Upgrade simpleaf and alevinqc

CITATIONS.md

@@ -18,6 +18,14 @@
 
   > Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct 1;32(19):3047-8. doi: 10.1093/bioinformatics/btw354. Epub 2016 Jun 16. PubMed PMID: 27312411; PubMed Central PMCID: PMC5039924.
 
+* [Alevin-fry](https://doi.org/10.1038/s41592-022-01408-3)
+
+  > He, D., Zakeri, M., Sarkar, H. et al. Alevin-fry unlocks rapid, accurate and memory-frugal quantification of single-cell RNA-seq data. Nat Methods 19, 316–322 (2022).
+
+* [Simpleaf](https://doi.org/10.1093/bioinformatics/btad614)
+
+  > He, D., Patro, R. simpleaf: a simple, flexible, and scalable framework for single-cell data processing using alevin-fry, Bioinformatics, Volume 39, Issue 10, October 2023, btad614.
+
 * [Alevin](https://doi.org/10.1186/s13059-019-1670-y)
 
   > Srivastava, A., Malik, L., Smith, T. et al. Alevin efficiently estimates accurate gene abundances from dscRNA-seq data. Genome Biol 20, 65 (2019).

docs/output.md

@@ -86,13 +86,13 @@ For details on how to load these into R and perform further downstream analysis,
 **Output directory: `results/alevin`**
 
 - `alevin`
-  - Contains the created Salmon Alevin pseudo-aligned output
+  - Contains the created alevin-fry pseudo-aligned output
 - `alevinqc`
   - Contains the QC report for the aforementioned Salmon Alevin output data
 
 **Output directory: `results/reference_genome`**
 
-- `salmon_index`
+- `simpleaf_index`
   - Contains the indexed reference transcriptome for Salmon Alevin
 - `alevin/txp2gene.tsv`
   - The transcriptome to gene mapping TSV file utilized by Salmon Alevin

docs/usage.md

@@ -39,15 +39,15 @@ An [example samplesheet](../assets/samplesheet.csv) has been provided with the p
 
 This parameter is currently supported by
 
-- [Salmon Alevin](https://salmon.readthedocs.io/en/latest/alevin.html#expectcells)
+- [Alevin-fry](https://alevin-fry.readthedocs.io/en/latest/generate_permit_list.html#:~:text=%2D%2Dexpect%2Dcells%20%3Cncells%3E)
 - [STARsolo](https://github.com/alexdobin/STAR/blob/master/docs/STARsolo.md)
 - [Cellranger](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
 
 Note that since cellranger v7, it is **not recommended** anymore to supply the `--expected-cells` parameter.
 
 ## Aligning options
 
-By default, the pipeline uses [Salmon Alevin](https://salmon.readthedocs.io/en/latest/alevin.html) (i.e. --aligner alevin) to perform pseudo-alignment of reads to the reference genome and to perform the downstream BAM-level quantification. Then QC reports are generated with AlevinQC.
+By default, the pipeline uses [Alevin-fry](https://alevin-fry.readthedocs.io/en/latest/) (i.e. --aligner alevin) via [Simpleaf](https://simpleaf.readthedocs.io/en/latest/) to perform pseudo-alignment of reads to the reference genome and to perform the downstream BAM-level quantification. Then QC reports are generated with AlevinQC.
 
 Other aligner options for running the pipeline are:
 
@@ -100,11 +100,11 @@ The command `kb --list` shows all supported, preconfigured protocols. Additional
 
 For more details, please refer to the [Kallisto/bustools documentation](https://pachterlab.github.io/kallisto/manual#bus).
 
-#### Alevin/fry
+#### Alevin-fry
 
-Alevin/fry also supports custom chemistries in a slighly different format, e.g. `1{b[16]u[12]x:}2{r:}`.
+Alevin-fry also supports custom chemistries in a slightly different format, e.g. `1{b[16]u[12]x:}2{r:}`.
 
-For more details, see the [simpleaf documentation](https://simpleaf.readthedocs.io/en/latest/quant-command.html#a-note-on-the-chemistry-flag)
+For more details, see the [simpleaf documentation](https://simpleaf.readthedocs.io/en/latest/quant-command.html#a-note-on-the-chemistry-flag) and the [language specification](https://hackmd.io/@PI7Og0l1ReeBZu_pjQGUQQ/rJMgmvr13).
 
 #### UniverSC
 

modules/local/alevinqc.nf

@@ -3,10 +3,10 @@ process ALEVINQC {
     label 'process_low'
 
     //The alevinqc 1.14.0 container is broken, missing some libraries - thus reverting this to previous 1.12.1 version
-    conda "bioconda::bioconductor-alevinqc=1.12.1"
+    conda "bioconda::bioconductor-alevinqc=1.18.0"
     container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
-        'https://depot.galaxyproject.org/singularity/bioconductor-alevinqc:1.12.1--r41h9f5acd7_0' :
-        'biocontainers/bioconductor-alevinqc:1.12.1--r41h9f5acd7_0' }"
+        'https://depot.galaxyproject.org/singularity/bioconductor-alevinqc:1.18.0--r43hf17093f_0' :
+        'biocontainers/bioconductor-alevinqc:1.18.0--r43hf17093f_0' }"
 
     input:
     tuple val(meta), path(alevin_results)
@@ -43,4 +43,4 @@ process ALEVINQC {
         "versions.yml"
     )
     """
-}
+}

modules/local/simpleaf_index.nf

@@ -2,10 +2,10 @@ process SIMPLEAF_INDEX {
     tag "$transcript_gtf"
     label "process_medium"
 
-    conda 'bioconda::simpleaf=0.10.0-1'
+    conda 'bioconda::simpleaf=0.17.2-0'
     container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
-        'https://depot.galaxyproject.org/singularity/simpleaf:0.10.0--h9f5acd7_1' :
-        'biocontainers/simpleaf:0.10.0--h9f5acd7_1' }"
+        'https://depot.galaxyproject.org/singularity/simpleaf:0.17.2--h919a2d8_0' :
+        'biocontainers/simpleaf:0.17.2--h919a2d8_0' }"
 
     input:
     path genome_fasta
@@ -14,7 +14,7 @@ process SIMPLEAF_INDEX {
 
     output:
     path "salmon/index"              , emit: index
-    path "salmon/ref/*_t2g_3col.tsv" , emit: transcript_tsv
+    path "salmon/ref/*t2g_3col.tsv" , emit: transcript_tsv
     path "versions.yml"              , emit: versions
     path "salmon"                    , emit: salmon
 
@@ -23,7 +23,8 @@ process SIMPLEAF_INDEX {
 
     script:
     def args = task.ext.args ?: ''
-    def seq_inputs = (params.transcript_fasta) ? "--refseq $transcript_fasta" : "--gtf $transcript_gtf"
+    def seq_inputs = (params.transcript_fasta) ? "--refseq $transcript_fasta" : "--fasta $genome_fasta --gtf $transcript_gtf"
+    def no_piscem = (params.no_piscem) ? '--no-piscem' : ''
     """
     # export required var
     export ALEVIN_FRY_HOME=.
@@ -36,8 +37,8 @@ process SIMPLEAF_INDEX {
     simpleaf \\
         index \\
         --threads $task.cpus \\
-        --fasta $genome_fasta \\
         $seq_inputs \\
+        $no_piscem \\
         $args \\
         -o salmon
 

modules/local/simpleaf_quant.nf

@@ -2,10 +2,10 @@ process SIMPLEAF_QUANT {
     tag "$meta.id"
     label 'process_high'
 
-    conda 'bioconda::simpleaf=0.10.0-1'
+    conda 'bioconda::simpleaf=0.17.2-0'
     container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
-        'https://depot.galaxyproject.org/singularity/simpleaf:0.10.0--h9f5acd7_1' :
-        'biocontainers/simpleaf:0.10.0--h9f5acd7_1' }"
+        'https://depot.galaxyproject.org/singularity/simpleaf:0.17.2--h919a2d8_0' :
+        'biocontainers/simpleaf:0.17.2--h919a2d8_0' }"
 
     input:
     //
@@ -29,6 +29,8 @@ process SIMPLEAF_QUANT {
     def args      = task.ext.args ?: ''
     def args_list = args.tokenize()
     def prefix    = task.ext.prefix ?: "${meta.id}"
+    // selective alignment is only available in salmon
+    def use_selective_alignment = (params.no_piscem && params.use_selective_alignment) ? '-s' : ''
 
     //
     // check if users are using one of the mutually excludable parameters:
@@ -70,6 +72,7 @@ process SIMPLEAF_QUANT {
         -c "$protocol" \\
         $expect_cells \\
         $unfiltered_command \\
+        $use_selective_alignment \\
         $args
 
     $save_whitelist

nextflow.config

@@ -25,7 +25,9 @@ params {
     // salmon alevin parameters (simpleaf)
     simpleaf_rlen     = 91
     barcode_whitelist = null
-    salmon_index      = null
+    simpleaf_index    = null
+    no_piscem         = false
+    use_selective_alignment = false
 
     // kallisto bustools parameters
     kallisto_index    = null

nextflow_schema.json

@@ -158,9 +158,9 @@
             "description": "",
             "default": "",
             "properties": {
-                "salmon_index": {
+                "simpleaf_index": {
                     "type": "string",
-                    "description": "This can be used to specify a precomputed Salmon index in the pipeline, in order to skip the generation of required indices by Salmon itself.",
+                    "description": "This can be used to specify a precomputed Simpleaf index in the pipeline, in order to skip the generation of required indices by Simpleaf itself.",
                     "fa_icon": "fas fa-fish",
                     "format": "path",
                     "exists": true
@@ -178,6 +178,16 @@
                     "default": 91,
                     "description": "It is the target read length the index will be built for, using simpleaf.",
                     "fa_icon": "fas fa-map-marked-alt"
+                },
+                "no_piscem": {
+                    "type": "boolean",
+                    "fa_icon": "fas fa-map-marked-alt",
+                    "description": "Don't use the default piscem mapper, instead use salmon-alevin"
+                },
+                "use_selective_alignment": {
+                    "type": "boolean",
+                    "fa_icon": "fas fa-map-marked-alt",
+                    "description": "Use selective-alignment for mapping instead of pseudoalignment with structural constraints (only if using salmon alevin as the underlying mapper)."
                 }
             }
         },

subworkflows/local/alevin.nf

@@ -16,7 +16,7 @@ workflow SCRNASEQ_ALEVIN {
     genome_fasta
     gtf
     transcript_fasta
-    salmon_index
+    simpleaf_index
     txp2gene
     barcode_whitelist
     protocol
@@ -26,16 +26,16 @@ workflow SCRNASEQ_ALEVIN {
     main:
     ch_versions = Channel.empty()
 
-    assert (genome_fasta && gtf && salmon_index && txp2gene) || (genome_fasta && gtf)  || (genome_fasta && gtf && transcript_fasta && txp2gene):
+    assert (genome_fasta && gtf && simpleaf_index && txp2gene) || (genome_fasta && gtf)  || (genome_fasta && gtf && transcript_fasta && txp2gene):
         """Must provide a genome fasta file ('--fasta') and a gtf file ('--gtf'), or a genome fasta file
         and a transcriptome fasta file ('--transcript_fasta`) if no index and txp2gene is given!""".stripIndent()
 
     /*
     * Build salmon index
     */
-    if (!salmon_index) {
+    if (!simpleaf_index) {
         SIMPLEAF_INDEX( genome_fasta, transcript_fasta, gtf )
-        salmon_index = SIMPLEAF_INDEX.out.index.collect()
+        simpleaf_index = SIMPLEAF_INDEX.out.index.collect()
         transcript_tsv = SIMPLEAF_INDEX.out.transcript_tsv.collect()
         ch_versions = ch_versions.mix(SIMPLEAF_INDEX.out.versions)
 
@@ -51,7 +51,7 @@ workflow SCRNASEQ_ALEVIN {
     */
     SIMPLEAF_QUANT (
         ch_fastq,
-        salmon_index,
+        simpleaf_index,
         txp2gene,
         protocol,
         barcode_whitelist

workflows/scrnaseq.nf

@@ -68,7 +68,7 @@ workflow SCRNASEQ {
     kb_workflow = params.kb_workflow
 
     //salmon params
-    ch_salmon_index = params.salmon_index ? file(params.salmon_index) : []
+    ch_simpleaf_index = params.simpleaf_index ? file(params.simpleaf_index) : []
 
     //star params
     star_index = params.star_index ? file(params.star_index, checkIfExists: true) : null
@@ -147,7 +147,7 @@ workflow SCRNASEQ {
             ch_genome_fasta,
             ch_filter_gtf,
             ch_transcript_fasta,
-            ch_salmon_index,
+            ch_simpleaf_index,
             ch_txp2gene,
             ch_barcode_whitelist,
             protocol_config['protocol'],