Trekker Bioinformatics Solutions

Overview

The comprehensive Trekker Bioinformatics Solutions include the Trekker Primary Analysis Pipeline and the Secondary Data Analysis Workflow.

Ways to run Trekker Bioinformatics Solutions:

Local: via installation on local workstations or clusters. Access installation and usage instructions by completing the form below.
Cloud: via cloud analysis on the Takara Bio Spatial Bioinformatics Portal.

The Trekker Primary Analysis Pipeline, which processes sequenced data from the Trekker Single-Cell Spatial Mapping Kit, extracts single-nuclei barcodes and spatial barcodes from Trekker FASTQ pairs. After barcode matching, spatial positioning is done using the spatial barcodes associated with each single nucleus, resulting in a spatial location assigned to each single nucleus. The Trekker Primary Analysis Pipeline additionally conducts quality control to help determine the next steps.

The Trekker Primary Analysis Pipeline requires:

FASTQ pairs from the Trekker spatial barcode library
Outputs selected from the bioinformatics pipeline of the single-nuclei sequencing library
Tile bead barcode file for the Trekker tile used
Sample sheet associating sample metadata with aforementioned inputs
- For the 'sc_platform' value and platform-specific samplesheet template, please see the table below

Table 1. 'sc_platform' value for each single-cell assay platform to be used in samplesheet.csv.

Single-cell assay platform	'sc_platform' value	Samplesheet template download link
10x GEM-X Flex Gene Expression Reagent Kits for Multiplexed Samples	TrekkerFX_FLEX
10x Chromium Next GEM Single Cell 3′ Kit v3.1	TrekkerU_C
10x Chromium GEM-X Single Cell 3′ Kit v4	TrekkerU_CX
BD Rhapsody System Single-Cell mRNA Whole Transcriptome Analysis	TrekkerU_R
10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression	TrekkerU_M
10x Chromium GEM-X Single Cell 5′ Reagent Kits v3	Trekker5C_CX
Illumina Single Cell 3′ RNA Prep (processed by DRAGEN)	TrekkerU_IL
Illumina Single Cell 3′ RNA Prep (processed by PIPSeeker)	TrekkerU_PIP
Parse Evercode WT v3	TrekkerQ_P
BD Rhapsody System Single-Cell TCR/BCR Next and mRNA Whole Transcriptome Analysis	TrekkerU_RVDJ
BD Rhapsody System Single-Cell ATAC-Seq and mRNA Whole Transcriptome Analysis	TrekkerU_RATAC

The Secondary Data Analysis workflow is a summary of popular tools developed by the scientific community to process the output of the Trekker Primary Analysis Pipeline and Seeker Primary Analysis Pipeline. This workflow provides guidelines specifically designed for Trekker and Seeker data.

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Supported operating systems

Linux platforms with ≥ 256 GB RAM and 12 cores.

IMPORTANT: The pipeline does NOT support MacOS or Windows operating systems.

Additional third-party software dependencies*

The pipeline can be executed through Docker, Singularity, or Conda

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

Table 2. Trekker Primary Analysis Pipeline version history

Version number	Release date	Notes
1.4.0	2026-01	Newly supported single-cell platforms 10x GEM-X Flex Gene Expression Reagent Kits for Multiplexed Samples (Trekker FX kit) 10x Chromium GEM-X Single Cell 5′ Reagent Kits v3 (Trekker 5C kit) Illumina Single Cell 3′ RNA Prep (processed by DRAGEN; Trekker U kit) New features Trekker FX nuclei positioning enhancement: added a function to assign spatial coordinates to nuclei with no spatial location previously assigned. Spatial coordinate disambiguation: for nuclei assigned to multiple spatial locations, the reported coordinate is now derived from the spatial cluster with the highest UMI count. If the highest-UMI coordinate is identified as a contaminant, it will be excluded. Flexibility in multiomic data handling: for 10x datasets containing multiple modalities (e.g., RNA-seq + ATAC-seq or RNA-seq + CRISPR), the pipeline can now process RNA-seq data independently.
1.3.0	2025-07	New features Now supports processing data from the following additional single-cell assay platforms: 10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Illumina Single Cell 3′ RNA Prep Parse Evercode WT v3 Added bead removal feature to convert nuclei from 2+ to 1 spatial location assigned, leading to increased % nuclei with 1 spatial location assigned. Added ATAC data analysis and visualization, and incorporated ATAC data in the pipeline output Added dimensional reduction and clustering using ATAC peaks Added spatial and UMAP visualization of ATAC data in the HTML report Incorporated ATAC data under the “ATAC” assay in all seurat objects generated in the pipeline output Affected platforms: 10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression BD Rhapsody System Single-Cell ATAC-Seq and mRNA Whole Transcriptome Analysis
1.2.0	2025-04	Bug fixes Can take relative path to sample sheet as input Can take Windows excel edited sample sheet as input Increased allocated memory for the analysis step for larger datasets New features Now support processing data generated from the following single cell assay platforms: BD Rhapsody System Single-Cell ATAC-Seq and mRNA Whole Transcriptome Analysis BD Rhapsody System Single-Cell TCR/BCR Next and mRNA Whole Transcriptome Analysis

Version number

Release date

Notes

1.4.0

2026-01

Newly supported single-cell platforms

10x GEM-X Flex Gene Expression Reagent Kits for Multiplexed Samples (Trekker FX kit)
10x Chromium GEM-X Single Cell 5′ Reagent Kits v3 (Trekker 5C kit)
Illumina Single Cell 3′ RNA Prep (processed by DRAGEN; Trekker U kit)

New features

Trekker FX nuclei positioning enhancement: added a function to assign spatial coordinates to nuclei with no spatial location previously assigned.
Spatial coordinate disambiguation: for nuclei assigned to multiple spatial locations, the reported coordinate is now derived from the spatial cluster with the highest UMI count. If the highest-UMI coordinate is identified as a contaminant, it will be excluded.
Flexibility in multiomic data handling: for 10x datasets containing multiple modalities (e.g., RNA-seq + ATAC-seq or RNA-seq + CRISPR), the pipeline can now process RNA-seq data independently.

1.3.0

2025-07

New features

Now supports processing data from the following additional single-cell assay platforms:
- 10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression
- Illumina Single Cell 3′ RNA Prep
- Parse Evercode WT v3
Added bead removal feature to convert nuclei from 2+ to 1 spatial location assigned, leading to increased % nuclei with 1 spatial location assigned.
Added ATAC data analysis and visualization, and incorporated ATAC data in the pipeline output
- Added dimensional reduction and clustering using ATAC peaks
- Added spatial and UMAP visualization of ATAC data in the HTML report
- Incorporated ATAC data under the “ATAC” assay in all seurat objects generated in the pipeline output
- Affected platforms:
  - 10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression
  - BD Rhapsody System Single-Cell ATAC-Seq and mRNA Whole Transcriptome Analysis

1.2.0

2025-04

Bug fixes

Can take relative path to sample sheet as input
Can take Windows excel edited sample sheet as input
Increased allocated memory for the analysis step for larger datasets

New features

Now support processing data generated from the following single cell assay platforms:

BD Rhapsody System Single-Cell ATAC-Seq and mRNA Whole Transcriptome Analysis
BD Rhapsody System Single-Cell TCR/BCR Next and mRNA Whole Transcriptome Analysis

Download the software

If you are interested in using the Trekker Bioinformatics Solutions, please review the End User License Agreement (EULA) before populating the required fields of the form.

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.

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