/Desktop
perl CSBB-v3.0_Linux.pl Generate-TPM-Counts-Matrix /Users/xx/Desktop/CSBB_RNA-SEQ human /Users/xx/Desktop
XVII) Running ExpressionPlot
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perl CSBB-v3.0_MacOS.pl ExpressionPlot Path_to_File
perl CSBB-v3.0_Linux.pl ExpressionPlot Path_to_File
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XVII) Running Process-ChIP-ATAC_SingleEnd
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perl CSBB-v3.0_MacOS.pl Process-ChIP-ATAC_SingleEnd Path_to_Fastq_File Species [human or mouse or xenopus-leavis or xenopus-trop] Output_Folder_path Phred_Quality_encoding [phred33 or phred64 or solexa] ExperimentType [ATAC or ChIP-TF or ChIP-Epigenetic]
perl CSBB-v3.0_Linux.pl Process-ChIP-ATAC_SingleEnd Path_to_Fastq_File Species [human or mouse or xenopus-leavis or xenopus-trop] Output_Folder_path Phred_Quality_encoding [phred33 or phred64 or solexa] ExperimentType [ATAC or ChIP-TF or ChIP-Epigenetic]
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Example :
perl CSBB-v3.0_MacOS.pl Process-ChIP-ATAC_SingleEnd /Users/xx/Desktop/my_fastq_pair1.fastq human /Users/xx/Desktop phred33 ChIP-TF
perl CSBB-v3.0_Linux.pl Process-ChIP-ATAC_SingleEnd /Users/xx/Desktop/my_fastq_pair1.fastq human /Users/xx/Desktop phred33 ChIP-TF
XVIII) Running Process-ChIP-ATAC_PairedEnd
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perl CSBB-v3.0_MacOS.pl Process-ChIP-ATAC_PairedEnd Path_to_Fastq_File_pair1 Path_to_Fastq_File_pair2 Species [human or mouse or xenopus-leavis or xenopus-trop] Output_Folder_path Phred_Quality_encoding [phred33 or phred64 or solexa] ExperimentType [ATAC or ChIP-TF or ChIP-Epigenetic]
perl CSBB-v3.0_Linux.pl Process-ChIP-ATAC_PairedEnd Path_to_Fastq_File_pair1 Path_to_Fastq_File_pair2 Species [human or mouse or xenopus-leavis or xenopus-trop] Output_Folder_path Phred_Quality_encoding [phred33 or phred64 or solexa] ExperimentType [ATAC or ChIP-TF or ChIP-Epigenetic]
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Example :
perl CSBB-v3.0_MacOS.pl Process-ChIP-ATAC_PairedEnd /Users/xx/Desktop/my_fastq_pair1.fastq /Users/xx/Desktop/my_fastq_pair2.fastq human /Users/xx/Desktop phred33 ChIP-TF
perl CSBB-v3.0_Linux.pl Process-ChIP-ATAC_PairedEnd /Users/xx/Desktop/my_fastq_pair1.fastq /Users/xx/Desktop/my_fastq_pair2.fastq human /Users/xx/Desktop phred33 ChIP-TF
################################################################ please read ##############################################
****** CSBB is learning and evolving. If you have questions or Bugs please contact Praneet Chaturvedi on https://github.com/csbbcompbio/CSBB-v3.0
############################################################## END OF README ####################################################