diff --git a/CHANGELOG.md b/CHANGELOG.md index 129b056..aed2e56 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -4,6 +4,23 @@ This file contains all notable changes to Bambu-Pipe. --- +## [v0.10.1] - 2026-08-26 + +### Added +- `--loupe_alignment` for Visium Spatial Gene Expression samples, taking the manual alignment `.json` exported after fiducial alignment and tissue detection in Loupe Browser. Required for `visium-v*` samples + - Out-of-tissue barcodes are filtered from the BAM before transcript discovery and quantification + - Introduce the `VISIUM_BUILD_TISSUE_POSITIONS` module, which builds the tissue positions file for `visium-v*` samples; the spatial metadata in this file is attached to the `colData` of the `SummarizedExperiment` objects + - Loupe alignment example (`examples/loupe_alignment_visium_example.json`), used by the `test_visium` smoke test +- `CB`/`UB` tags in aligned BAM files (minimap2 `-y`), carrying the barcode and UMI from the FASTQ header comments + +### Changed +- The spatial metadata attached to Visium `SummarizedExperiment` objects now follows the Space Ranger tissue positions format (`barcode`, `in_tissue`, `array_row`, `array_col`, `pxl_row_in_fullres`, `pxl_col_in_fullres`), replacing the `x_coordinate`/`y_coordinate` columns +- `FILTER_BARCODED_BAM` moved to `modules/prepare_input/shared/` and is used by both the standard Visium and Visium HD workflows; it now fails when no reads remain after filtering +- User-supplied BAM files must carry the barcode and UMI in the `CB`/`UB` tags; barcodes encoded in the read name are no longer supported + +### Fixed +- flexiplex-filter's knee detection could discard most in-tissue barcodes for Visium samples; the inflection search now covers the whole barcode rank curve (`-u 0`) for `visium-v*` chemistries + ## [v0.10.0] - 2026-08-17 ### Added diff --git a/README.md b/README.md index 9cd166e..c8bafd1 100644 --- a/README.md +++ b/README.md @@ -4,13 +4,14 @@ - [Installation](#installation) - [General Usage](#general-usage) - [Parameters](#parameters) +- [Single Cell Analysis](#single-cell-analysis) - [Spatial Analysis](#spatial-analysis) - [Visium Spatial Gene Expression](#visium-spatial-gene-expression) - [Visium HD](#visium-hd) +- [Fusion Transcript Analysis (Under Development)](#fusion-transcript-analysis-under-development) - [Output](#output) - [Advanced Usage](#advanced-usage) - [Additional Information](#additional-information) -- [Release History](#release-history) - [Citation](#citation) - [Contributors](#contributors) @@ -36,28 +37,28 @@ This pipeline performs context-aware transcript discovery and quantification fro - Cluster-level EM: Gene expression-based cell clustering with [Seurat](https://github.com/satijalab/seurat) across all samples, followed by per-sample cluster-level transcript quantification - Single-cell EM: Transcript quantification for each cell, or for each spot in spatial samples -### **Installation** +### **Installation** Install the following dependencies before running the pipeline: - [Nextflow](https://www.nextflow.io/docs/latest/install.html) ≥ 26.04.0 -- [Docker](https://docs.docker.com/engine/install/ubuntu/) (or [Singularity](https://docs.sylabs.io/guides/3.0/user-guide/installation.html) if you do not have user permissions for Docker). +- [Docker](https://docs.docker.com/engine/install/ubuntu/) (or [Singularity](https://docs.sylabs.io/guides/3.0/user-guide/installation.html) if you do not have user permissions for Docker). -### **General Usage** -To run the pipeline, you must provide a samplesheet, reference genome, and reference annotation file as input. The pipeline performs transcript discovery and quantification on either a single sample or multiple samples based on the number of samples specified in the samplesheet. Refer to the [Parameters](#parameters) and Samplesheet (CSV) sections below for more details. +### **General Usage** +To run the pipeline, you must provide a samplesheet, reference genome, and reference annotation file as input. The pipeline performs transcript discovery and quantification on either a single sample or multiple samples based on the number of samples specified in the samplesheet. Refer to the [Parameters](#parameters) and Samplesheet (CSV) sections below for more details. **Running the pipeline** -Use the command below to run the pipeline on the test data provided in `examples/` -``` +Use the command below to run the pipeline on the test data provided in `examples/`. +``` nextflow run main.nf \ --input examples/samplesheet_test_sc_fastq.csv \ --genome examples/GRCh38.primary_assembly.genome.chr21.fa.gz \ --annotation examples/gencode.v49.primary_assembly.annotation.chr21.gtf.gz \ -profile singularity,hpc -``` +``` **Samplesheet (CSV)** -The pipeline requires a `.csv` formatted samplesheet to define the input data. This file is mandatory, regardless of the number of samples being processed. Each row in the samplesheet represents a single sample and its corresponding file path and metadata. +The pipeline requires a `.csv` formatted samplesheet to define the input data. This file is mandatory, regardless of the number of samples being processed. Each row in the samplesheet represents a single sample and its corresponding file path and metadata. *Required Columns* @@ -106,9 +107,9 @@ For the following chemistries, the pipeline handles the full workflow — FASTQ - `visium-v2` (Visium Spatial Gene Expression Slide 6.5 mm; serial prefix V2) - `visium-v3` (Visium Spatial Gene Expression Slide 6.5 mm; serial prefix V3) - `visium-v4` (Visium CytAssist Spatial Gene Expression Slide 6.5 mm; serial prefix V4) -- `visium-v5` (Visium CytAssist Spatial Gene Expression Slide 11mm; serial prefix V5) +- `visium-v5` (Visium CytAssist Spatial Gene Expression Slide 11 mm; serial prefix V5) -> **Note:** Visium Spatial Gene Expression samples (`visium-v*`) must be run one at a time. Multi-sample runs are not supported for these chemistries. +> **Note:** Visium Spatial Gene Expression samples (`visium-v*`) must be run one at a time and require a Loupe manual alignment file. See the [Visium Spatial Gene Expression](#visium-spatial-gene-expression) section for details. *Custom Chemistries* @@ -135,18 +136,18 @@ To configure the executor and container, pass profile types via the `-profile` a ### **Parameters** **Mandatory** -- `--input` [string]: Path to the samplesheet `.csv` file -- `--genome` [string]: Path to the reference genome `.fa`, `.fasta`, or `.fa.gz` file -- `--annotation` [string]: Path to the reference annotation `.gtf`, `.gff`, `.gtf.gz`, or `.gff.gz` file. When restarting the pipeline with `--manual_clustering`, provide the `extended_annotations.rds` file instead +- `--input` [string]: Path to the samplesheet `.csv` file +- `--genome` [string]: Path to the reference genome `.fa`, `.fasta`, or `.fa.gz` file +- `--annotation` [string]: Path to the reference annotation `.gtf`, `.gff`, `.gtf.gz`, or `.gff.gz` file. When restarting the pipeline with `--manual_clustering`, provide the `extended_annotations.rds` file instead **Optional** - `--output_dir` [string, default: 'output']: Path to the output directory -- `--chemistry` [string, default: null]: Specify if all samples in the samplesheet share the same library chemistry +- `--chemistry` [string, default: null]: Specify if all samples in the samplesheet share the same library chemistry - `--technology` [string, default: null]: Specify if all samples in the samplesheet share the same sequencing technology - `--bam_only` [boolean, default: false]: If true, stops the pipeline after genome alignment and saves BAM files only (see Advanced Usage section) - `--qscore_filtering` [boolean, default: true]: Enable or disable quality score filtering of reads - `--ndr` [float, default: null]: NDR threshold for Bambu transcript discovery. If not set, Bambu will recommend a suitable value -- `--deduplicate_umis` [boolean, default: true]: If true, Bambu will perform UMI deduplication +- `--deduplicate_umis` [boolean, default: true]: If true, Bambu will perform UMI deduplication - `--quantification_mode` [string, default: "clusteredEM"]: Quantification mode for transcript counts. Available options are: - "no_quant": Transcript quantification is not performed - "EM": Performs transcript quantification for each cell/spatial coordinate @@ -154,32 +155,61 @@ To configure the executor and container, pass profile types via the `-profile` a - `--seurat_resolution` [float, default: 0.8]: Seurat clustering resolution - `--manual_clustering` [boolean, default: false]: If true, skips clustering and quantifies from cluster assignments generated outside the pipeline (see Advanced Usage section) +**Visium** + +See the [Visium Spatial Gene Expression](#visium-spatial-gene-expression) section for more details. +- `--loupe_alignment` [string, default: null]: Path to the manual alignment `.json` file exported from Loupe Browser. Required for `visium-v*` samples + **Visium HD** See the [Visium HD](#visium-hd) section for the required input files and samplesheet format. - `--visium_hd` [boolean, default: false]: Enable the Visium HD workflow - `--bins` [string, default: null]: Path to a `.csv` listing all the resolutions at which the unique counts and gene counts are computed. Required when `--visium_hd` is set - `--clustering_bin` [integer, default: 8]: Bin size (µm) to cluster at. Must be one of the values in `--bins`. -- `--barcode_mappings` [string, default: null]: Path to the Spaceranger `barcode_mappings.parquet`. Required whenever a bin coarser than 2 µm is listed in `--bins` (e.g. 8 µm) +- `--barcode_mappings` [string, default: null]: Path to the Space Ranger `barcode_mappings.parquet`. Required whenever a bin coarser than 2 µm is listed in `--bins` (e.g. 8 µm) - `--banksy` [boolean, default: true]: If true, clusters with [Banksy](https://github.com/prabhakarlab/Banksy) using both expression and spatial position. If false, clusters on gene expression alone - `--banksy_lambda` [float, default: 0.8]: Weight given to spatial information - `--banksy_k_geom` [integer, default: 50]: Number of spatial neighbours per bin +### **Single Cell Analysis** + +By default, the pipeline runs the single-cell workflow described in the [Overview](#overview) and [General Usage](#general-usage) sections. All supported 10x Single Cell chemistries (`10x3v*`, `10x5v*`) follow this workflow, with no additional parameters or input files required. + ### **Spatial Analysis** #### **Visium Spatial Gene Expression** -The pipeline applies the same processing steps to both 10x Single Cell and Visium Spatial Gene Expression (`visium-v*`) samples. The only difference is that the generated `SummarizedExperiment` objects are attached with spatial mapping information, which is stored in `colData`. +The pipeline applies the same processing steps to both 10x Single Cell and Visium Spatial Gene Expression (`visium-v*`) samples, with two additions for Visium samples: barcodes outside the tissue are filtered out, and spatial metadata is attached to the results. -*Example: Spatial Mapping Information* +*Manual Fiducial Alignment and Tissue Detection (Loupe Browser)* -In the `SummarizedExperiment` object, each row in `colData` contains a spatial barcode together with its X and Y coordinates on the slide. +For Visium Spatial Gene Expression samples (`visium-v*`), perform manual fiducial alignment and tissue detection in [Loupe Browser](https://www.10xgenomics.com/support/software/loupe-browser/latest) before running the pipeline, and supply the exported alignment `.json` file via the `--loupe_alignment` parameter. For more information, please refer to the [Manual Fiducial Alignment](https://www.10xgenomics.com/support/software/space-ranger/latest/analysis/inputs/image-fiducial-alignment) documentation. -| barcode | x_coordinate | y_coordinate | -|:---|:---|:---| -| AAACAACGAATAGTTC | 17 | 1 | -| AAACAAGTATCTCCCA | 103 | 51 | -| AAACAATCTACTAGCA | 44 | 4 | +Using the alignment `.json` file supplied, the pipeline removes out-of-tissue barcodes from the BAM file before transcript discovery and quantification, and attaches spatial metadata information to the results generated by Bambu (see *Spatial Metadata* below). + +*Running the pipeline* + +```bash +nextflow run main.nf \ + --input examples/samplesheet_test_visium.csv \ + --genome examples/GRCh38.primary_assembly.genome.chr21.fa.gz \ + --annotation examples/gencode.v49.primary_assembly.annotation.chr21.gtf.gz \ + --loupe_alignment examples/loupe_alignment_visium_example.json \ + -profile singularity,hpc +``` +*Spatial Metadata* + +The spatial metadata attached to the results follows the [Space Ranger Tissue Positions](https://www.10xgenomics.com/support/software/space-ranger/latest/analysis/outputs/spatial-outputs) format: the spatial barcode, the in-tissue flag, its array coordinates, and its pixel position in the full-resolution tissue image. The metadata is stored in the `colData` of the `SummarizedExperiment` objects described in the [Output](#output) section. + +| barcode | in_tissue | array_row | array_col | pxl_row_in_fullres | pxl_col_in_fullres | +|:---|:---|:---|:---|:---|:---| +| CCAAGCTTGATCTCCT | 1 | 0 | 18 | 15260.561 | 2350.7175 | +| GAGCGCTATGTCAGGC | 1 | 0 | 20 | 15027.729 | 2349.6375 | +| CTTCGTGCCCGCATCG | 1 | 0 | 22 | 14794.896 | 2348.5576 | + +*Clustering* + +The pipeline currently clusters Visium spots on gene expression alone, using the same Seurat workflow as single-cell samples. Spatially aware clustering (e.g. using Banksy) for `visium-v*` samples will be added in a future release. #### **Visium HD** @@ -190,9 +220,9 @@ This pipeline does not demultiplex Visium HD reads. Barcode assignment and align *Required Input Files* -Alongside the BAM file, Visium HD runs require two Spaceranger outputs: +Alongside the BAM file, Visium HD runs require two Space Ranger outputs: -| Spaceranger output | Description | +| Space Ranger output | Description | |---|---| | `tissue_positions.parquet` | One file per resolution, from `binned_outputs/square_XXXum/spatial/`| | `barcode_mappings.parquet` | Maps each 2 µm barcode to its bin at every resolution | @@ -201,7 +231,7 @@ For more information, please refer to the [Space Ranger Spatial Outputs](https:/ *Samplesheet (CSV)* -Visium HD runs take exactly one sample and only needs the `sample` and `path` columns in the input samplesheet. +Visium HD runs take exactly one sample and only need the `sample` and `path` columns in the input samplesheet. ```csv sample,path visium_hd_example,examples/visium_hd_example.bam @@ -236,14 +266,21 @@ nextflow run main.nf \ For more information on the Visium HD parameters, please refer to the [Parameters](#parameters) section. +*Spatial Metadata* + +The spatial metadata attached to the results follows the same format described in the [Visium Spatial Gene Expression](#visium-spatial-gene-expression) section. For each resolution, the metadata is extracted from that resolution's `tissue_positions.parquet` file. + *Clustering* Clustering is performed at a single resolution only, set by `--clustering_bin`, when `--quantification_mode` is set to `clusteredEM`. By default it uses Seurat with [Banksy](https://github.com/prabhakarlab/Banksy), which combines gene expression with spatial position. Set `--banksy false` to cluster on gene expression alone. When clustering is skipped under `--quantification_mode EM` (i.e., spot-level EM), transcript counts are produced at every resolution listed in `--bins`. +### **Fusion Transcript Analysis (Under Development)** +This feature is still under development and will be released in a future update. + ### **Output** -All outputs from the pipeline are written to the directory specified by the `--output_dir` parameter. The pipeline produces per-sample alignment files and the combined transcript discovery and quantification results. +All outputs from the pipeline are written to the directory specified by the `--output_dir` parameter. The pipeline produces per-sample alignment files and the combined transcript discovery and quantification results. *Output Structure* ``` @@ -256,10 +293,10 @@ output/ │ ├── execution_trace.txt │ └── pipeline_dag.svg │ -├── bam/ +├── bam/ │ ├── _demultiplexed.bam │ └── _demultiplexed.bam.bai -│ # (one pair per sample for multi-sample runs) +│ # (one pair per sample for multi-sample runs) │ ├── extended_annotations.gtf │ @@ -346,9 +383,9 @@ output/ ``` **Description of the Output Files** -| File | Description +| File | Description |---|--- -| `_demultiplexed.bam` | BAM file containing demultiplexed, trimmed and aligned reads +| `_demultiplexed.bam` | BAM file containing all demultiplexed, trimmed and aligned reads, with the barcode and UMI stored in the `CB`/`UB` tags | `_demultiplexed.bam.bai` | BAM index for the corresponding BAM file | `extended_annotations.gtf` | A `.gtf` file containing the novel transcripts discovered by Bambu as well as the reference annotations provided by the user. | `seurat_obj.rds` | A [SeuratObject](https://satijalab.github.io/seurat-object/reference/Seurat-class.html) containing normalised counts, PCA embeddings, and cluster assignments. For multi-sample runs, also contains Harmony-integrated embeddings corrected for sequencing technology and capture chemistry. For Visium HD runs, holds the bins of the `--clustering_bin` resolution and, with `--banksy`, the BANKSY assay and embeddings. UMAP has not been computed. Only produced when `--quantification_mode` is set to `clusteredEM`. @@ -377,20 +414,16 @@ The [RangedSummarizedExperiment](https://www.rdocumentation.org/packages/Summari - `counts`: expression estimates - `CPM`: sequencing depth normalised estimates - `fullLengthCounts`: estimates of read counts mapped as full length reads for each transcript -- `uniqueCounts`: counts of reads that are uniquely mapped to each transcript +- `uniqueCounts`: counts of reads that are uniquely mapped to each transcript > **Note:** In `se_unique_counts.rds`, unique counts are stored under the `counts` assay, not `uniqueCounts`. -### **Fusion Transcript Analysis (Under Development)** -This feature is still under development and will be released in a future update. - - ### **Advanced Usage** **Running Pipeline with a Custom Chemistry or Pre-aligned BAM** -If your dataset uses a chemistry not listed under Supported 10x Library Chemistries, or if you prefer to perform FASTQ preprocessing and genome alignment manually, start the pipeline directly from a pre-processed, demultiplexed BAM file. The BAM file must have the barcode and UMI information encoded either in the `CB`/`UB` column, or in the read name using the format `_#`. +If your dataset uses a chemistry not listed under Supported 10x Library Chemistries, or if you prefer to perform FASTQ preprocessing and genome alignment manually, start the pipeline directly from a pre-processed, demultiplexed BAM file. The BAM file must have the barcode and UMI information encoded in the `CB`/`UB` tags. *Samplesheet (Custom Chemistry)* @@ -502,7 +535,7 @@ nextflow run . -profile singularity \ *Visium HD* -The same two stages described above applies. Cluster the `gene_counts/gene_counts_/` matrix of whichever resolution you want, then restart with `--visium_hd` and set `--clustering_bin` to that resolution: +The same two stages described above apply. Cluster the `gene_counts/gene_counts_/` matrix of whichever resolution you want, then restart with `--visium_hd` and set `--clustering_bin` to that resolution: ```bash nextflow run . -profile singularity \ @@ -569,7 +602,7 @@ Sim, A., Ling, M. H., Chen, Y., Lu, H., See, Y. X., Perrin, A., Leng Agnes, O. B The following are citations for the other tools used in this pipeline: #### Banksy -Singhal, V., Chou, N., Lee, J., Yue, Y., Liu, J., Chock, W. K., Lin, L., Chang, Y.-C., Teo, E. M. L., Aow, J., Lee, H. K., Chen, K. H., & Prabhakar, S. (2024, February 27). Banksy unifies cell typing and tissue domain segmentation for scalable spatial omics data analysis. Nature News. https://www.nature.com/articles/s41588-024-01664-3 +Singhal, V., Chou, N., Lee, J., Yue, Y., Liu, J., Chock, W. K., Lin, L., Chang, Y.-C., Teo, E. M. L., Aow, J., Lee, H. K., Chen, K. H., & Prabhakar, S. (2024, February 27). Banksy unifies cell typing and tissue domain segmentation for scalable spatial omics data analysis. Nature Genetics. https://www.nature.com/articles/s41588-024-01664-3 #### Chopper De Coster Wouter, & Rademakers, R. (2023). NanoPack2: Population scale evaluation of long-read sequencing data. Bioinformatics, 39(5). https://doi.org/10.1093/bioinformatics/btad311 diff --git a/bin/visium_build_tissue_positions.py b/bin/visium_build_tissue_positions.py new file mode 100755 index 0000000..f3b9488 --- /dev/null +++ b/bin/visium_build_tissue_positions.py @@ -0,0 +1,58 @@ +#!/usr/bin/env python3 +"""Build the tissue positions CSV (Space Ranger format) for visium-v* samples from the +Loupe manual alignment JSON and the chemistry's coordinates file. Also writes the +in-tissue barcode list used by samtools to filter out-of-tissue barcodes from the BAM. +""" +import argparse +import csv +import json +import sys + + +def main(): + parser = argparse.ArgumentParser(description=__doc__) + parser.add_argument("loupe_alignment", help="Loupe manual alignment .json") + parser.add_argument("spatial_coordinates", help="chemistry coordinates file (barcode, x, y; 1-based)") + parser.add_argument("tissue_positions", help="output tissue positions CSV") + parser.add_argument("tissue_barcodes", help="output in-tissue barcode list") + args = parser.parse_args() + + with open(args.loupe_alignment) as f: + oligos = json.load(f)["oligo"] + spots = {(o["row"], o["col"]): o for o in oligos} + + coords = {} + with open(args.spatial_coordinates) as f: + for line in f: + barcode, x, y = line.split() + coords[(int(y) - 1, int(x) - 1)] = barcode + + # the JSON grid and the (shifted) coordinates grid must coincide exactly + if set(spots) != set(coords): + sys.exit( + f"error: the {len(spots)} spots in {args.loupe_alignment} do not match the " + f"{len(coords)}-spot layout in {args.spatial_coordinates} -- check that the " + "alignment JSON was made for this slide chemistry" + ) + + n_tissue = 0 + with open(args.tissue_positions, "w", newline="") as fc, open(args.tissue_barcodes, "w") as fb: + writer = csv.writer(fc) + writer.writerow(["barcode", "in_tissue", "array_row", "array_col", + "pxl_row_in_fullres", "pxl_col_in_fullres"]) + for (row, col), oligo in sorted(spots.items(), key=lambda item: item[0]): + in_tissue = int(bool(oligo.get("tissue"))) + writer.writerow([coords[(row, col)], in_tissue, row, col, + oligo["imageY"], oligo["imageX"]]) + if in_tissue: + fb.write(coords[(row, col)] + "\n") + fb.write(coords[(row, col)] + "-1\n") + n_tissue += 1 + + if n_tissue == 0: + sys.exit("error: no spot in the alignment JSON is marked as tissue") + print(f"{n_tissue} of {len(spots)} spots are in tissue") + + +if __name__ == "__main__": + main() diff --git a/conf/smoke_test.config b/conf/smoke_test.config index 4d87e50..dece1f2 100644 --- a/conf/smoke_test.config +++ b/conf/smoke_test.config @@ -57,8 +57,9 @@ profiles { test_visium { params { - input = "${projectDir}/examples/samplesheet_test_visium.csv" - output_dir = "${projectDir}/.smoke_test/test_visium/output" + input = "${projectDir}/examples/samplesheet_test_visium.csv" + loupe_alignment = "${projectDir}/examples/loupe_alignment_visium_example.json" + output_dir = "${projectDir}/.smoke_test/test_visium/output" } } diff --git a/examples/loupe_alignment_visium_example.json b/examples/loupe_alignment_visium_example.json new file mode 100644 index 0000000..2399d4f --- /dev/null +++ b/examples/loupe_alignment_visium_example.json @@ -0,0 +1 @@ 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\ No newline at end of file diff --git a/lib/Validation.groovy b/lib/Validation.groovy index 4b4300b..bba56d3 100644 --- a/lib/Validation.groovy +++ b/lib/Validation.groovy @@ -69,6 +69,18 @@ class Validation { throw new Exception("params.barcode_mappings '${params.barcode_mappings}' must be a .parquet file") } } + + // Loupe alignment checks (standard Visium only) + if (params.loupe_alignment != null) { + if (params.visium_hd) + throw new Exception("params.loupe_alignment is not used by the Visium HD workflow — tissue filtering uses the tissue_positions parquet files instead") + + if (!params.loupe_alignment.exists()) + throw new Exception("params.loupe_alignment '${params.loupe_alignment}' does not exist") + + if (params.loupe_alignment.extension != 'json') + throw new Exception("params.loupe_alignment '${params.loupe_alignment}' must be a .json file") + } } static def validateVisiumHDRows(rows) { @@ -107,10 +119,13 @@ class Validation { throw new Exception("params.clustering_bin '${clusteringBin}' is not listed in params.bins '${bins}' — available bins: ${resolutions.join(', ')}") } - static def validateVisiumSampleCount(samples) { + static def validateVisiumSampleCount(samples, loupeAlignment) { def has_visium = samples.any { sample, path, meta -> meta.chemistry.startsWith('visium') } if (has_visium && samples.size() > 1) throw new Exception("Visium chemistry requires exactly 1 sample, but found ${samples.size()}") + + if (loupeAlignment != null && !has_visium) + throw new Exception("params.loupe_alignment was provided but no sample uses a visium chemistry") } static def validateRow(row, params, log) { @@ -130,6 +145,9 @@ class Validation { if (!params.valid_technologies.contains(row.technology)) throw new Exception("Sample '${row.sample}' has invalid technology '${row.technology}' — must be one of: ${params.valid_technologies.join(', ')}") + + if (row.chemistry.startsWith('visium') && params.loupe_alignment == null) + throw new Exception("Visium sample '${row.sample}' requires params.loupe_alignment — a Loupe manual alignment .json with the tissue selection") } } diff --git a/main.nf b/main.nf index 9237623..dbe2b2c 100644 --- a/main.nf +++ b/main.nf @@ -24,6 +24,7 @@ include { EXTRACT_SPOT_BARCODES } from './modules/bambu/visium_hd include { MAP_CLUSTERS_TO_2UM_SPOTS } from './modules/bambu/visium_hd/map_clusters_to_2um_spots.nf' include { CONVERT_BARCODE_MAPPINGS } from './modules/prepare_input/visium_hd/convert_barcode_mappings.nf' include { SPOT_BIN_MAPPINGS } from './modules/prepare_input/visium_hd/spot_bin_mappings.nf' +include { FILTER_BARCODED_BAM } from './modules/prepare_input/shared/filter_barcoded_bam.nf' include { SEURAT_VISIUM_HD } from './modules/seurat/visium_hd/clustering.nf' params { @@ -39,6 +40,7 @@ params { deduplicate_umis: Boolean quantification_mode: String seurat_resolution: Float + loupe_alignment: Path? visium_hd: Boolean barcode_mappings: Path? bins: Path? @@ -56,6 +58,7 @@ workflow STANDARD { ch_annotation: Path ndr: Float? manual_clustering: Boolean + loupe_alignment: Path? main: if (!manual_clustering) { @@ -68,7 +71,7 @@ workflow STANDARD { ch_n_samples = ch_rows.count() - PREPARE_INPUT_STANDARD(ch_rows, ch_barcode_coordinate_config) + PREPARE_INPUT_STANDARD(ch_rows, ch_barcode_coordinate_config, loupe_alignment) // input files are split by type (fastq, bam) ch_input_fastq = PREPARE_INPUT_STANDARD.out.fastq @@ -82,6 +85,17 @@ workflow STANDARD { if (!params.bam_only) { // process bam samples ch_bam_files = ALIGNMENT.out.bam.mix(ch_input_bam) + + // remove out-of-tissue barcodes from visium BAMs using the Loupe alignment's tissue selection + if (loupe_alignment != null) { + ch_bam_branched = ch_bam_files.branch { _sample, _path, meta -> + visium: meta.chemistry.startsWith('visium') + other: true + } + FILTER_BARCODED_BAM(ch_bam_branched.visium, PREPARE_INPUT_STANDARD.out.tissue_barcodes) + ch_bam_files = FILTER_BARCODED_BAM.out.bam.mix(ch_bam_branched.other) + } + BAMBU_PREPARE_ANNOTATION(ch_annotation) BAMBU_CONSTRUCT_READ_CLASS(ch_bam_files, ch_genome, BAMBU_PREPARE_ANNOTATION.out.annotation) @@ -231,7 +245,7 @@ workflow { if (params.visium_hd) { VISIUM_HD(ch_rows, ch_genome, ch_annotation, params.ndr, params.manual_clustering) } else { - STANDARD(ch_rows, ch_genome, ch_annotation, params.ndr, params.manual_clustering) + STANDARD(ch_rows, ch_genome, ch_annotation, params.ndr, params.manual_clustering, params.loupe_alignment) } channel.topic('versions').collectFile(name: 'software_versions.yml', storeDir: "${params.output_dir}") diff --git a/modules/alignment/align.nf b/modules/alignment/align.nf index 9f78ab8..851fb10 100644 --- a/modules/alignment/align.nf +++ b/modules/alignment/align.nf @@ -23,7 +23,7 @@ process MINIMAP_ALIGNMENT{ preset="splice" fi - minimap2 -ax \$preset -uf --junc-bed $bed -t $task.cpus $ref_mmi $newfastq | \ + minimap2 -ax \$preset -y -uf --junc-bed $bed -t $task.cpus $ref_mmi $newfastq | \ samtools sort -@ $task.cpus -o ${sample}_demultiplexed.bam samtools index -@ $task.cpus ${sample}_demultiplexed.bam diff --git a/modules/prepare_input/visium_hd/filter_barcoded_bam.nf b/modules/prepare_input/shared/filter_barcoded_bam.nf similarity index 78% rename from modules/prepare_input/visium_hd/filter_barcoded_bam.nf rename to modules/prepare_input/shared/filter_barcoded_bam.nf index 9b9720b..a7fc3a8 100644 --- a/modules/prepare_input/visium_hd/filter_barcoded_bam.nf +++ b/modules/prepare_input/shared/filter_barcoded_bam.nf @@ -19,6 +19,11 @@ process FILTER_BARCODED_BAM { samtools view -@ $task.cpus -D CB:$barcodes -o ${sample}_filtered.bam $bam samtools index -@ $task.cpus ${sample}_filtered.bam + if [[ \$(samtools view -c -@ $task.cpus ${sample}_filtered.bam) -eq 0 ]]; then + echo "No reads remain after barcode filtering -- check that the BAM carries CB tags matching $barcodes" >&2 + exit 1 + fi + cat <<-END_VERSIONS > versions.yml "${task.process}": samtools: \$(samtools --version 2>&1 | head -1) diff --git a/modules/prepare_input/standard/extract_spatial_coordinates.nf b/modules/prepare_input/standard/extract_spatial_coordinates.nf index 4120a3c..3f91e85 100644 --- a/modules/prepare_input/standard/extract_spatial_coordinates.nf +++ b/modules/prepare_input/standard/extract_spatial_coordinates.nf @@ -7,14 +7,13 @@ process EXTRACT_10X_SPATIAL_COORDINATES { path(barcode_coordinate_config) output: - tuple val(chemistry), path("${chemistry}_spatial_coordinates.txt"), emit: spatial_coordinates + path("spatial_coordinates.txt"), emit: spatial_coordinates script: """ # extract spatial coordinate file path from config csv IFS=',' read -r _ _ sc_filename < <(awk -F',' -v chem=$chemistry '\$1 == chem' $barcode_coordinate_config) - cp $params.cellranger_dir/\$sc_filename ./${chemistry}_spatial_coordinates.txt - sed -i '1ibarcode\tx_coordinate\ty_coordinate' ./${chemistry}_spatial_coordinates.txt + cp $params.cellranger_dir/\$sc_filename ./spatial_coordinates.txt """ } diff --git a/modules/prepare_input/standard/visium_build_tissue_positions.nf b/modules/prepare_input/standard/visium_build_tissue_positions.nf new file mode 100644 index 0000000..37b0302 --- /dev/null +++ b/modules/prepare_input/standard/visium_build_tissue_positions.nf @@ -0,0 +1,27 @@ +process VISIUM_BUILD_TISSUE_POSITIONS { + publishDir "$params.output_dir/intermediate_visium", mode: 'copy', pattern: 'tissue_positions.csv', enabled: params.save_intermediates + publishDir "$params.output_dir/intermediate_visium", mode: 'copy', pattern: 'tissue_barcodes.txt', enabled: params.save_intermediates + label "pyarrow_pandas" + label "low_cpu" + label "low_mem" + label "short" + + input: + path(spatial_coordinates) + path(loupe_alignment) + + output: + path('tissue_positions.csv'), emit: tissue_positions + path('tissue_barcodes.txt'), emit: tissue_barcodes + path "versions.yml", topic: 'versions' + + script: + """ + visium_build_tissue_positions.py $loupe_alignment $spatial_coordinates tissue_positions.csv tissue_barcodes.txt + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + python: \$(python3 --version 2>&1) + END_VERSIONS + """ +} diff --git a/modules/preprocess_fastq.nf b/modules/preprocess_fastq.nf index 912372a..9bf4d27 100644 --- a/modules/preprocess_fastq.nf +++ b/modules/preprocess_fastq.nf @@ -105,8 +105,15 @@ process PREPROCESS_FASTQ { flank_seq="-x \$left_flank -b \$barcode -u \$umi -x \$right_flank" # Flexiplex filter (barcode filtering) + # For visium chemistries, search the whole rank curve instead + # (previous runs using the default parameters failed to search the whole rank curve for the inflection point) + if [[ $meta.chemistry == visium-v* ]]; then + max_rank_arg="-u 0" + else + max_rank_arg="" + fi flexiplex -p $task.cpus \$flank_seq -f 0 ${sample}_chopper_out.fastq - flexiplex-filter -w $whitelist --outfile my_filtered_barcode_list.txt flexiplex_barcodes_counts.txt + flexiplex-filter \$max_rank_arg -w $whitelist --outfile my_filtered_barcode_list.txt flexiplex_barcodes_counts.txt # Chain commands: Flexiplex demultiplexing -> (Optional) Save intermediate file after flexiplex -> Cutadapt trimming of reverse primer -> \ # Cutadapt re-search for all primer and TSO sequences to remove non-standard reads -> Reverse complementation of reads for 3' and visium chemistry -> Compression with pigz diff --git a/nextflow.config b/nextflow.config index ad6d272..725fc66 100644 --- a/nextflow.config +++ b/nextflow.config @@ -2,7 +2,7 @@ manifest { name = 'Bambu-Pipe' description = 'Context-aware transcript quantification from long-read single-cell and spatial transcriptomics data' author = 'Andre Sim, Chin Hao Lee, Min Hao Ling' - version = 'v0.9-beta' + version = 'v0.10.1' mainScript = 'main.nf' nextflowVersion = '!>=26.04.0' } @@ -47,6 +47,13 @@ params { */ manual_clustering = false // boolean + /* + Optional: Visium + Note: Set --loupe_alignment to point to the manual alignment .json file exported from + Loupe Browser. Required for visium-v* samples. + */ + loupe_alignment = null + /* Optional: Visium HD Note: Set --visium_hd true for a single-sample run starting from a pre-aligned, barcode-tagged BAM file. diff --git a/subworkflows/prepare_input_standard.nf b/subworkflows/prepare_input_standard.nf index 8e01e07..b4e6489 100644 --- a/subworkflows/prepare_input_standard.nf +++ b/subworkflows/prepare_input_standard.nf @@ -1,10 +1,12 @@ include { EXTRACT_10X_BARCODES } from '../modules/prepare_input/standard/extract_barcodes.nf' include { EXTRACT_10X_SPATIAL_COORDINATES } from '../modules/prepare_input/standard/extract_spatial_coordinates.nf' +include { VISIUM_BUILD_TISSUE_POSITIONS } from '../modules/prepare_input/standard/visium_build_tissue_positions.nf' workflow PREPARE_INPUT_STANDARD { take: ch_rows ch_barcode_coordinate_config + loupe_alignment main: // parse samplesheet rows into channel of tuples (sample, path, metadata) @@ -15,8 +17,8 @@ workflow PREPARE_INPUT_STANDARD { [row.sample, sample_path, meta] } - // validate: ensure only one visium sample is processed at a time - ch_samples.collect(flat: false).map { samples -> Validation.validateVisiumSampleCount(samples) } + // validate: for visium runs, ensure only one sample is processed at a time + ch_samples.collect(flat: false).map { samples -> Validation.validateVisiumSampleCount(samples, loupe_alignment) } // extract distinct chemistries from metadata ch_unique_chem = ch_samples.map { _sample, _path, meta -> meta.chemistry }.unique() @@ -28,23 +30,31 @@ workflow PREPARE_INPUT_STANDARD { ch_barcodes = EXTRACT_10X_BARCODES.out.barcodes .mix(ch_unique_custom.map { chem -> [chem, null] }) - // extract spatial coordinates for visium chemistries only; add placeholder for non-visium samples - ch_unique_visium = ch_unique_chem.filter { chem -> chem.startsWith('visium') } - ch_unique_non_visium = ch_unique_chem.filter { chem -> !chem.startsWith('visium') } + // extract spatial coordinates for visium chemistries only + ch_unique_visium = ch_unique_chem.filter { chem -> chem.startsWith('visium') } EXTRACT_10X_SPATIAL_COORDINATES(ch_unique_visium, ch_barcode_coordinate_config) - ch_spatial_coordinates = EXTRACT_10X_SPATIAL_COORDINATES.out.spatial_coordinates - .mix(ch_unique_non_visium.map { chem -> [chem, null] }) - // update metadata with barcode and spatial coordinate paths - ch_updated_samples = ch_samples.map { sample, path, meta -> [meta.chemistry, sample, path, meta] } + // update sample tuples with the chemistry's barcode whitelist + ch_keyed_samples = ch_samples.map { sample, path, meta -> [meta.chemistry, sample, path, meta] } .combine(ch_barcodes, by: 0) - .combine(ch_spatial_coordinates, by: 0) - .map { _chem, sample, path, meta, bc, sc -> - def updated_meta = meta + [ - barcode: bc, - spatial_metadata: sc - ] - return [sample, path, updated_meta] + + // build the tissue positions (spatial metadata) from the Loupe manual alignment file and the spatial + // coordinate whitelist file. Also generate the in-tissue barcode list which will be used to filter + // the BAM file. + if (loupe_alignment != null) { + VISIUM_BUILD_TISSUE_POSITIONS(EXTRACT_10X_SPATIAL_COORDINATES.out.spatial_coordinates, loupe_alignment) + ch_tissue_barcodes = VISIUM_BUILD_TISSUE_POSITIONS.out.tissue_barcodes.first() + ch_updated_samples = ch_keyed_samples + .combine(VISIUM_BUILD_TISSUE_POSITIONS.out.tissue_positions.first()) + .map { _chem, sample, path, meta, bc, sc -> + [sample, path, meta + [barcode: bc, spatial_metadata: sc]] + } + } else { + ch_tissue_barcodes = channel.empty() + ch_updated_samples = ch_keyed_samples + .map { _chem, sample, path, meta, bc -> + [sample, path, meta + [barcode: bc, spatial_metadata: null]] + } } // split samples by file type @@ -55,4 +65,5 @@ workflow PREPARE_INPUT_STANDARD { emit: fastq = ch_fastq bam = ch_bam + tissue_barcodes = ch_tissue_barcodes } \ No newline at end of file diff --git a/subworkflows/prepare_input_visium_hd.nf b/subworkflows/prepare_input_visium_hd.nf index 4866c7c..bb4d891 100644 --- a/subworkflows/prepare_input_visium_hd.nf +++ b/subworkflows/prepare_input_visium_hd.nf @@ -1,4 +1,4 @@ -include { FILTER_BARCODED_BAM } from '../modules/prepare_input/visium_hd/filter_barcoded_bam.nf' +include { FILTER_BARCODED_BAM } from '../modules/prepare_input/shared/filter_barcoded_bam.nf' include { CONVERT_BARCODE_MAPPINGS } from '../modules/prepare_input/visium_hd/convert_barcode_mappings.nf' include { CONVERT_TISSUE_POSITIONS } from '../modules/prepare_input/visium_hd/convert_tissue_positions.nf'