update pipeline number

torres-alexis · torres-alexis · commit 6c92e0eef999 · 2025-01-28T14:44:22.000-08:00
diff --git a/RNAseq/Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-G.md b/RNAseq/Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-G.md
@@ -4,7 +4,7 @@
 
 ---
 
-**Date:** January 24, 2025  
+**Date:** January 28, 2025  
 **Revision:** G  
 **Document Number:** GL-DPPD-7101-G  
 
@@ -23,14 +23,14 @@ Lauren Sanders (GeneLab Project Scientist)
 
 ## Updates from previous version  
 
-Added separate pipeline document: [GL-DPPD-7XXX.md](../Pipeline_GL-DPPD-7XXX_Versions/GL-DPPD-7XXX.md) to document the pipeline steps for Bowtie2 alignment, used when the `--microbes` parameter is specified. In short, reads are aligned to a reference genome using Bowtie2 rather than STAR, gene counts are quantified using FeatureCounts rather than RSEM. Other steps remain unchanged.
+Added separate pipeline document: [GL-DPPD-7115.md](../Pipeline_GL-DPPD-7115_Versions/GL-DPPD-7115.md) to document the pipeline steps for Bowtie2 alignment, used when the `--microbes` parameter is specified. In short, reads are aligned to a reference genome using Bowtie2 rather than STAR, gene counts are quantified using FeatureCounts rather than RSEM. Other steps remain unchanged.
 
 Added "_GLbulkRNAseq" suffix to output files to prevent naming conflicts with files relevant to other assays.
 
-Updated [Ensembl Reference Files](../../GeneLab_Reference_Annotations/Pipeline_GL-DPPD-7110_Versions/GL-DPPD-7110/GL-DPPD-7110_annotations.csv) to use:
-- Animals: Ensembl release 111 → 112
-- Plants: Ensembl plants release 57 → 59  
-- Bacteria: Ensembl bacteria release 57 → 59  
+Updated [Ensembl Reference Files](../../GeneLab_Reference_Annotations/Pipeline_GL-DPPD-7110_Versions/GL-DPPD-7110-A/GL-DPPD-7110-A_annotations.csv) now use:
+- Animals: Ensembl release 112
+- Plants: Ensembl plants release 59
+- Bacteria: Ensembl bacteria release 59
 
 Software Updates:
 
diff --git a/RNAseq/Pipeline_GL-DPPD-7115_Versions/GL-DPPD-7115.md b/RNAseq/Pipeline_GL-DPPD-7115_Versions/GL-DPPD-7115.md
@@ -4,8 +4,8 @@
 
 ---
 
-**Date:** January 24, 2025  
-**Document Number:** GL-DPPD-7XXX  
+**Date:** January 28, 2025  
+**Document Number:** GL-DPPD-7115   
 
 **Submitted by:**  
 Alexis Torres (GeneLab Data Processing Team)  
@@ -93,6 +93,7 @@ Differences with default workflow:
 |Cutadapt|4.2|[https://cutadapt.readthedocs.io/en/stable/](https://cutadapt.readthedocs.io/en/stable/)|
 |TrimGalore!|0.6.10|[https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/](https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)|
 |Bowtie 2|2.5.4|[https://github.com/BenLangmead/bowtie2](https://github.com/BenLangmead/bowtie2)|
+|subread|2.0.8|[https://subread.sourceforge.net/](https://subread.sourceforge.net/)|
 |Samtools|1.21|[http://www.htslib.org/](http://www.htslib.org/)|
 |infer_experiment|5.0.4|[http://rseqc.sourceforge.net/#infer-experiment-py](http://rseqc.sourceforge.net/#infer-experiment-py)|
 |geneBody_coverage|5.0.4|[http://rseqc.sourceforge.net/#genebody-coverage-py](http://rseqc.sourceforge.net/#genebody-coverage-py)|
diff --git a/RNAseq/README.md b/RNAseq/README.md
@@ -17,7 +17,7 @@ RNAseq (Prokaryotes):
 
   - Contains the current and previous GeneLab RNAseq consensus processing pipeline (RCP) versions documentation
 
-* [**Pipeline_GL-DPPD-7XXX_Versions**](Pipeline_GL-DPPD-7XXX_Versions)
+* [**Pipeline_GL-DPPD-7115_Versions**](Pipeline_GL-DPPD-7115_Versions)
 
   - Contains the current and previous GeneLab RNAseq (Prokaryotes) consensus processing pipeline (RCP) versions documentation
 
diff --git a/RNAseq/Workflow_Documentation/NF_RCP/CHANGELOG.md b/RNAseq/Workflow_Documentation/NF_RCP/CHANGELOG.md
@@ -5,15 +5,21 @@ All notable changes to this project will be documented in this file.
 The format is based on [Keep a Changelog](https://keepachangelog.com/en/1.0.0/),
 and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html).
 
-## [2.0.0](https://github.com/nasa/GeneLab_Data_Processing/tree/NF_RCP-G_2.0.0/RNAseq/Workflow_Documentation/NF_RCP) - 2025-01-24
+## [2.0.0](https://github.com/nasa/GeneLab_Data_Processing/tree/NF_RCP-G_2.0.0/RNAseq/Workflow_Documentation/NF_RCP) - 2025-01-28
 
 ### Added
 
-- Prokaryotes pipeline support via `--microbes` parameter
-  - In short, reads are aligned to a reference genome using Bowtie 2 rather than STAR, gene counts are quantified using featureCounts rather than RSEM. Other steps remain unchanged.
-- Unaligned reads FASTQ output from STAR
-- Variance-stabilizing transformation (VST) counts output
-- Parallel rRNA-removed DGE analysis and results. Additional 04-DESeq2_NormCounts_rRNArm/ and 05-DESeq2_DGE_rRNArm/ directories are created for rRNA-removed DGE results.
+- Prokaryotes pipeline support via `--microbes` parameter:  
+  - Reads are aligned to a reference genome using Bowtie 2 rather than STAR, and gene counts are quantified using featureCounts instead of RSEM. Other steps remain unchanged.  
+  - Added software versions:  
+    - Bowtie 2 2.5.4  
+    - subread 2.0.8  
+- Read alignment now outputs unaligned reads as FASTQ files.  
+- Added Variance-stabilizing transformation (VST) counts table.**  
+- Incorporated rRNA removal into gene counts and differential gene expression (DGE) analysis.  
+  - Separate results are generated for rRNA-removed DGE analysis, with new output directories:  
+    - `04-DESeq2_NormCounts_rRNArm/`  
+    - `05-DESeq2_DGE_rRNArm/`
 
 ### Changed
 
@@ -39,10 +45,10 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0
   - matplotlib 3.8.3
   - numpy 1.26.4
   - scipy 1.14.1
-- Updated Ensembl reference files:
-  - Animals: release 111 → 112
-  - Plants: release 57 → 59
-  - Bacteria: release 57 → 59
+- Updated [Ensembl Reference Files](../../../GeneLab_Reference_Annotations/Pipeline_GL-DPPD-7110_Versions/GL-DPPD-7110-A/GL-DPPD-7110-A_annotations.csv) now use: 
+  - Animals: Ensembl release 112
+  - Plants: Ensembl plants release 59
+  - Bacteria: Ensembl bacteria release 59
 - Added "_GLbulkRNAseq" suffix to output files
 - RSeQC inner_distance minimum value now dynamically set based on read length
 - DESeq2 analysis now handles technical replicates
diff --git a/RNAseq/Workflow_Documentation/NF_RCP/README.md b/RNAseq/Workflow_Documentation/NF_RCP/README.md
@@ -1,10 +1,10 @@
-# NF_RCP-F Workflow Information and Usage Instructions <!-- omit in toc -->
+# NF_RCP Workflow Information and Usage Instructions <!-- omit in toc -->
 
 ## General Workflow Info <!-- omit in toc -->
 
 ### Implementation Tools <!-- omit in toc -->
 
-The current GeneLab RNAseq consensus processing pipeline (RCP), [GL-DPPD-7101-F](../../Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-F.md), is implemented as a [Nextflow](https://nextflow.io/) DSL2 workflow and utilizes [Singularity](https://docs.sylabs.io/guides/3.10/user-guide/introduction.html) to run all tools in containers. This workflow (NF_RCP-F) is run using the command line interface (CLI) of any unix-based system.  While knowledge of creating workflows in Nextflow is not required to run the workflow as is, [the Nextflow documentation](https://nextflow.io/docs/latest/index.html) is a useful resource for users who want to modify and/or extend this workflow.   
+The current GeneLab RNAseq consensus processing pipeline (RCP), [GL-DPPD-7101-G](../../Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-G.md), is implemented as a [Nextflow](https://nextflow.io/) DSL2 workflow and utilizes [Singularity](https://docs.sylabs.io/guides/3.10/user-guide/introduction.html) to run all tools in containers. This workflow (NF_RCP-F) is run using the command line interface (CLI) of any unix-based system.  While knowledge of creating workflows in Nextflow is not required to run the workflow as is, [the Nextflow documentation](https://nextflow.io/docs/latest/index.html) is a useful resource for users who want to modify and/or extend this workflow.   
 
 ### Workflow & Subworkflows <!-- omit in toc -->
 
@@ -17,20 +17,20 @@ The current GeneLab RNAseq consensus processing pipeline (RCP), [GL-DPPD-7101-F]
 </p>
 
 ---
-The NF_RCP-F workflow is composed of three subworkflows as shown in the image above.
-Below is a description of each subworkflow and the additional output files generated that are not already indicated in the [GL-DPPD-7101-F pipeline 
-document](../../Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-F.md):
+The NF_RCP-G workflow is composed of three subworkflows as shown in the image above.
+Below is a description of each subworkflow and the additional output files generated that are not already indicated in the [GL-DPPD-7101-G pipeline 
+document](../../Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-G.md):
 
 1. **Analysis Staging Subworkflow**
 
    - Description:
      - This subworkflow extracts the metadata parameters (e.g. organism, library layout) needed for processing from the OSD/GLDS ISA archive and retrieves the raw reads files hosted on the [Open Science Data Repository (OSDR)](https://osdr.nasa.gov/bio/repo/).
        > *OSD/GLDS ISA archive*: ISA directory containing Investigation, Study, and Assay (ISA) metadata files for a respective GLDS dataset - the *ISA.zip file is located in the [OSDR](https://osdr.nasa.gov/bio/repo/) under 'Files' -> 'Study Metadata Files' for any GeneLab Data Set (GLDS) in the OSDR.
 
-2. **RNASeq Consensus Pipeline Subworkflow**
+2. **RNAseq Consensus Pipeline Subworkflow**
 
    - Description:
-     - This subworkflow uses the staged raw data and metadata parameters from the Analysis Staging Subworkflow to generate processed data using [version F of the GeneLab RCP](../../Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-F.md).
+     - This subworkflow uses the staged raw data and metadata parameters from the Analysis Staging Subworkflow to generate processed data using [version G of the GeneLab RCP](../../Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-G.md).
 
 3. **V&V Pipeline Subworkflow**
 
@@ -97,13 +97,13 @@ We recommend installing Singularity on a system wide level as per the associated
 
 ### 2. Download the Workflow Files
 
-All files required for utilizing the NF_RCP-F GeneLab workflow for processing RNASeq data are in the [workflow_code](workflow_code) directory. To get a 
+All files required for utilizing the NF_RCP-F GeneLab workflow for processing RNAseq data are in the [workflow_code](workflow_code) directory. To get a 
 copy of latest NF_RCP-F version on to your system, the code can be downloaded as a zip file from the release page then unzipped after downloading by running the following commands: 
 
 ```bash
-wget https://github.com/nasa/GeneLab_Data_Processing/releases/download/NF_RCP-F_1.0.4/NF_RCP-F_1.0.4.zip
+wget https://github.com/nasa/GeneLab_Data_Processing/releases/download/NF_RCP-G_2.0.0/NF_RCP-G_2.0.0.zip
 
-unzip NF_RCP-F_1.0.4.zip
+unzip NF_RCP-G_2.0.0.zip
 ```
 
 <br>
@@ -114,11 +114,11 @@ unzip NF_RCP-F_1.0.4.zip
 
 Although Nextflow can fetch Singularity images from a url, doing so may cause issues as detailed [here](https://github.com/nextflow-io/nextflow/issues/1210).
 
-To avoid this issue, run the following command to fetch the Singularity images prior to running the NF_RCP-F workflow:
-> Note: This command should be run in the location containing the `NF_RCP-F_1.0.4` directory that was downloaded in [step 2](#2-download-the-workflow-files) above. Depending on your network speed, fetching the images will take ~20 minutes.  
+To avoid this issue, run the following command to fetch the Singularity images prior to running the NF_RCP-G workflow:
+> Note: This command should be run in the location containing the `NF_RCP-G_2.0.0` directory that was downloaded in [step 2](#2-download-the-workflow-files) above. Depending on your network speed, fetching the images will take ~20 minutes.  
 
 ```bash
-bash NF_RCP-F_1.0.4/bin/prepull_singularity.sh NF_RCP-F_1.0.4/config/software/by_docker_image.config
+bash NF_RCP-G_2.0.0/bin/prepull_singularity.sh NF_RCP-G_2.0.0/config/software/by_docker_image.config
 ```
 
 
@@ -134,15 +134,15 @@ export NXF_SINGULARITY_CACHEDIR=$(pwd)/singularity
 
 ### 4. Run the Workflow
 
-While in the location containing the `NF_RCP-F_1.0.4` directory that was downloaded in [step 2](#2-download-the-workflow-files), you are now able to run the workflow. Below are three examples of how to run the NF_RCP-F workflow:
+While in the location containing the `NF_RCP-G_2.0.0` directory that was downloaded in [step 2](#2-download-the-workflow-files), you are now able to run the workflow. Below are three examples of how to run the NF_RCP-F workflow:
 > Note: Nextflow commands use both single hyphen arguments (e.g. -help) that denote general nextflow arguments and double hyphen arguments (e.g. --ensemblVersion) that denote workflow specific parameters.  Take care to use the proper number of hyphens for each argument.
 
 <br>
 
 #### 4a. Approach 1: Run the workflow on a GeneLab RNAseq dataset with automatic retrieval of Ensembl reference fasta and gtf files
 
 ```bash
-nextflow run NF_RCP-F_1.0.4/main.nf \ 
+nextflow run NF_RCP-G_2.0.0/main.nf \ 
    -profile singularity \
    --gldsAccession OSD-194 
 ```
@@ -154,7 +154,7 @@ nextflow run NF_RCP-F_1.0.4/main.nf \
 > Note: The `--ref_source` and `--ensemblVersion` parameters should match the reference source and version number of the local reference fasta and gtf files used
 
 ```bash
-nextflow run NF_RCP-F_1.0.4/main.nf \ 
+nextflow run NF_RCP-G_2.0.0/main.nf \ 
    -profile singularity \
    --gldsAccession OSD-194 \
    --ensemblVersion 107 \
@@ -170,7 +170,7 @@ nextflow run NF_RCP-F_1.0.4/main.nf \
 > Note: Specifications for creating a runsheet manually are described [here](examples/runsheet/README.md).
 
 ```bash
-nextflow run NF_RCP-F_1.0.4/main.nf \ 
+nextflow run NF_RCP-G_2.0.0/main.nf \ 
    -profile singularity \
    --gldsAccession output_directory \
    --runsheetPath </path/to/runsheet> 
@@ -180,7 +180,7 @@ nextflow run NF_RCP-F_1.0.4/main.nf \
 
 **Required Parameters For All Approaches:**
 
-* `NF_RCP-F_1.0.4/main.nf` - Instructs Nextflow to run the NF_RCP-F workflow 
+* `NF_RCP-G_2.0.0/main.nf` - Instructs Nextflow to run the NF_RCP-F workflow 
 
 * `-profile` - Specifies the configuration profile(s) to load, `singularity` instructs Nextflow to setup and use singularity for all software called in the workflow
 
@@ -230,7 +230,7 @@ nextflow run NF_RCP-F_1.0.4/main.nf \
 All parameters listed above and additional optional arguments for the RCP workflow, including debug related options that may not be immediately useful for most users, can be viewed by running the following command:
 
 ```bash
-nextflow run NF_RCP-F_1.0.4/main.nf --help
+nextflow run NF_RCP-G_2.0.0/main.nf --help
 ```
 
 See `nextflow run -h` and [Nextflow's CLI run command documentation](https://nextflow.io/docs/latest/cli.html#run) for more options and details common to all nextflow workflows.
@@ -242,7 +242,7 @@ See `nextflow run -h` and [Nextflow's CLI run command documentation](https://nex
 ### 5. Additional Output Files
 
 The outputs from the Analysis Staging and V&V Pipeline Subworkflows are described below:
-> Note: The outputs from the RNASeq Consensus Pipeline Subworkflow are documented in the [GL-DPPD-7101-F](../../Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-F.md) processing protocol.
+> Note: The outputs from the RNAseq Consensus Pipeline Subworkflow are documented in the [GL-DPPD-7101-F](../../Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-F.md) processing protocol.
 
 **Analysis Staging Subworkflow**
 
