wf-transcriptomes v2.0.0

.gitlab-ci.yml

@@ -16,9 +16,7 @@ variables:
               PYTEST_TESTS_PATH: "bin/workflow_glue/tests/common"
               RTEST_CONTAINER_NAME: "wf-transcriptomes-core"
               RTEST_CONTAINER_CONFIG_KEY: "container_sha"
-              WF_TEMPLATE_ENFORCEMENT_BRANCH: "ruo-next"
+              WF_TEMPLATE_ENFORCEMENT_REF: "v6.1.0-rc1"
-              RELEASE_WORKFLOW: "no" # do not release this workflow!
-              RELEASE_WITHOUT_DEMO: "yes"
 
 pytest_wfcontainer:
     extends: pytest
@@ -74,7 +72,8 @@ docker-run:
             - MATRIX_NAME: [
                 "int_discover_dna", "int_fixed_rna", "int_de_control_vs_control",
                 "smoke_discover", "smoke_fixed", "smoke_direct_rna", "smoke_de",
-                "mouse_de_0countquant", "mods_bigwig_igv", "mismatch-sample-alias"
+                "mouse_de_0countquant", "mods_bigwig_igv", "mismatch-sample-alias",
+                "mouse_splice_error", "mouse_splice_realign"
             ]
     rules:
         # NOTE As we're overriding the rules block for the included docker-run
@@ -202,6 +201,26 @@ docker-run:
                 --ref_annotation ${CI_PROJECT_NAME}/data/gencode.v22.annotation.chr20.gtf \
                 --sample_sheet ${CI_PROJECT_NAME}/data/mods_rna_subset/sample_sheet.csv \
                 --igv"
+        - if: $MATRIX_NAME == "mouse_splice_error"
+          variables:
+              NF_BEFORE_SCRIPT: "mkdir -p ${CI_PROJECT_NAME}/data/ && wget https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-transcriptomes/mouse_splice_fail.tar.gz -O ${CI_PROJECT_NAME}/data/mouse_splice_fail.tar.gz && tar -xzvf ${CI_PROJECT_NAME}/data/mouse_splice_fail.tar.gz  -C ${CI_PROJECT_NAME}/data/" 
+              NF_WORKFLOW_OPTS: "--bam ${CI_PROJECT_NAME}/data/mouse_splice_fail/samples \
+                  --de_analysis --ref_genome ${CI_PROJECT_NAME}/data/mouse_splice_fail/mouse_subset.fa \
+                  --ref_annotation ${CI_PROJECT_NAME}/data/mouse_splice_fail/mouse_subset.gtf.gz \
+                  --sample_sheet ${CI_PROJECT_NAME}/data/mouse_splice_fail/sample_sheet.csv"
+              ASSERT_NEXTFLOW_FAILURE: "1"
+              AFTER_NEXTFLOW_CMD: >
+                  grep -qF 'Cannot proceed with mixed splice-aware CIGAR evidence in input BAMs.' .nextflow.log
+        - if: $MATRIX_NAME == "mouse_splice_realign"
+          variables:
+              NF_BEFORE_SCRIPT: "mkdir -p ${CI_PROJECT_NAME}/data/ && wget https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-transcriptomes/mouse_splice_realign.tar.gz -O ${CI_PROJECT_NAME}/data/mouse_splice_realign.tar.gz && tar -xzvf ${CI_PROJECT_NAME}/data/mouse_splice_realign.tar.gz  -C ${CI_PROJECT_NAME}/data/" 
+              NF_WORKFLOW_OPTS: "--bam ${CI_PROJECT_NAME}/data/mouse_splice_realign/samples \
+                  --de_analysis --ref_genome ${CI_PROJECT_NAME}/data/mouse_splice_realign/mouse_subset.fa \
+                  --ref_annotation ${CI_PROJECT_NAME}/data/mouse_splice_realign/mouse_subset.gtf.gz \
+                  --sample_sheet ${CI_PROJECT_NAME}/data/mouse_splice_realign/sample_sheet.csv"
+              AFTER_NEXTFLOW_CMD: >
+                  grep -q 'bam_alignment' ${CI_PROJECT_NAME}/execution/trace.txt && grep -q 'No input BAMs appear to contain splice-aware CIGAR evidence. The workflow will realign all inputs.' .nextflow.log
+        
 
 
 singularity-run:

CHANGELOG.md

@@ -4,7 +4,8 @@ All notable changes to this project will be documented in this file.
 The format is based on [Keep a Changelog](https://keepachangelog.com/en/1.1.0/),
 and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html).
 
-## [v2.0.0-rc1]
+
+## [v2.0.0]
 
 This release refreshes `wf-transcriptomes` around a new reference-guided transcriptomics workflow built on `bambu`, with `SQANTI3` transcript classification and QC, `DESeq2` for differential gene expression, `DEXSeq` for differential transcript usage, and per-sample modified base summarisation with `modkit` when modification tags are present in aligned BAMs.
 
@@ -32,9 +33,10 @@ This release refreshes `wf-transcriptomes` around a new reference-guided transcr
     - Sample-level hierarchical clustering, PCA and distance heatmap plots
     - Contrast-level interactive volcano plots
 ### Removed
+- Dependence on the older `StringTie/GffCompare/Salmon` transcriptomics pathway.
 - `--transcriptome_source` parameter; use `--transcriptome_mode` instead.
 - `--ref_transcriptome` parameter; use `--transcriptome_mode fixed_annotation` together with `--ref_genome` and `--ref_annotation`.
-- Dependence on the older `StringTie/GffCompare/Salmon` transcriptomics pathway.
+- `--threads` parameter; advanced users may use Nextflow process selectors to override the preset per-process CPU and memory limits.
 
 ## [v1.7.2]
 This patch release of wf-transcriptomes updates internal workflow naming, and does not affect any workflow outputs.

README.md

@@ -112,6 +112,24 @@ the following command:
 nextflow pull epi2me-labs/wf-transcriptomes
 ```
 
+A demo dataset is provided for testing of the workflow.
+It can be downloaded and unpacked using the following commands:
+```
+wget https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-transcriptomes/v2/wf-transcriptomes-demo.tar.gz
+tar -xzvf wf-transcriptomes-demo.tar.gz
+```
+The workflow can then be run with the downloaded demo data using:
+```
+nextflow run epi2me-labs/wf-transcriptomes \
+	--bam 'wf-transcriptomes-demo/samples' \
+	--de_analysis \
+	--direct_rna \
+	--ref_annotation 'wf-transcriptomes-demo/gencode.v22.annotation.chr20.gtf' \
+	--ref_genome 'wf-transcriptomes-demo/hg38_chr20.fa' \
+	--sample_sheet 'wf-transcriptomes-demo/sample_sheet.csv' \
+	-profile standard
+```
+
 
 
 
@@ -351,7 +369,6 @@ The workflow's analysis is controlled by a user provided genome, annotation, and
 
 | Nextflow parameter name  | Type | Description | Help | Default |
 |--------------------------|------|-------------|------|---------|
-| threads | integer | Thread count to use for the core workflow processes. |  | 4 |
 | mod_codes | string | Comma-separated modified base codes to pass to modkit pileup. | Provide values accepted by `modkit pileup --modified-bases`, for example `A:a,C:m`. If omitted, the workflow infers `primary_base:mod_code` pairs from the BAM with `modkit modbam check-tags`. |  |
 | force_alignment | boolean | Force re-alignment of input BAM files. | Read alignment is skipped if the existing sequence names in the aligned BAM match the provided reference. Enable this if the existing alignments used incorrect minimap2 presets (e.g. missing --splice or direct RNA settings). | False |
 | ndr | number | Optional bambu novel discovery rate override. | Lower values are more conservative (higher precision), while higher values are more permissive (higher novel-discovery sensitivity). See the [`bambu` repository](https://github.com/GoekeLab/bambu) for method details. |  |

bin/workflow_glue/check_valid_modbam.py

@@ -1,36 +0,0 @@
-#!/usr/bin/env python
-"""Check whether the input is a modbam."""
-
-import os
-import sys
-
-import pysam
-
-from .util import get_named_logger, wf_parser  # noqa: ABS101
-
-
-def main(args):
-    """Run the entry point."""
-    logger = get_named_logger("check_valid_modbam")
-    logger.info(f'Checking file: {args.bam}')
-
-    # Check the first 10K reads of the input bam for ML/MM fields
-    valid_reads = 0
-    fields = ['mm', 'ml']
-    for i, alignment in enumerate(pysam.AlignmentFile(args.bam)):
-        n_tags = len([
-            tag for (tag, val) in alignment.get_tags() if tag.lower() in fields])
-        if n_tags == 2:
-            valid_reads += 1
-            break
-        if i >= 9999:
-            break
-    if valid_reads == 0:
-        sys.exit(os.EX_DATAERR)
-
-
-def argparser():
-    """Argument parser for entrypoint."""
-    parser = wf_parser("check_valid_modbam")
-    parser.add_argument("bam", help="Input bam file")
-    return parser

bin/workflow_glue/report.py

@@ -386,21 +386,28 @@ def _mod_summary_matrix_style():
     """
 
 
-def _contrast_results(de_dir, filename, n=None):
+def _contrast_results(de_dir, filename):
     """Return a dict of per-contrast result DataFrames read from filename."""
     tables = {}
     for contrast_dir in sorted(Path(de_dir).iterdir()):
         if not contrast_dir.is_dir():
             continue
-        table = _read_table(contrast_dir / filename)
+        # Enforce str dtype in case of all Nan values.
+        table = _read_table(
+            contrast_dir / filename,
+            dtype={'gene_name': str, 'transcript_name': str}
+        )
         if table is None or table.empty:
             continue
-        data = table
+
-        if n is not None:
+        if 'gene_name' in table.columns:
-            data = data.head(n)
+            table["gene_name"] = table["gene_name"].fillna("-")
-        if "padj" in data.columns:
+        if 'transcript_name' in table.columns:
-            data.sort_values("padj", ascending=True, inplace=True)
+            table["transcript_name"] = table["transcript_name"].fillna("-")
-        tables[contrast_dir.name] = data
+
+        if "padj" in table.columns:
+            table.sort_values("padj", ascending=True, inplace=True)
+        tables[contrast_dir.name] = table
 
     return tables
 
@@ -548,17 +555,23 @@ def _heatmap_style():
 
 def _volcano_style():
     return """
-        .volcano-table-grid {
+        .volcano-plot-grid {
             display: grid;
-            grid-template-columns: repeat(2, minmax(0, 1fr));
+            grid-template-columns: minmax(0, 1fr) minmax(430px, 34%);
-            gap: 20px 10px;
+            gap: 18px;
             align-items: start;
         }
-        .volcano-table-grid > * {
+        .volcano-plot-grid > *,
+        .volcano-side-panel > * {
             min-width: 0;
         }
-        @media screen and (max-width: 1000px) {
+        .volcano-side-panel {
-            .volcano-table-grid {
+            display: grid;
+            gap: 10px;
+            align-items: start;
+        }
+        @media screen and (max-width: 1100px) {
+            .volcano-plot-grid {
                 grid-template-columns: 1fr;
             }
         }
@@ -1417,10 +1430,11 @@ def main(args):
 
                     h3("Gene expression volcano Plot")
                     gn_vol, gn_class_table, gn_selected_table = volcano(table)
-                    EZChart(gn_vol, width="100%", height="550")
+                    with div(_class="volcano-plot-grid"):
-                    with div(_class="volcano-table-grid"):
+                        EZChart(gn_vol, width="100%", height="550")
-                        EZChart(gn_class_table, width="100%", height="auto")
+                        with div(_class="volcano-side-panel"):
-                        EZChart(gn_selected_table, width="100%", height="auto")
+                            EZChart(gn_class_table, width="100%", height="auto")
+                            EZChart(gn_selected_table, width="100%", height="auto")
 
         with report.add_section("Differential transcript usage", "DTU"):
             p(
@@ -1505,10 +1519,11 @@ def main(args):
 
                         h3("Transcript expression volcano Plot")
                         tr_vol, tr_class_table, tr_selected_table = volcano(dtu_table)
-                        EZChart(tr_vol, width="100%", height="550")
+                        with div(_class="volcano-plot-grid"):
-                        with div(_class="volcano-table-grid"):
+                            EZChart(tr_vol, width="100%", height="550")
-                            EZChart(tr_class_table, width="100%", height="auto")
+                            with div(_class="volcano-side-panel"):
-                            EZChart(tr_selected_table, width="100%", height="auto")
+                                EZChart(tr_class_table, width="100%", height="auto")
+                                EZChart(tr_selected_table, width="100%", height="auto")
                     else:
                         p("No DTU results available for this contrast.")
 

bin/workflow_glue/tests/common/test_check_sample_sheet.py

@@ -26,13 +26,12 @@ def _run_check_sample_sheet(sample_sheet_path, params_json, capsys):
     [
         [
             "condition", "batch_1", "site.2",
-            "S01", "a1", "sample_group",
+            "S01", "a1", "sample_group"
-            "01", "1batch", "2.site-3"
         ],
     ],
 )
 def test_validate_r_formula_names_accepts_valid_names(names):
-    """R formula names accept alnum-led names with safe punctuation."""
+    """R formula names accept letter-led names with safe punctuation."""
     _validate_r_formula_names(names)
 
 
@@ -45,6 +44,9 @@ def test_validate_r_formula_names_accepts_valid_names(names):
         ["size:batch"],
         ["size|batch"],
         ["sueño"],
+        ["01"],
+        ["1batch"],
+        ["2.site-3"],
         [""]
     ],
 )
@@ -240,7 +242,7 @@ def test_check_sample_sheet_rejects_unsafe_covariate_values(
     """Configured covariate values must use safe contrast/design characters."""
     sample_sheet_path = tmp_path / "sample_sheet_invalid_covariate_value.csv"
     sample_sheet_path.write_text(
-        "sample_name,condition,good-batch,site\n"
+        "sample_name,condition,good_batch,site\n"
         "test_name1,control,b1,s1\n"
         "test_name2,control,b2,s2\n"
         "test_name3,treated,b 1,s1\n"
@@ -252,7 +254,7 @@ def test_check_sample_sheet_rejects_unsafe_covariate_values(
         json.dumps({
             "de_analysis": True,
             "condition_column": "condition",
-            "covariates": "good-batch,site",
+            "covariates": "good_batch,site",
         })
     )
 
@@ -260,16 +262,16 @@ def test_check_sample_sheet_rejects_unsafe_covariate_values(
 
     assert out.startswith(
         "Covariate value names must be safe for R formulas. "
-        "Invalid names: b 1. Names must start with a letter or number and "
+        "Invalid names: b 1. Names must start with a letter and "
-        "contain only letters, numbers, underscores, dots, and hyphens. "
+        "contain only letters, numbers, underscores, and dots. "
-        "(column: good-batch, line: 3)"
+        "(column: good_batch, line: 3)"
     )
 
 
 def test_check_sample_sheet_rejects_unsafe_column_names(
         tmp_path, capsys
 ):
-    """Configured DE condition and covariate column names may include hyphens."""
+    """Configured DE design names may not include hyphens or spaces."""
     sample_sheet_path = tmp_path / "sample_sheet_invalid_design_name.csv"
     sample_sheet_path.write_text(
         "sample_name,condition-column,bad covariate\n"
@@ -289,12 +291,14 @@ def test_check_sample_sheet_rejects_unsafe_column_names(
     )
 
     out = _run_check_sample_sheet(sample_sheet_path, params_json, capsys)
-    assert out.startswith(
+    assert out.splitlines() == [
+        "Design column names must be safe for R formulas. "
+        "Invalid names: condition-column. Names must start with a letter and "
+        "contain only letters, numbers, underscores, and dots.",
         "Covariate column names must be safe for R formulas. "
-        "Invalid names: bad covariate. Names must start with a letter or "
+        "Invalid names: bad covariate. Names must start with a letter and "
-        "number and contain only letters, numbers, underscores, dots, and "
+        "contain only letters, numbers, underscores, and dots.",
-        "hyphens."
+    ]
-    )
 
 
 def test_check_sample_sheet_rejects_unsafe_condition_values(
@@ -323,13 +327,13 @@ def test_check_sample_sheet_rejects_unsafe_condition_values(
 
     assert out.splitlines() == [
         "Condition value names must be safe for R formulas. "
-        "Invalid names: control/group. Names must start with a letter or "
+        "Invalid names: control/group. Names must start with a letter and "
-        "number and contain only letters, numbers, underscores, dots, and "
+        "contain only letters, numbers, underscores, and dots. "
-        "hyphens. (column: condition, line: 1)",
+        "(column: condition, line: 1)",
         "Condition value names must be safe for R formulas. "
-        "Invalid names: control/group. Names must start with a letter or "
+        "Invalid names: control/group. Names must start with a letter and "
-        "number and contain only letters, numbers, underscores, dots, and "
+        "contain only letters, numbers, underscores, and dots. "
-        "hyphens. (column: condition, line: 2)",
+        "(column: condition, line: 2)",
     ]
 
 

bin/workflow_glue/tests/common/test_check_valid_modbam.py

@@ -1,66 +0,0 @@
-"""Tests for modified-base BAM validation."""
-
-import os
-
-import pytest
-from workflow_glue import check_valid_modbam
-
-
-class FakeAlignment:
-    """Minimal alignment stub exposing pysam's get_tags API."""
-
-    def __init__(self, tags):
-        """Store the synthetic SAM tags returned by ``get_tags``."""
-        self._tags = tags
-
-    def get_tags(self):
-        """Return the synthetic tag list for this fake alignment."""
-        return self._tags
-
-
-def _args(*argv):
-    return check_valid_modbam.argparser().parse_args(list(argv))
-
-
-def test_main_accepts_bam_with_mm_and_ml_tags(monkeypatch):
-    """A read carrying both MM and ML tags should pass validation."""
-    monkeypatch.setattr(
-        check_valid_modbam.pysam,
-        "AlignmentFile",
-        lambda _: [
-            FakeAlignment([("MM", "A+a.,0;"), ("ML", [255])]),
-        ],
-    )
-
-    check_valid_modbam.main(_args("input.bam"))
-
-
-def test_main_rejects_bam_without_mod_tags(monkeypatch):
-    """A BAM with no modified-base tags should exit with EX_DATAERR."""
-    monkeypatch.setattr(
-        check_valid_modbam.pysam,
-        "AlignmentFile",
-        lambda _: [
-            FakeAlignment([]),
-            FakeAlignment([("NM", 0)]),
-        ],
-    )
-
-    with pytest.raises(SystemExit) as exc_info:
-        check_valid_modbam.main(_args("input.bam"))
-
-    assert exc_info.value.code == os.EX_DATAERR
-
-
-def test_main_accepts_when_mod_tags_appear_later_in_scan(monkeypatch):
-    """Validation should continue scanning until it finds a tagged read."""
-    monkeypatch.setattr(
-        check_valid_modbam.pysam,
-        "AlignmentFile",
-        lambda _: [
-            FakeAlignment([("NM", 0)]),
-            FakeAlignment([("mm", "C+m.,0;"), ("ml", [200])]),
-        ],
-    )
-
-    check_valid_modbam.main(_args("input.bam"))

bin/workflow_glue/volcano.py

@@ -1,6 +1,6 @@
 """Interactive volcano plot visualization for differential expression analysis."""
 
-from bokeh.events import Tap
+from bokeh.events import DocumentReady, Tap
 from bokeh.layouts import column as bokeh_column, row as bokeh_row
 from bokeh.models import (
     AutocompleteInput,
@@ -102,15 +102,7 @@ def _volcano_source_data(data, fold_threshold=1, p_threshold=0.05):
     if not has_gene_name:
         data["gene_name"] = ""
 
-    data["gene_group"] = data["GENEID"] if "GENEID" in data.columns else ""
+    data["gene_group"] = data["GENEID"]
-    if is_transcript_plot:
-        if "gene_name" in data.columns:
-            named_group = data["gene_name"].astype(bool)
-            data.loc[named_group, "gene_group"] = data.loc[named_group, "gene_name"]
-        else:
-            named_group = data["GENEID"].astype(bool)
-            data.loc[named_group, "gene_group"] = data.loc[named_group, "GENEID"]
-
     data["selected_label"] = ""
 
     # Transformations and thresholds
@@ -306,6 +298,25 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
     _pos_means = source_data["mean_expression"][source_data["mean_expression"] > 0]
     mean_min_log = float(_pos_means.min()) if len(_pos_means) > 0 else 0.01
     y_threshold = -np.log10(p_threshold)
+    responsive_slider_title_stylesheet = """
+    @media screen and (max-width: 1100px) {
+        .bk-slider-title {
+            display: block;
+            max-width: 100%;
+            overflow: hidden;
+            text-overflow: ellipsis;
+            white-space: nowrap;
+        }
+        .bk-slider-title:hover {
+            overflow: visible;
+            text-overflow: clip;
+            white-space: normal;
+            position: relative;
+            z-index: 1;
+            background: white;
+        }
+    }
+    """
 
     source = ColumnDataSource(source_data)
     selection_state = ColumnDataSource({"indices": [[]]})
@@ -577,8 +588,9 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
         source=selected_source,
         columns=table_columns,
         autosize_mode="force_fit",
-        height=310,
+        height=355,
         index_position=None,
+        visible=False,
     )
     legend_table = DataTable(
         source=legend_source,
@@ -654,6 +666,7 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
         step=0.1,
         show_value=False,
         sizing_mode="stretch_width",
+        stylesheets=[responsive_slider_title_stylesheet],
     )
     fold_input = TextInput(
         title="",
@@ -668,6 +681,7 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
         step=0.1,
         show_value=False,
         sizing_mode="stretch_width",
+        stylesheets=[responsive_slider_title_stylesheet],
     )
     p_input = TextInput(
         title="",
@@ -681,6 +695,7 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
             selection_state=selection_state,
             highlight_source=highlight_source,
             selected_source=selected_source,
+            selected_table=selected_table,
             legend_source=legend_source,
             fold_slider=fold_slider,
             fold_input=fold_input,
@@ -783,6 +798,8 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
             selectedData.volcano_class.push(data.volcano_class[index]);
         });
         selected_source.data = selectedData;
+        selected_table.visible = selected.length > 0;
+        selected_table.height = window.innerWidth <= 1100 ? 180 : 355;
         if (view_toggle.active) {
             const highlightData = {
                 log2FoldChange: [],
@@ -1060,7 +1077,8 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
             "Search by gene/transcript…" if is_transcript_plot else "Search by gene…"),
         completions=[],
         min_characters=1,
-        width=240,
+        height=32,
+        width=225,
     )
     search_input.js_on_change("value_input", CustomJS(
         args=dict(
@@ -1169,7 +1187,8 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
     clear_button = Button(
         label="Clear selection",
         button_type="default",
-        width=130,
+        height=32,
+        width=120,
     )
     clear_button.js_on_click(CustomJS(
         args=dict(
@@ -1186,6 +1205,7 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
     export_button = Button(
         label="\u2B07 TSV",
         button_type="light",
+        height=32,
         width=55
     )
 
@@ -1232,8 +1252,15 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
     controls = bokeh_row(
         fold_slider, fold_input, p_slider, p_input, sizing_mode="stretch_width")
     toggle_row = bokeh_row(
-        plot_mode_toggle, view_toggle,
+        plot_mode_toggle,
-        gene_select_toggle, select_significant_button
+        view_toggle,
+        gene_select_toggle,
+        select_significant_button,
+        sizing_mode="stretch_width",
+        styles={
+            "flex-wrap": "wrap",
+            "row-gap": "6px",
+        },
     )
     volcano_ma_plot._fig = bokeh_column(
         bokeh_row(controls, sizing_mode="stretch_width"),
@@ -1255,8 +1282,6 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
         sizing_mode="stretch_width",
         styles={
             "padding-top": "4px",
-            "padding-left": "35px",
-            "padding-right": "20px"
         },
     )
     selected_plot = BokehPlot()
@@ -1265,8 +1290,27 @@ def volcano(data, fold_threshold=1, p_threshold=0.05):
             bokeh_row(
                 search_input, clear_button, export_button)
         ),
-        selected_table, sizing_mode="stretch_width",
+        selected_table,
+        sizing_mode="stretch_width",
         styles={
-            "padding-top": "25px",
+            "padding-top": "8px",
         })
+    selected_plot._fig.js_on_event(DocumentReady, CustomJS(
+        args=dict(
+            selected_source=selected_source,
+            selected_table=selected_table,
+        ),
+        code="""
+        const listenerKey = "volcano_selected_table_resize_" + selected_source.id;
+        function updateSelectedTableLayout() {
+            selected_table.visible = selected_source.data.source_index.length > 0;
+            selected_table.height = window.innerWidth <= 1100 ? 180 : 355;
+        }
+        updateSelectedTableLayout();
+        if (!window[listenerKey]) {
+            window[listenerKey] = true;
+            window.addEventListener("resize", updateSelectedTableLayout);
+        }
+        """,
+    ))
     return volcano_ma_plot, classes_table, selected_plot

bin/workflow_glue/wfg_helpers/check_bam_headers_in_dir.py

@@ -8,37 +8,69 @@ import pysam
 from ..util import get_named_logger, wf_parser  # noqa: ABS101
 
 
-def extract_header_info(xam_file, has_reads, check_ref=False):
+READ_SCAN_LIMIT = 10000
+
+
+def alignment_has_cigar_op(alignment, cigar_op):
+    """Return whether an alignment has a splice/reference-skip CIGAR op."""
+    return any(op == cigar_op for op, _ in (alignment.cigartuples or []))
+
+
+def alignment_has_tags(alignment, tags):
+    """Return whether an alignment carries all tags in a list of SAM tags."""
+    alignment_tags = {tag.upper() for tag, _ in alignment.get_tags()}
+    return all(tag in alignment_tags for tag in tags)
+
+
+def check_n_reads(
+    alignment_file,
+    check_modbase_tags=False,
+    check_splice_cigars=False,
+    read_scan_limit=READ_SCAN_LIMIT,
+):
+    """Extract bounded read-level facts from an open XAM file."""
+    read_facts = {
+        "has_reads": False,
+    }
+    if check_splice_cigars:
+        read_facts["has_splice_cigars"] = False
+    if check_modbase_tags:
+        read_facts["has_modbase_tags"] = False
+
+    for i, alignment in enumerate(alignment_file.fetch(until_eof=True)):
+        if i >= read_scan_limit:
+            break
+        read_facts["has_reads"] = True
+        if check_splice_cigars and not read_facts["has_splice_cigars"]:
+            read_facts["has_splice_cigars"] = alignment_has_cigar_op(
+                alignment, cigar_op=3)
+        if check_modbase_tags and not read_facts["has_modbase_tags"]:
+            read_facts["has_modbase_tags"] = alignment_has_tags(
+                alignment, tags=["MM", "ML"])
+
+        if all(read_facts.values()):
+            break
+
+    return read_facts
+
+
+def check_header(alignment_file, check_ref=False):
     """Extract header information from a BAM/CRAM file."""
-    try:
+    # Extract SQ lines, comparing only SN/LN/M5 elements
-        f = pysam.AlignmentFile(xam_file, check_sq=False)
+    # (see CW-4842 - ignore different SQ.UR values)
-    except (ValueError, IOError):
+    sq_lines = [{
-        return None, None, None, False
+        "SN": sq["SN"],
+        "LN": sq["LN"],
+        "M5": sq.get("M5"),
+    } for sq in alignment_file.header.get("SQ", [])]
 
-    with f:
+    hd_lines = alignment_file.header.get("HD")
-        # Extract SQ lines, comparing only SN/LN/M5 elements
-        # (see CW-4842 - ignore different SQ.UR values)
-        sq_lines = [{
-            "SN": sq["SN"],
-            "LN": sq["LN"],
-            "M5": sq.get("M5"),
-        } for sq in f.header.get("SQ", [])]
 
-        hd_lines = f.header.get("HD")
+    xam_reflen = None
+    if check_ref:
+        xam_reflen = set(zip(alignment_file.references, alignment_file.lengths))
 
-        xam_reflen = None
+    return sq_lines, hd_lines, xam_reflen
-        if check_ref:
-            xam_reflen = set(zip(f.references, f.lengths))
-        # Whilst we have file open check for atleast 1 reads
-        # unless reads already found
-        if not has_reads:
-            try:
-                next(f.fetch(until_eof=True))
-                has_reads = True
-            except StopIteration:
-                has_reads = False
-
-    return sq_lines, hd_lines, xam_reflen, has_reads
 
 
 def compare_ref_lengths(xam_reflen, ref_reflen, logger):
@@ -107,16 +139,37 @@ def main(args):
     requires_realign = False
     any_sq_lines = False  # Track if any file has @SQ lines
     has_reads = False
+    # a bit hacky to check these specific XAM properties here but this is a very good
+    # place to do it. in future i'd like to abstract this out to be more like how sample
+    # sheets are checked, with delegation to the workflow scope for determining checks
+    has_splice_cigars = False
+    has_modbase_tags = False
 
     for xam_file in target_files:
-        sq_lines, hd_lines, xam_reflen, has_reads = extract_header_info(
+        try:
-            xam_file, has_reads, check_ref=bool(args.ref))
+            xam_fh = pysam.AlignmentFile(xam_file, check_sq=False)
-
+        except (ValueError, IOError):
-        if sq_lines is None:
             # File couldn't be opened
             logger.error(f"Failed to open {xam_file}")
             continue
 
+        with xam_fh:
+            sq_lines, hd_lines, xam_reflen = check_header(
+                xam_fh,
+                check_ref=bool(args.ref),
+            )
+            if not (has_reads and has_splice_cigars and has_modbase_tags):
+                read_facts = check_n_reads(
+                    xam_fh,
+                    check_splice_cigars=not has_splice_cigars,
+                    check_modbase_tags=not has_modbase_tags,
+                )
+                has_reads = has_reads or read_facts["has_reads"]
+                has_splice_cigars = (
+                    has_splice_cigars or read_facts["has_splice_cigars"])
+                has_modbase_tags = (
+                    has_modbase_tags or read_facts["has_modbase_tags"])
+
         if sq_lines:
             any_sq_lines = True
 
@@ -157,9 +210,11 @@ def main(args):
         f"IS_UNALIGNED={int(is_unaligned)};" +
         f"MIXED_SQ_HEADERS={int(mixed_sq_headers)};" +
         f"IS_SORTED={int(sorted_xam)};" +
-        f"HAS_READS={int(has_reads)};"
+        f"HAS_READS={int(has_reads)};" +
+        f"HAS_SPLICE_CIGARS={int(has_splice_cigars)};" +
+        f"HAS_MODBASE_TAGS={int(has_modbase_tags)};"
     )
-    logger.info(f"Checked (u)BAM headers in '{args.input_path}'.")
+    logger.info(f"Checked (u)BAM heads in '{args.input_path}'.")
 
 
 def argparser():

bin/workflow_glue/wfg_helpers/validators/wf.py

@@ -5,7 +5,7 @@ import re
 
 from .default import SampleSheetValidator  # noqa: ABS101
 
-_R_FORMULA_NAME_RE = re.compile(r"^[A-Za-z0-9][A-Za-z0-9._-]*$")
+_R_FORMULA_NAME_RE = re.compile(r"^[A-Za-z][A-Za-z0-9_.]*$")
 
 
 def _split_covariates(value):
@@ -31,8 +31,8 @@ def _validate_r_formula_names(names, label="Column"):
             details.append(f"Invalid names: {', '.join(invalid)}")
         raise ValueError(
             f"{label} names must be safe for R formulas. {'; '.join(details)}. "
-            "Names must start with a letter or number and contain "
+            "Names must start with a letter and contain "
-            "only letters, numbers, underscores, dots, and hyphens."
+            "only letters, numbers, underscores, and dots."
         )
 
 

bin/workflow_glue_r/R/common.R

@@ -1,24 +1,3 @@
-workflow_glue_r_is_r_formula_name <- function(name) {
-    is.character(name) &&
-        length(name) == 1 &&
-        grepl("^[A-Za-z][A-Za-z0-9_.]*$", name)
-}
-
-workflow_glue_r_validate_r_formula_names <- function(names, label = "Column") {
-    invalid <- names[!vapply(names, workflow_glue_r_is_r_formula_name, logical(1))]
-    if (length(invalid) > 0) {
-        stop(
-            sprintf(
-                "%s names must be safe for R formulas. Invalid names: %s. Names must start with a letter and contain only letters, numbers, underscores, and dots.",
-                label,
-                paste(invalid, collapse = ", ")
-            ),
-            call. = FALSE
-        )
-    }
-    invisible(names)
-}
-
 workflow_glue_r_normalise_tsv_value <- function(value) {
     if (length(value) == 0 || all(is.na(value))) {
         return(NA_character_)

bin/workflow_glue_r/R/de_analysis.R

@@ -59,10 +59,6 @@ de_analysis_arg_parser <- function() {
 
 de_validate_inputs <- function(tx_se, gene_se, sample_df, argv) {
     covariates <- workflow_glue_r_parse_csv_list(argv$covariates)
-    workflow_glue_r_validate_r_formula_names(
-        c(argv$condition_column, covariates),
-        label = "Design column"
-    )
 
     if (any(duplicated(colnames(tx_se)))) {
         stop("Transcript RDS sample names must be unique.", call. = FALSE)

bin/workflow_glue_r/tests/testthat/test_de_analysis.R

@@ -13,39 +13,6 @@ testthat::test_that("covariates parsed and trimmed", {
     )
 })
 
-# R formulas break on spaces/hyphens in column names (e.g., ~ `time point` produces errors).
-# Validate condition_column and covariate names are safe (alphanumeric + underscore).
-testthat::test_that("formula-unsafe column names rejected", {
-    tx_se <- make_test_tx_se()
-    gene_se <- make_test_gene_se()
-    sample_df <- data.frame(
-        alias = colnames(tx_se),
-        condition = rep(c("control", "treated"), each = 3),
-        batch = rep(c("b1", "b2", "b1"), 2),
-        stringsAsFactors = FALSE
-    )
-    argv <- list(
-        transcript_rds = "transcripts.rds",
-        gene_rds = "genes.rds",
-        sample_sheet = "sample_sheet.csv",
-        condition_column = "time point",
-        covariates = "batch",
-        reference_level = NULL
-    )
-    argv <- workflow_glue_r_normalise_args(argv, de_analysis_arg_spec())
-
-    testthat::expect_error(
-        de_validate_inputs(tx_se, gene_se, sample_df, argv),
-        "Design column names must be safe for R formulas"
-    )
-
-    argv$condition_column <- "condition"
-    argv$covariates <- "batch-id"
-    testthat::expect_error(
-        de_validate_inputs(tx_se, gene_se, sample_df, argv),
-        "Invalid names: batch-id"
-    )
-})
 
 testthat::test_that("DTU transcript output renames contrast-specific log2fold column", {
     contrast_name <- "treated_control"

docs/04_install_and_run.md

@@ -37,3 +37,21 @@ the following command:
 ```
 nextflow pull epi2me-labs/wf-transcriptomes
 ```
+
+A demo dataset is provided for testing of the workflow.
+It can be downloaded and unpacked using the following commands:
+```
+wget https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-transcriptomes/v2/wf-transcriptomes-demo.tar.gz
+tar -xzvf wf-transcriptomes-demo.tar.gz
+```
+The workflow can then be run with the downloaded demo data using:
+```
+nextflow run epi2me-labs/wf-transcriptomes \
+	--bam 'wf-transcriptomes-demo/samples' \
+	--de_analysis \
+	--direct_rna \
+	--ref_annotation 'wf-transcriptomes-demo/gencode.v22.annotation.chr20.gtf' \
+	--ref_genome 'wf-transcriptomes-demo/hg38_chr20.fa' \
+	--sample_sheet 'wf-transcriptomes-demo/sample_sheet.csv' \
+	-profile standard
+```

docs/07_input_parameters.md

@@ -48,7 +48,6 @@
 
 | Nextflow parameter name  | Type | Description | Help | Default |
 |--------------------------|------|-------------|------|---------|
-| threads | integer | Thread count to use for the core workflow processes. |  | 4 |
 | mod_codes | string | Comma-separated modified base codes to pass to modkit pileup. | Provide values accepted by `modkit pileup --modified-bases`, for example `A:a,C:m`. If omitted, the workflow infers `primary_base:mod_code` pairs from the BAM with `modkit modbam check-tags`. |  |
 | force_alignment | boolean | Force re-alignment of input BAM files. | Read alignment is skipped if the existing sequence names in the aligned BAM match the provided reference. Enable this if the existing alignments used incorrect minimap2 presets (e.g. missing --splice or direct RNA settings). | False |
 | ndr | number | Optional bambu novel discovery rate override. | Lower values are more conservative (higher precision), while higher values are more permissive (higher novel-discovery sensitivity). See the [`bambu` repository](https://github.com/GoekeLab/bambu) for method details. |  |

evaluation/tests.sh

@@ -1,60 +0,0 @@
-#!/usr/bin/env bash
-
-# A few simple tests with different combinations of CLI options
-# Run from within an appropriate active conda environment
-
-if [[ "$#" -lt 1 ]]; then
-    echo "usage: tests.sh <outdir> [nextflow.config]"
-    exit 1
-fi
-
-if [[ "$#" -eq 1 ]]; then
-    config=''
-fi
-
-if [[ "$#" -eq 2 ]]; then
-  config="-c $2";
-fi
-
-SCRIPT_DIR="$( cd -- "$( dirname -- "${BASH_SOURCE[0]}" )" &> /dev/null && pwd )"
-cd $SCRIPT_DIR/../;
-
-singledir="test_data/fastq"
-multisampledir="test_data/demultiplexed_fastq"
-
-# This is for when using SIRV dataset with non-canonical spice junctions
-#"--minimap2_opts '-uf --splice-flank=no'"
-results=()
-
-# Reference based tests
-OUTPUT=$1/reference_single_dir;
-nextflow run . --fastq $singledir $config --ref_genome test_data/SIRV_150601a.fasta --minimap2_opts '-uf --splice-flank=no' \
---ref_annotation test_data/SIRV_isofroms.gtf -profile local --out_dir ${OUTPUT} -w ${OUTPUT}/workspace -resume;
-r=$?
-results+=("$(basename $OUTPUT): $r")
-
-OUTPUT=$1/multiple_samples;
-nextflow run . --fastq $multisampledir $config --ref_genome test_data/SIRV_150601a.fasta --minimap2_opts '-uf --splice-flank=no'\
---ref_annotation test_data/SIRV_isofroms.gtf -profile local --out_dir ${OUTPUT} -w ${OUTPUT}/workspace \
---sample_sheet test_data/sample_sheet -resume;
-r=$?
-results+=("$(basename $OUTPUT): $r")
-
-OUTPUT=$1/reference_no_ref_annotation;
-nextflow run . --fastq $singledir $config --ref_genome test_data/SIRV_150601a.fasta --minimap2_opts '-uf --splice-flank=no'\
- -profile local --out_dir ${OUTPUT} -w ${OUTPUT}/workspace -resume;
-r=$?
-results+=("$(basename $OUTPUT): $r")
-
-# Force split_bam to make multiple alignment bundles
-OUTPUT=$1/reference_frce_split_bam;
-nextflow run . --fastq $singledir  $config --ref_genome test_data/SIRV_150601a.fasta --minimap2_opts '-uf --splice-flank=no'\
---ref_annotation test_data/SIRV_isofroms.gtf -profile local --out_dir ${OUTPUT} -w ${OUTPUT}/workspace \
---bundle_min_reads 5 -resume;
-r=$?
-results+=("$(basename $OUTPUT): $r")
-
-echo "Exit status codes for each test"
-for value in "${results[@]}"; do
-     echo "${value}"
-done

lib/common.nf

@@ -67,7 +67,7 @@ process minimap2_alignment {
     cpus { bsargs["alignment_threads"] }
     memory { bsargs["minimap2_memory"][task.attempt - 1] }
     maxRetries { bsargs["minimap2_memory"].size() - 1 }
-    errorStrategy = {task.exitStatus in [137,140] ? 'retry' : 'finish'}
+    errorStrategy "retry"  // retry on any error as we may not exit with appropriate codes when exceeding memory
     input:
         tuple val(meta), path(reads, stageAs: 'reads/*'), path(reference), path(ref_index)
         tuple val(align_ext), val(index_ext) // either [bam, bai] or [cram, crai]

lib/ingress.nf

@@ -340,6 +340,9 @@ def fastq_ingress(Map arguments, aln_ref_ch = null)
        `cram` outputs a CRAM file with index (.cram, .crai)
  *  - "sort_threads": number of threads to use for sorting aligned reads (default: 1)
  *  - "force_alignment": boolean. Run alignment regardless of inputs.
+ *  - "checked_bam_hook": closure. Optional hook called after all BAM header/read checks
+ *     complete. Receives a list of `[meta, paths]` tuples and must return replacement
+ *     tuples of the same shape. Can warn, error, or add routing metadata.
  * @param aln_ref_ch: optional channel with a reference tuple (ref, ref_idx) to align against.
  *  If provided alignment will be attempted if inputs are unaligned or not already aligned to this ref.
  * @return: channel of `[Map(alias, barcode, type, ...), Path|null, Path|null]`.
@@ -361,8 +364,13 @@ def xam_ingress(Map arguments, aln_ref_ch = null)
             "fastcat_extra_args": "",
             "fastq_chunk": null,
             "force_alignment": false,
+            "checked_bam_hook": null,
         ]
     )
+    // immediately separate hooks from margs as they cannot be serialised
+    // and are currently only needed in this scope
+    def checked_bam_hook = margs.remove("checked_bam_hook")
+
     margs["fastq_chunk"] ?= 0  // cant pass null through channel
 
     if (margs["return_fastq"] && aln_ref_ch){
@@ -408,14 +416,24 @@ def xam_ingress(Map arguments, aln_ref_ch = null)
         [meta + [src_xam: src_xam, src_xai: src_xai], paths]
     }
     | combine(aln_ref)
-    | checkBamHeaders
+    | checkBamHeads
-    | map { meta, paths, is_unaligned_env, mixed_sq_headers_env, is_sorted_env, has_reads_env ->
+    | map {
+        meta,
+        paths,
+        is_unaligned_env,
+        mixed_sq_headers_env,
+        is_sorted_env,
+        has_reads_env,
+        has_splice_cigars_env,
+        has_modbase_tags_env ->
         // convert the env. variables from strings ('0' or '1') into bools
         boolean is_unaligned = is_unaligned_env as int as boolean
         boolean mixed_sq_headers = mixed_sq_headers_env as int as boolean
         boolean is_sorted = is_sorted_env as int as boolean
         // if no reads, no error (multisample may have some empty bams) but do not attempt alignment
         boolean has_reads = has_reads_env as int as boolean
+        boolean has_splice_cigars = has_splice_cigars_env as int as boolean
+        boolean has_modbase_tags = has_modbase_tags_env as int as boolean
         // throw an error if there was a sample with mixed headers
         if (mixed_sq_headers) {
             error "Found mixed headers in (u)BAM files of sample '${meta.alias}'."
@@ -423,7 +441,24 @@ def xam_ingress(Map arguments, aln_ref_ch = null)
         // add `is_unaligned` to the metamap (note the use of `+` to create a copy of
         // `meta` to avoid modifying every item in the channel;
         // https://github.com/nextflow-io/nextflow/issues/2660)
-        [meta + [is_unaligned: is_unaligned, is_sorted: is_sorted, has_reads: has_reads], paths]
+        [
+            meta + [
+                is_unaligned: is_unaligned,
+                is_sorted: is_sorted,
+                has_reads: has_reads,
+                has_splice_cigars: has_splice_cigars,
+                has_modbase_tags: has_modbase_tags,
+            ],
+            paths
+        ]
+    }
+
+    if (aln_ref_ch && checked_bam_hook) {
+        ch_check_bams = ch_check_bams
+        | collect(flat: false)
+        | flatMap { checked_bams ->
+            checked_bam_hook(checked_bams)
+        }
     }
 
     // Handle alignment
@@ -431,7 +466,7 @@ def xam_ingress(Map arguments, aln_ref_ch = null)
         alignment_fork = ch_check_bams
         | branch {
             meta, paths ->
-                to_align: (meta.is_unaligned || margs.force_alignment) && meta.has_reads
+                to_align: (meta.is_unaligned || meta.requires_alignment || margs.force_alignment) && meta.has_reads
                 noalign: true
         }
         if (margs["output_xam_fmt"] == "bam"){
@@ -448,14 +483,18 @@ def xam_ingress(Map arguments, aln_ref_ch = null)
         // Update meta is unaligned
         mm2_aln_final = mm2_aln.alignment.map{
             meta, xam, xai, stats ->
-                // remove has reads from meta as no longer required
+                // remove alignment routing metadata that is no longer required
-                def newmeta = meta.findAll { k, v -> k != 'has_reads' }
+                def newmeta = meta.findAll {
+                    k, v -> !(k in ['has_reads', 'requires_alignment'])
+                }
                 [newmeta + [is_unaligned: false], xam, xai, stats]
         }
         // Process BAM files that do not require realignment by passing them through the standard downstream steps (merging, sorting, indexing, etc.)
         ch_result_tmp = alignment_fork.noalign.map{
             meta, paths -> 
-                def newmeta = meta.findAll { k, v -> k != 'has_reads' }
+                def newmeta = meta.findAll {
+                    k, v -> !(k in ['has_reads', 'requires_alignment'])
+                }
                 [newmeta, paths]
         }
     } else {
@@ -769,7 +808,7 @@ process fastcat {
     """
 }
 
-process checkBamHeaders {
+process checkBamHeads {
     label "ingress"
     label "wf_common"
     cpus 1
@@ -784,6 +823,8 @@ process checkBamHeaders {
             env(MIXED_SQ_HEADERS),
             env(IS_SORTED),
             env(HAS_READS),
+            env(HAS_SPLICE_CIGARS),
+            env(HAS_MODBASE_TAGS),
         )
     script:
     String ref_arg = ref.fileName.name == OPTIONAL_FILE.name ? "" : "--ref $ref --ref_idx $ref_index"

main.nf

@@ -20,6 +20,8 @@ process getVersions {
     publishDir "${params.out_dir}", mode: 'copy', pattern: "versions.txt"
     cpus 1
     memory "2 GB"
+    input:
+        path "additional_versions.txt"
     output:
         path "versions.txt"
     script:
@@ -28,6 +30,36 @@ process getVersions {
     samtools --version | head -n 1 | sed 's/ /,/' >> versions.txt
     gffread --version | sed 's/^/gffread,/' >> versions.txt || true
     Rscript -e 'pkgs <- c("bambu", "DESeq2", "DEXSeq"); for (pkg in pkgs) {cat(pkg, as.character(packageVersion(pkg)), sep = ","); cat("\\n")}' >> versions.txt
+    cat additional_versions.txt >> versions.txt
+    """
+}
+
+
+process getSqantiVersion {
+    label "wf_transcriptomes_sqanti"
+    cpus 1
+    memory "2 GB"
+    output:
+        path "versions.txt"
+    script:
+    """
+    sqanti3_qc.py --version | sed 's/ /,/' >> versions.txt
+    """
+}
+
+
+process getModkitVersion {
+    label "modkit"
+    cpus 1
+    memory "2 GB"
+    input:
+        path "old_versions.txt"
+    output:
+        path "versions.txt"
+    script:
+    """
+    cp old_versions.txt versions.txt
+    modkit --version | sed 's/ /,/' >> versions.txt
     """
 }
 
@@ -100,7 +132,7 @@ workflow wf {
         ref_genome
         ref_annotation
     main:
-        software_versions = getVersions()
+        software_versions = getVersions(getModkitVersion(getSqantiVersion()))
         workflow_params = getParams()
 
         transcriptome_results = transcriptome(reads, ref_genome, ref_annotation, sample_sheet)
@@ -235,6 +267,70 @@ workflow {
         "sort_threads": 3,
     ]
 
+    def checked_bam_hook = { checked_bams ->
+        if (params.force_alignment) {
+            // short-circuit this hook to allow for forced realignment
+            return checked_bams
+        }
+
+        // determine BAMs with and without splice-aware CIGAR evidence
+        ArrayList aliases_with_splice_cigars = []
+        ArrayList aliases_without_splice_cigars = []
+        checked_bams.each {
+            def meta = it[0]
+            if (meta.has_reads) {
+                if (meta.has_splice_cigars) {
+                    aliases_with_splice_cigars.add(meta.alias)
+                } else {
+                    aliases_without_splice_cigars.add(meta.alias)
+                }
+            }
+        }
+        boolean any_with_splice_cigars = aliases_with_splice_cigars
+        boolean any_without_splice_cigars = aliases_without_splice_cigars
+
+        // explode on mixed splice-aware status
+        if (any_with_splice_cigars && any_without_splice_cigars) {
+            log.error """
+                Cannot proceed with mixed splice-aware CIGAR evidence in input BAMs.
+
+                This workflow requires splice-aware alignment. It cannot safely realign
+                only the BAMs that appear to lack splice-aware CIGARs, because the
+                original alignment parameters for the other BAMs are unknown.
+
+                It is unlikely for a splice-aware aligned transcriptome BAM to have no
+                reads with N CIGAR operations. Inspect your BAMs to understand why their
+                splice-aware alignment evidence differs. Either remove the BAMs without
+                splice-aware CIGARs from the analysis, or use --force_alignment to realign
+                all inputs.
+
+                Samples with splice CIGARs: ${aliases_with_splice_cigars.join(', ')}
+                Samples without splice CIGARs: ${aliases_without_splice_cigars.join(', ')}
+                """.stripIndent().trim()
+            error "Mixed splice-aware CIGAR evidence in input BAMs."
+        }
+
+        // all read-containing BAMs have splice-aware CIGAR evidence, or there are no reads
+        if (!any_without_splice_cigars) {
+            return checked_bams
+        }
+
+        // if we're here, all read-containing BAMs lack splice-aware CIGAR evidence
+        // fiddle the metamap to set requires_alignment (if there are reads to align)
+        log.warn "No input BAMs appear to contain splice-aware CIGAR evidence. " +
+            "The workflow will realign all inputs."
+        checked_bams.collect {
+            def meta = it[0]
+            def paths = it[1]
+            [
+                meta + [
+                    requires_alignment: meta.has_reads,
+                ],
+                paths
+            ]
+        }
+    }
+
     if (params.fastq) {
         samples = fastq_ingress([
             "input": params.fastq,
@@ -243,6 +339,7 @@ workflow {
         samples = xam_ingress([
             "input": params.bam,
             "force_alignment": params.force_alignment,
+            "checked_bam_hook": checked_bam_hook,
         ] + ingress_args, ref_genome)
     }
 

nextflow.config

@@ -22,7 +22,6 @@ params {
     analyse_unclassified = false
     analyse_fail = false
 
-    threads = 4
     mod_codes = null
     force_alignment = false
     ndr = null
@@ -44,9 +43,9 @@ params {
         per_read_stats = false
         allow_multiple_basecall_models = false
         example_cmd = [
+            "--bam 'wf-transcriptomes-demo/samples'",
             "--de_analysis",
             "--direct_rna",
-            "--fastq 'wf-transcriptomes-demo/differential_expression_fastq'",
             "--ref_annotation 'wf-transcriptomes-demo/gencode.v22.annotation.chr20.gtf'",
             "--ref_genome 'wf-transcriptomes-demo/hg38_chr20.fa'",
             "--sample_sheet 'wf-transcriptomes-demo/sample_sheet.csv'",
@@ -68,7 +67,7 @@ manifest {
     description     = 'Long-read transcript discovery, quantification, differential expression, QC and mod counting.'
     mainScript      = 'main.nf'
     nextflowVersion = '>=23.04.2'
-    version         = 'v2.0.0-rc1'
+    version         = 'v2.0.0'
 }
 
 process {

nextflow_schema.json

@@ -4,6 +4,8 @@
   "title": "epi2me-labs/wf-transcriptomes",
   "workflow_title": "Transcriptomes",
   "description": "Long-read transcript discovery, quantification, differential expression, QC and mod counting.",
+  "demo_url": "https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-transcriptomes/v2/wf-transcriptomes-demo.tar.gz",
+  "aws_demo_url": "https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-transcriptomes/wf-transcriptomes-demo/transcriptomes.v2.aws.nextflow.config",
   "url": "https://github.com/epi2me-labs/wf-transcriptomes",
   "type": "object",
   "definitions": {
@@ -167,11 +169,6 @@
       "type": "object",
       "description": "Performance tuning and tool-specific advanced parameters.",
       "properties": {
-        "threads": {
-          "type": "integer",
-          "default": 4,
-          "description": "Thread count to use for the core workflow processes."
-        },
         "mod_codes": {
           "type": "string",
           "description": "Comma-separated modified base codes to pass to modkit pileup.",

subworkflows/differential_expression.nf

@@ -3,7 +3,7 @@ nextflow.enable.dsl = 2
 
 process runDifferentialAnalysis {
     label "wf_transcriptomes"
-    cpus { params.threads ?: 4 }
+    cpus 4
     memory "32 GB"
     input:
         path transcript_rds

subworkflows/mods.nf

@@ -1,35 +1,6 @@
-// Check that the bam has modifications
-process validate_modbam {
-    label "wf_common"
-    cpus 1
-    memory 4.GB
-    input:
-        tuple val(meta),
-            path(alignment),
-            path(alignment_index),
-            val(alignment_stats)
-    output:
-        tuple val(meta),
-            path(alignment),
-            path(alignment_index),
-            val(alignment_stats),
-            env(valid)
-
-    script:
-    """
-    valid=0
-    workflow-glue check_valid_modbam ${alignment} || valid=\$?
-
-    # Allow EX_OK and EX_DATAERR, otherwise explode
-    if [ \$valid -ne 0 ] && [ \$valid -ne 65 ]; then
-        exit 1
-    fi
-    """
-}
-
 process runModkitPileup {
     label "modkit"
-    cpus { params.threads ?: 4 }
+    cpus 4
     memory "16 GB"
     input:
         tuple val(alias),
@@ -158,15 +129,12 @@ workflow mods {
         xams
         ref_genome
     main:
-        // Check inputs have modtags, we'll early abort mod analysis for any that don't
+        xams.branch {
-        validate_modbam(xams)
+            nomods: it[0].has_modbase_tags != true
-            .branch {
+                return it[0].alias
-                nomods: it[-1] == '65'
+            mods: it[0].has_modbase_tags == true
-                    return it[0].alias
+                return [it[0].alias] + it  // prepend alias for joining
-                mods: it[-1] == '0'
+        }.set{xams_with}
-                    return [it[0].alias] + it[0..-2]  // prepend alias for joining and drop exit_code marker
-            }
-            .set{xams_with}
 
         // warn for samples without mods
         xams_with.nomods.subscribe {

subworkflows/transcriptome.nf

@@ -138,7 +138,7 @@ process buildSampleTranscriptomeFasta {
 
 process runJointSqanti {
     label "wf_transcriptomes_sqanti"
-    cpus { params.threads ?: 4 }
+    cpus 2
     memory "24 GB"
     publishDir "${params.out_dir}/cohort",
         mode: "copy",
@@ -172,7 +172,7 @@ process runJointSqanti {
 
 process runPerSampleSqanti {
     label "wf_transcriptomes_sqanti"
-    cpus { params.threads ?: 4 }
+    cpus 2
     memory "24 GB"
     publishDir "${params.out_dir}/samples/${meta.alias}",
         mode: "copy",