nf-core · egreenberg7 · Oct 24, 2025 · Oct 24, 2025 · Oct 24, 2025 · Oct 24, 2025
diff --git a/CHANGELOG.md b/CHANGELOG.md
@@ -10,6 +10,19 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0
 ### Enhancements and fixes
 
 - [PR #1608](https://github.com/nf-core/rnaseq/pull/1608) - Bump version after release 3.21.0
+- [PR #1616](https://github.com/nf-core/rnaseq/pull/1616) - Add Sylph for contamination detection.
+
+| Old parameter | New parameter      |
+| ------------- | -------------------|
+|               | `--sylph_db`       |
+|               | `--sylph_taxonomy` |
+
+### Software dependencies
+
+| Dependency | Old version | New version |
+| -----------| ----------- | ----------- |
+| `sylph`    |             | 0.7.0       |
+| `sylph-tax`|             | 1.2.0       |
 
 ## [[3.21.0](https://github.com/nf-core/rnaseq/releases/tag/3.21.0)] - 2025-09-18
 

diff --git a/CITATIONS.md b/CITATIONS.md
@@ -88,6 +88,10 @@
 
   > Kovaka S, Zimin AV, Pertea GM, Razaghi R, Salzberg SL, Pertea M. Transcriptome assembly from long-read RNA-seq alignments with StringTie2 Genome Biol. 2019 Dec 16;20(1):278. doi: 10.1186/s13059-019-1910-1. PubMed PMID: 31842956; PubMed Central PMCID: PMC6912988.
 
+- [Sylph](https://pubmed.ncbi.nlm.nih.gov/39379646/)
+
+  > Shaw J, Yu YW. Rapid species-level metagenome profiling and containment estimation with sylph. Nat Biotechnol. 2025 Aug;43(8):1348-1359. doi: 10.1038/s41587-024-02412-y. Epub 2024 Oct 8. PMID: 39379646; PMCID: PMC12339375.
+
 - [Trim Galore!](https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)
 
 - [UMI-tools](https://pubmed.ncbi.nlm.nih.gov/28100584/)

diff --git a/README.md b/README.md
@@ -48,7 +48,9 @@
     3. [`dupRadar`](https://bioconductor.org/packages/release/bioc/html/dupRadar.html)
     4. [`Preseq`](http://smithlabresearch.org/software/preseq/)
     5. [`DESeq2`](https://bioconductor.org/packages/release/bioc/html/DESeq2.html)
-    6. [`Kraken2`](https://ccb.jhu.edu/software/kraken2/) -> [`Bracken`](https://ccb.jhu.edu/software/bracken/) on unaligned sequences; _optional_
+    6. Contamination detection on unaligned sequences;  _optional_
+        1. [`Kraken2`](https://ccb.jhu.edu/software/kraken2/) -> [`Bracken`](https://ccb.jhu.edu/software/bracken/)
+        2. [`Sylph`](https://sylph-docs.github.io/)
 15. Pseudoalignment and quantification ([`Salmon`](https://combine-lab.github.io/salmon/) or ['Kallisto'](https://pachterlab.github.io/kallisto/); _optional_)
 16. Present QC for raw read, alignment, gene biotype, sample similarity, and strand-specificity checks ([`MultiQC`](http://multiqc.info/), [`R`](https://www.r-project.org/))
 

diff --git a/docs/output.md b/docs/output.md
@@ -57,6 +57,7 @@ The pipeline is built using [Nextflow](https://www.nextflow.io/) and processes d
     - [featureCounts](#featurecounts)
     - [DESeq2](#deseq2)
     - [Kraken2/Bracken](#kraken2bracken)
+    - [Sylph](#Sylph)
     - [MultiQC](#multiqc)
   - [Pseudoalignment and quantification](#pseudoalignment-and-quantification)
     - [Pseudoalignment](#pseudoalignment)
@@ -737,6 +738,21 @@ The plot on the left hand side shows the standard PC plot - notice the variable
 
 ![MultiQC - Bracken top species plot](images/bracken-top-n-plot.png)
 
+### Sylph
+
+<details markdown="1">
+<summary>Output files</summary>
+
+- `<ALIGNER>/contaminants/sylph`
+  - `*.tsv` Summary of containment ANI and abundances of detected species in the sample. See the [Sylph documentation](https://sylph-docs.github.io/Output-format/) for full details on the output format. 
+  - `*.sylphmpa` Taxonomic report of unaligned reads from `sylph-tax`. See the [Sylph documentation](https://sylph-docs.github.io/sylph-tax-output-format/) for full details on the output format.
+
+</details>
+
+[Sylph](https://sylph-docs.github.io/) is a metagenomic profiler that determines the species present in reads by statistically estimating containment ANI. These algorithms are run on unaligned sequences to detect potential contamination of samples. MultiQC TBD. 
+
+#TODO add MultiQC info
+
 ### MultiQC
 
 <details markdown="1">

diff --git a/docs/usage.md b/docs/usage.md
@@ -401,11 +401,15 @@ By default, the input GTF file will be filtered to ensure that sequence names co
 The `--contaminant_screening` option is not currently available using ARM architecture ('-profile arm')
 :::
 
-The pipeline provides the option to scan unaligned reads for contamination from other species using [Kraken2](https://ccb.jhu.edu/software/kraken2/), with the possibility of applying corrections from [Bracken](https://ccb.jhu.edu/software/bracken/). Since running Bracken is not computationally expensive, we recommend always using it to refine the abundance estimates generated by Kraken2.
+The pipeline provides the option to scan unaligned reads for contamination from other species using either [Sylph](https://sylph-docs.github.io/) or the [Kraken2](https://ccb.jhu.edu/software/kraken2/)/[Bracken](https://ccb.jhu.edu/software/bracken/) suite.
 
-It is important to note that the accuracy of Kraken2 is [highly dependent on the database](https://doi.org/10.1099/mgen.0.000949) used. Specifically, it is [crucial](https://doi.org/10.1128/mbio.01607-23) to ensure that the host genome is included in the database. If you are particularly concerned about certain contaminants, it may be beneficial to use a smaller, more focused database containing primarily those contaminants instead of the full standard database. Various pre-built databases [are available for download](https://benlangmead.github.io/aws-indexes/k2), and instructions for building a custom database can be found in the [Kraken2 documentation](https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown). Additionally, genomes of contaminants detected in previous sequencing experiments are available on the [OpenContami website](https://openlooper.hgc.jp/opencontami/help/help_oct.php).
+Sylph is a [faster and much more memory-efficient tool](https://doi.org/10.1038/s41587-024-02412-y) with about equal precision in species detection to Kraken2/Bracken. Sylph also has lower rates of false positives. However, Sylph does not assign specific reads to species; it only provides overall abundance estimates. Sylph abundance estimates also [cannot assign a certain percentage of reads as unclassified](https://github.com/bluenote-1577/sylph/issues/49). 
 
-While Kraken2 is capable of detecting low-abundance contaminants in a sample, false positives can occur. Therefore, if only a very small number of reads from a contaminating species are detected, these results should be interpreted with caution.
+Pre-constructed sylph databases can be found [here](https://sylph-docs.github.io/pre%E2%80%90built-databases/) and taxonomies [here](https://sylph-docs.github.io/sylph-tax/). The [documentation](https://sylph-docs.github.io/sylph-tax/) also has instructions on creating custom databases/taxonomies. As a newer tool, the effect of database choice on Sylph's performance has not been explored as thoroughly as for Kraken2 or Bracken. However, the following comments on choosing databases for Kraken2 are very likely still applicable to an extent for Sylph.
+
+The accuracy of Kraken2 is [highly dependent on the database](https://doi.org/10.1099/mgen.0.000949) used. Specifically, it is [crucial](https://doi.org/10.1128/mbio.01607-23) to ensure that the host genome/transcriptome is included in the database. (Note that the pre-built sylph databases do _not_ appear to contain the human genome/transcriptome). If you are particularly concerned about certain contaminants, it may be beneficial to use a smaller, more focused database containing primarily those contaminants instead of the full standard database. Various pre-built databases [are available for download](https://benlangmead.github.io/aws-indexes/k2), and instructions for building a custom database can be found in the [Kraken2 documentation](https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown). Additionally, genomes of contaminants detected in previous sequencing experiments are available on the [OpenContami website](https://openlooper.hgc.jp/opencontami/help/help_oct.php).
+
+While Kraken2 is capable of detecting low-abundance contaminants in a sample, false positives can occur. Therefore, if only a very small number of reads from a contaminating species are detected, these results should be interpreted with caution. Lastly, while Kraken2 can be used without Bracken, since running Bracken is not computationally expensive, we recommend always using it to refine the abundance estimates generated by Kraken2.
 
 ## Running the pipeline
 

diff --git a/modules.json b/modules.json
@@ -262,6 +262,16 @@
                         "git_sha": "1f008221e451e7a4738226c49e69aaa2eb731369",
                         "installed_by": ["modules", "quantify_pseudo_alignment"]
                     },
+                    "sylph/profile": {
+                        "branch": "master",
+                        "git_sha": "41dfa3f7c0ffabb96a6a813fe321c6d1cc5b6e46",
+                        "installed_by": ["modules"]
+                    },
+                    "sylphtax/taxprof": {
+                        "branch": "master",
+                        "git_sha": "41dfa3f7c0ffabb96a6a813fe321c6d1cc5b6e46",
+                        "installed_by": ["modules"]
+                    },
                     "trimgalore": {
                         "branch": "master",
                         "git_sha": "05954dab2ff481bcb999f24455da29a5828af08d",

diff --git a/modules/nf-core/sylph/profile/environment.yml b/modules/nf-core/sylph/profile/environment.yml
diff --git a/modules/nf-core/sylph/profile/main.nf b/modules/nf-core/sylph/profile/main.nf
diff --git a/modules/nf-core/sylph/profile/meta.yml b/modules/nf-core/sylph/profile/meta.yml
diff --git a/modules/nf-core/sylph/profile/nextflow.config b/modules/nf-core/sylph/profile/nextflow.config
diff --git a/modules/nf-core/sylph/profile/tests/main.nf.test b/modules/nf-core/sylph/profile/tests/main.nf.test
diff --git a/modules/nf-core/sylph/profile/tests/main.nf.test.snap b/modules/nf-core/sylph/profile/tests/main.nf.test.snap
diff --git a/modules/nf-core/sylphtax/taxprof/environment.yml b/modules/nf-core/sylphtax/taxprof/environment.yml