Cogent™ NGS Analysis Pipeline

Version 3.3

Overview

Cogent NGS Analysis Pipeline (also referred to as CogentAP) is bioinformatic software for analyzing single-cell RNA-seq, bulk RNA-seq, and single-cell DNA-seq data. Functionalities include gene or transcript expression counting, in silico ribodepletion, gene fusion detection, immune profiling analysis, and more. Please refer to the table in the bioinformatics resource portal for the full list of validated Takara Bio reagent kits supported for use with the pipeline. CogentAP inputs raw sequencing data and outputs an HTML report and other files, such as a gene matrix, to continue further analysis. Output also includes R data objects, which may be used as input for Cogent NGS Discovery Software.

Install the software to access the following features:

Command-line interface program
Mini-dataset files (WellList.txt and FASTQ files)—used to test the CogentAP functionality
Sample output reports and stats—used to compare to the analysis results of the mini-dataset test

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Supported operating systems

CogentAP can be installed on the following versions of Linux:

CentOS 8 or higher
Red Hat 8 or higher
Ubuntu 18.4 or higher

Hardware requirements

For analyzing Illumina NextSeq® High-Output data:

24-core CPU
64 GB+ RAM
1 TB available hard drive space*

Analyzing MiniSeq® or MiSeq® data may be possible on servers with less computational power than the specifications above. Analysis of HiSeq® or NovaSeq™ data would require better hardware than is listed.

*Data generated using the Shasta Total RNA-Seq Kit or Shasta Whole-Genome Amplification Kit requires at least six times the size of the input FASTQ files of free hard drive space for analyses with default parameters, and at least ten times the size of the input FASTQ files of free hard drive space for analyses including ribodepletion, immune profiling, and fusion analyses.

Additional third-party software dependencies*

Conda
bcl2fastq or BCL Convert for conversion of sequencer BCL files to undetermined FASTQ files

*Not included with the software; must be downloaded and installed separately.

Supported applications

See a list of supported applications.

Additional product information

More information is available in the Cogent NGS Analysis Pipeline User Manual or Quick Start Guide.

Cogent NGS Analysis Pipeline version history

Version number	Release date	Notes
CogentAP v3.3	2026-04-22	New features: Added a primary analysis workflow for demultiplexing, alignment, and gene counting for Long Read Sequencing Kit v1 Added primary RNA-Seq analysis support for C. elegans and Rhesus macaque (M. mulatta) Added support for gene body coverage plots for non-Shasta Total RNA kits in short read RNA-Seq analysis. Added support for exporting gene and transcript-level TPM matrices for short read RNA-Seq kits except Shasta Total RNA kits Added support for predicted ploidy calculation per barcode and reporting in the CogentAP output for single-cell DNA-Seq (Shasta WGA CNV) analysis Added support for UMI-aware immune clonotype profiling for stranded_umi RNA-Seq kits Added support for local file addition via the add_genome command Added a read statistics plot showing informative and unwanted read percentages, together with a QC metrics table, to support assessment of experiment performance and sample data quality for stranded and stranded_umi kits Improvements: Improved gene quantification by resolving many-to-one GeneID:GeneSymbol mappings to better preserve gene locus identity Enabled shared use of CogentAP from a centrally installed, pre-configured environment, allowing multiple users to analyze datasets without needing chmod 777 for reporting Added error handling to detect UDI (i7+i5) index length mismatches between the well-list and FASTQ files, with guidance on the appropriate parameter to automatically update the well-list Updated cogent rna analyze down sampling behavior: barcodes below ‘--downsample_depth’ are now excluded by default unless ‘--include_low_depth_fastqs’ is provided Bug fixes: Fixed a failure during analysis statistics generation when ribodepletion was skipped or when Shasta Total RNA datasets were analyzed using an RNA demux + analyze workflow Fixed a failure in post-process fusion analysis when ‘--immune’ was not used during analyze, by ensuring that generated junction files are retained Fixed a failure in the DNA QC report for Shasta WGA datasets when the UDI index length exceeded 16 bp Fixed an issue where the user's local Python environment could interfere with the Python packages used by certain processes
CogentAP v3.2.31	2025-11-17	New features: Support for rat genome (rn7) in RNA-Seq analysis Report number of duplicate reads for both UMI and non-UMI RNA-Seq kits Support for 51 bp read length in Shasta WGA analysis Option to downsample reads per barcode to a set number of reads in RNA-Seq analysis Improvements: Updated stats tables of preliminary RNA analysis html reports for better user readability Summary of Experiment Sequencing Metrics Mapping Metrics Updated color scheme for Shasta WGA samples analyzed using the cogent dna analyze workflow Bug fixes: Fixed old CRAN links with updated R Cloud links to maintain compatibility with package installation Fixed the detection of number of genes and transcripts when the default threshold was changed in the config file, which could lead to an underestimation of genes and transcripts Fixed the reporting of junctions and spanning fragments for detected fusions that could lead to an underestimate of junctions and/or spanning fragments for some fusions Fixed the reporting of Mitochondrial reads for UMI kits that would lead to an overestimation of the number of mitochondrial reads in the final analysis stats Fixed the early termination of analyze_direct in some rare cases when an unmapped read was encountered as the first read in the first chunk being analyzed Fixed the retention of reads mapping to satellite-DNA alignment hot spots in mm39, ensuring such reads are now properly discarded during Shasta WGA analysis Documentation: User Manual Quick Start Guide
CogentAP v3.2	2025-08-01	Added a new postprocess analysis in the DNA-seq module to perform single-nucleotide variant (SNV) calling Added an option for analysis of Shasta Total RNA-Seq kit data using a single pair of FASTQ files with more efficient memory usage and increased processing speed Added an option to generate gene body coverage plots for Shasta Total RNA-Seq analysis (human and mouse genomes) Support for optional depletion of globin and RN7SL2 during RNA-seq analysis from human genome Added automated validation of input sample sheet/well-list files Updated stats for UMIs and other chemistries Documentation: User Manual Quick Start Guide
CogentAP v3.1.27	2025-04-23	Minor bug fixes: Corrections to the mitochondrial gene database for both human and mouse genomes Issue causing some ‘smartseq_fla_umi’ analysis runs to stop unexpectedly before the end of the run Issue where only R2 fastq reads were analyzed using FASTQC Issue with Shasta WGA analysis trim alignment default settings causing lower than expected alignment rates Issue on the WGA reporting that omitted some statistics from the HTML report if their value was zero (0) No changes to documentation Documentation: User Manual Quick Start Guide
CogentAP v3.1.23	2025-04-02	Minor bug fixes: Issue with demultiplexing which could lead to some barcodes/cells not being selected in rare cases when the number of reads for an experiment is very low and a given barcode represents a small fraction of the reads Issue with analysis where the incorrect reference file was used for the mouse genome for ribosomal depletion during the SortMeRNA process. This only affects analysis when the mouse genome is used as the reference. No changes to documentation
CogentAP v3.1	2025-01-15	Changes to install and upgrade procedure New pipeline framework (Nextflow) New DNA-seq analysis workflow, including CNV calling with Ginkgo New DNA-seq demux option Option to perform ribodepletion of data using SortMeRNA New kit data input options: Shasta Total RNA-Seq Kit and the Shasta Whole-Genome Amplification Kit
CogentAP 2.0.1	2023-05-26	Updated launch procedure Documentation: User Manual Quick Start Guide
CogentAP 2.0	2022-08-17	New plate-based kit data input option: SMART-Seq mRNA LP (with UMIs), SMART-Seq mRNA, and SMART-Seq mRNA LP (refer to the bioinformatics resources portal for a full list of supported kits) Documentation: User Manual Quick Start Guide
CogentAP 1.5.1	2022-03-25	New plate-based kit data input options Adds Nextera®- and TruSeq®-based adapter sequences for trimming New default gene fusion analysis filtering level to improve identification of true fusions New variable to customize filtering levels for gene fusion analysis (CLI-only) Minor bug fixes Documentation: User Manual Quick Start Guide
CogentAP 1.5	2021-12-03	Prerequisites: Requires Miniconda3 version 4.10.2 or higher Improved support for full-length transcriptomics, with additional options for transcript, gene fusion, and immune profiling analysis New kit data input options: SMART-Seq Pro Application Kit - 2 Chip Documentation: User Manual Quick Start Guide
CogentAP 1.0	2020-09-11	Prerequisites: Requires Miniconda3 version 4.8.2 or higher Requires bcl2fastq New kit data input options: ICELL8 Single-Cell System 3' DE for UMI workflow, SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (includes UMIs) Documentation: User Manual Quick Start Guide
mappa Analyzer 1.0	2019-06-07	Prerequisites: Requires Miniconda3 version 4.5.0 or later Kit data input options: ICELL8 system sequencing results of full-length transcriptome or single-cell differential expression (3' DE or 5' DE) workflows, no UMI support Documentation: User Guide

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After checking the box to indicate acceptance of the EULA and submitting the completed form, your browser will be directed to a page where you can download the software. Installation instructions can be found in the Cogent NGS Analysis Pipeline User Manual or Quick Start Guide.

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