https://github.com/nschan/nf-genomeassembly
Nextflow pipeline to assemble genomes from long reads.
https://github.com/nschan/nf-genomeassembly
annotation arabidopsis arabidopsis-thaliana genome genome-assembly genome-sequencing hifi long-read-sequencing long-reads nanopore nextflow nextflow-pipeline nextflow-pipelines oxford-nanopore pacbio pacbio-hifi-sequencing-reads scaffolding
Last synced: 3 months ago
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Nextflow pipeline to assemble genomes from long reads.
- Host: GitHub
- URL: https://github.com/nschan/nf-genomeassembly
- Owner: nschan
- License: mit
- Created: 2024-04-15T08:18:04.000Z (about 1 year ago)
- Default Branch: master
- Last Pushed: 2024-11-06T12:46:50.000Z (7 months ago)
- Last Synced: 2024-12-26T13:26:04.002Z (5 months ago)
- Topics: annotation, arabidopsis, arabidopsis-thaliana, genome, genome-assembly, genome-sequencing, hifi, long-read-sequencing, long-reads, nanopore, nextflow, nextflow-pipeline, nextflow-pipelines, oxford-nanopore, pacbio, pacbio-hifi-sequencing-reads, scaffolding
- Language: Nextflow
- Homepage:
- Size: 996 KB
- Stars: 3
- Watchers: 2
- Forks: 0
- Open Issues: 0
-
Metadata Files:
- Readme: README.md
- License: LICENSE
Awesome Lists containing this project
README
[](https://zenodo.org/doi/10.5281/zenodo.10972895)
The goal of [`nf-genomeassembly`](https://github.com/nschan/nf-genomeassembly) and [`nf-annotate`](https://github.com/nschan/nf-annotate) is to make to genome assembly and annotation workflows accessible for a broader community, particularily for plant-sciences. Long-read sequencing technologies are already cheap and will continue to drop in price, genome sequencing will soon be available to many researchers without a strong bioinformatic background.
The assembly is naturally quite organisms agnostic, but the annotation pipeline contains some steps that may not make sense for other eukaryotes, unless there is a particular interest in NB-LRR genes.
> I am currently preparing `nf-genomeassembly` to be added into nf-co.re/genomeassembler. Please see here for the latest version: [`genomeassembler`](https://github.com/nschan/genomeassembler)
# nf-genomeassembly
Assembly pipeline for genomes from long-read sequencing written in [`nextflow`](https://nextflow.io/).
The pipeline supports for assembly Oxford Nanopore, Pacbio HiFi, combinations of ONT and pacbio HiFi, and can take short-reads for quality control and / or polishing.
# Procedure
Preprocessisng:
- For nanopore:
* Extract all fastq.gz files in the readpath folder into a single fastq file. By default this is skipped, enable with `--collect`.
* Barcodes and adaptors will be removed using [`porechop`](https://github.com/rrwick/Porechop). By default this is skipped, enable with `--porechop`.
> NB: flye claims to work well on raw, un-trimmed reads
* Read QC is done via [`nanoq`](https://github.com/esteinig/nanoq)
- For pacbio:
* [`lima`](https://lima.how/) to remove primers.
Assembly
* k-mer based assessment of ONT reads via [`Jellyfish`](https://github.com/gmarcais/Jellyfish) and [`genomescope`](https://github.com/schatzlab/genomescope/)
* Assemblies are performed with [`flye`](https://github.com/fenderglass/Flye),
* or [`hifiasm`](https://github.com/chhylp123/hifiasm)
Polishing:
* Polishing of ONT assemblies done using [`medaka`](https://github.com/nanoporetech/medaka)
* Optional short-read polishing can be done using [`pilon`](https://github.com/broadinstitute/pilon)
Scaffolding:
* [`LINKS`](https://github.com/bcgsc/LINKS)
* [`longstitch`](https://github.com/bcgsc/longstitch)
* [`ragtag`](https://github.com/malonge/RagTag)
Annotation:
* Annotations are lifted from reference using [`liftoff`](https://github.com/agshumate/Liftoff).
QC:
* Quality of each stage is assessed using [`QUAST`](https://github.com/ablab/quast) and [`BUSCO`](https://gitlab.com/ezlab/busco) (standalone),
* k-mer spectra can be used for further QC with [`yak`](https://github.com/lh3/yak),
* if short-reads are provided [`merqury`](https://github.com/lh3/yak) is run to compare k-mer spectra between assemblies (or scaffolds) and short-reads.
# Tubemap
# Usage
Clone this repo:
```bash
git clone https://github.com/nschan/nf-genomeassembly/
```
Run via nextflow:
The samplesheet is a `.csv` file with a header. It _must_ adhere to this format, including the header row. Please note the absence of spaces after the commas:
```
sample,ontreads,hifireads,ref_fasta,ref_gff
sampleName,path/to/reads,path/to/hifi.fastq.gz,path/to/reference.fasta,path/to/reference.gff
```
To run the default pipeline with a samplesheet on biohpc_gen using charliecloud:
```bash
nextflow run nf-genomeassembly --samplesheet 'path/to/sample_sheet.csv' \
-profile charliecloud,biohpc_gen
```
# Parameters
See also [schema.md](schema.md)
| Parameter | Effect |
| --- | --- |
| **General parameters** | |
| `--samplesheet` | Path to samplesheet |
| `--use_ref` | Use a refence genome? (default: `true`) |
| `--lift_annotations` | Lift annotations from reference using [`liftoff`](https://github.com/agshumate/Liftoff)? Default: `true` |
| `--out` | Results directory, default: `'./results'` |
| `--ont` | ONT reads are available? These should go into the `ontreads` column of the samplesheet. Default: `false` |
| `--hifi` | Pacbio hifi reads are available? These should go into the `hifireads` column of the samplesheet. default: `false` |
| **ONT Preprocessing** | |
| `--collect` | Are the provided reads a folder (`true`) or a single fq files (default: `false` ) |
| `--porechop` | Run [`porechop`](https://github.com/rrwick/Porechop) on ONT reads? (default: `false`) |
| **pacbio Preprocessing** | |
| `--lima` | Run [`lima`](https://lima.how/) on pacbio reads? default: `false`|
| `--pacbio_primers` | Primers to be used with [`lima`](https://lima.how/) (required if `--lima` is used)? default: `null`|
| **Assembly** | |
| `--assembler` | Assembler to use. Valid choices are: `'hifiasm'`, `'flye'`, or `'flye_on_hifiasm'`. `flye_on_hifiasm` will scaffold flye assembly (ont) on hifiasm (hifi) assembly using [`ragtag`](https://github.com/malonge/RagTag). Defaul: `'flye'`|
| **Assembly** | _`flye` specific arguments_ |
| `--flye_args` | The mode to be used by [`flye`](https://github.com/fenderglass/Flye); default: `"--nano-hq"`, options are: `"--pacbio-raw"`, `"--pacbio-corr"`, `"--pacbio-hifi"`, `"--nano-raw"`, `"--nano-corr"`, `"--nano-hq"` |
| `--kmer_length` | kmer size for [`Jellyfish`](https://github.com/gmarcais/Jellyfish)? (default: 21) |
| `--read_length` | Read length for [`genomescope`](https://github.com/schatzlab/genomescope/)? If this is `null` (default), the median read length estimated by [`nanoq`](https://github.com/esteinig/nanoq). will be used. If this is not `null`, the given value will be used for _all_ samples. |
| `--genome_size` | Expected genome size for [`flye`](https://github.com/fenderglass/Flye). If this is `null` (default), the haploid genome size for each sample will be estimated via [`genomescope`](https://github.com/schatzlab/genomescope/). If this is not `null`, the given value will be used for _all_ samples. |
| `--flye_args` | Arguments to be passed to [`flye`](https://github.com/fenderglass/Flye), default: `none`. Example: `--flye_args '--genome-size 130g --asm-coverage 50'` |
| **Assembly** | _`hifiasm` specific arguments_ |
| `--hifi_ont` | Use hifi and ONT reads with `hifiasm --ul`? default: `false`|
| `--hifiasm_args` | Extra arguments passed to [`hifiasm`](https://github.com/chhylp123/hifiasm). default: `''`|
| **Polishing** | |
| `--polish_medaka` | Polish using [`medaka`](https://github.com/nanoporetech/medaka), default: `false` |
| `--medaka_model` | Model used by [`medaka`](https://github.com/nanoporetech/medaka), default: '[email protected]:consesus' |
| `--polish_pilon` | Polish with short reads (see below) using [`pilon`](https://github.com/broadinstitute/pilon)? Sefault: `false` |
| **Scaffolding** | |
| `--scaffold_ragtag` | Scaffolding with [`ragtag`](https://github.com/malonge/RagTag)? Default: `false` |
| `--scaffold_links` | Scaffolding with [`LINKS`](https://github.com/bcgsc/LINKS)? Default: `false` |
| `--scaffold_longstitch` | Scaffolding with [`longstitch`](https://github.com/bcgsc/longstitch)? Default: `false` |
| **QC** | |
| `--short_reads` | Short reads available? These should go into `shortread_F` and `shortread_R` columns and the `paired` column should be true if both are filled. If only single-end reads are available, `shortread_R` remains empty, and `paired` is false. If short-reads are supplied, k-mer spectra will be used to assess quality of the assembly(s). Default: `false` |
| `--trim_short_reads` | Trim short reads with [`trimgalore`](https://github.com/FelixKrueger/TrimGalore)? Default: `true` |
| `--meryl_k` | Value of k for meryl k-mers. Default: `21` |
| `--qc_reads` | Long reads that should be used for QC when both ONT and HiFi reads are provided. Options are `'ONT'` or `'HIFI'`. Default: `'ONT'` |
| `--busco` | Run [`BUSCO`](https://gitlab.com/ezlab/busco)? Default: `'true'` |
| `--busco_db` | Path to local [`BUSCO`](https://gitlab.com/ezlab/busco) db? Default: `""` |
| `--busco_lineage` | [`BUSCO`](https://gitlab.com/ezlab/busco) lineage to use. Default: `brassicales_odb10` |
| `--quast`| Run [`QUAST`](https://github.com/ablab/quast)? Default: `true` |
| **Skipping steps** | |
| `--skip_assembly` | Skip assembly? Requires different samplesheet (!). Default: `false` |
| `--skip_alignments` | Skip alignments with [`minimap2`](https://github.com/lh3/minimap2)? Requires different samplesheet (!). Default: `false` |
# Included profiles
This pipelines comes with some profiles to modify run behaviour independent of infrastructure configs, which can be used via `-profile`.
| Name | Contents |
| --- | --- |
| `ont_flye` | Assemble ONT reads with `flye` |
| `hifi_flye` | Assemble pac-bio hifi reads with `flye` |
| `hifi_hifiasm` | Assemble pac-bio hifi reads with `hifiasm` |
| `hifi_ul` | Assemble ONT and HiFI reads via `hifiasm` |
| `ont_on_hifi` | Assemble HiFi (via `hifiasm`) and ONT (via `flye`) and subsequent scaffolding of the ONT assembly onto HiFi assembly with `ragtag` |
# Short reads: QC with yak
If short reads are available, [`yak`](https://github.com/lh3/yak) can be used to perform additional quality control based on kmer spectra.
This can be enabled using `--short_reads` and a samplesheet that looks like this:
```
sample,ontreads,hifireads,ref_fasta,ref_gff,shortread_F,shortread_R,paired
sampleName,ontreads.fa.gz,hifireads.fa.gz,assembly.fasta.gz,reference.fasta,reference.gff,short_F1.fastq,short_F2.fastq,true
```
If there are only single-end reads, shortread_R should remain empty, and paired should be `false`
# Short reads: Polishing with pilon
The assemblies can be polished using available short-reads using [`pilon`](https://github.com/broadinstitute/pilon).
`--polish_pilon`
This requires additional information in the samplesheet: `shortread_F`, `shortread_R` and `paired`:
```
sample,ontreads,ref_fasta,ref_gff,shortread_F,shortread_R,paired
sampleName,reads,assembly.fasta.gz,reference.fasta,reference.gff,short_F1.fastq,short_F2.fastq,true
```
In a case where only single-reads are available, `shortread_R` should be empty, and `paired` should be false.
# Scaffolding
[`LINKS`](https://github.com/bcgsc/LINKS), [`longstitch`](https://github.com/bcgsc/longstitch) and / or [`ragtag`](https://github.com/malonge/RagTag) can be used for scaffolding.
# Using liftoff
If `--lift_annotations` is used (default), the annotations from the reference genome will be mapped to assemblies and scaffolds using liftoff.
This will happen at each step of the pipeline where a new genome fasta is created, i.e. after assembly, after polishing and after scaffolding.
# No refence genome
If there is no reference genome available use `--use_ref false` to disable the reference genome.
Liftoff should not be used without a reference, QUAST will no longer compare to reference.
# Skipping Assembly
In case you already have an assembly and would only like to check it with QUAST and polish use
`--skip_assembly true`
This mode requires a different samplesheet:
```
sample,readpath,assembly,ref_fasta,ref_gff
sampleName,path/to/reads,assembly.fasta.gz,reference.fasta,reference.gff
```
When skipping flye the original reads will be mapped to the assembly and the reference genome.
# Skipping Flye and mappings
In case you have an assembly and have already mapped your reads to the assembly and the reference genome you can use
`--skip_assembly true --skip_alignments true`
This mode requires a different samplesheet:
```
sample,readpath,assembly,ref_fasta,ref_gff,assembly_bam,assembly_bai,ref_bam
sampleName,reads,assembly.fasta.gz,reference.fasta,reference.gff,reads_on_assembly.bam,reads_on_assembly.bai,reads_on_reference.bam
```
# QUAST
[`QUAST`](https://github.com/ablab/quast) will run with the following additional parameters:
```
--eukaryote \\
--glimmer \\
--conserved-genes-finding \\
```
# Acknowledgements
This pipeline builds on [modules](https://github.com/nf-core/modules) developed by [`nf-core`](https://nf-co.re).