AliNe is a pipeline written in Nextflow that aims to efficiently align reads against a reference genome using the tools of your choice.
AliNe is a pipeline written in Nextflow that aims to efficiently align reads against a reference genome.
- Can handle short reads paired or single, pacbio and ont (nanopore) data (see list of aligner in Table 1).
- A QC with FastQC is made at each step if option activated.
- A trimming is feasible before alignment if option activated.
- The pipeline deals automatically with all quality encoding ('sanger', 'solexa', 'illumina-1.3+', 'illumina-1.5+', 'illumina-1.8+'). All fastq will be standardised in Phred+33 for downstream alignments by seqkit.
- Deal automatically with the type of library used: stranded or not, firstrand, secondstrand etc... (see list of aligner in Table 2)
- Can deal with annotation file (see list of aligner in Table 3) You can choose to run one or several aligner in parallel.
Table 1 Here is the list of implemented aligners and the type of reads accepted:
| Tool | Single End (short reads) | Paired end (short reads) | Pacbio | ONT |
|---|---|---|---|---|
| bbmap | ✅ | ✅ | ||
| bowtie2 | ✅ | ✅ | ||
| bwaaln | ✅ | ✅ R1 and R2 independently aligned then merged with bwa sampe | ✅ | ✅ |
| bwamem | ✅ | ✅ | ||
| bwasw | ✅ | ✅ | ||
| graphmap2 | ✅ | ✅ | ||
| hisat2 | ✅ | ✅ | ||
| minimap2 | ✅ | ✅ | ||
| ngmlr | 🚫 | ✅ | ✅ | |
| novoalign | ✅ | ✅ | ✅ | |
| nucmer | ✅ | ✅ R1 and R2 are concatenated then aligned | ||
| star | ✅ | ✅ | ||
| star 2pass mode | ✅ | ✅ | ||
| subread | ✅ | ✅ | ||
| sublong | 🚫 | ✅ | ✅ | |
| tophat | ✅ | ✅ | 🚫 | 🚫 |
Legend
✅ Recommended
🚫 Not applicable
It is possible to bypass the default authorized read type using the AliNe --relax parameter.
The pipeline deals automatically with the library types. It extract 10 000 reads by default and run salmon to guess the library type. It is then translated to the correct option in the following aligners:
| Tool | tool option | Library type by salmon | Comment |
|---|---|---|---|
| bbmap | xs=fr / xs=ss / xs=us | ISF ISR / OSF OSR / U | strand information |
| bbmap | - / rcs=f / | ISF ISR IU / OSF OSR OU MSF MSR MU | read orientation |
| bowtie2 | --fr / --rf / --ff | ISF ISR IU / OSF OSR OU / MSF MSR MU | read orientation |
| bwaaln | 🚫 | 🚫 | 🚫 |
| bwamem | 🚫 | 🚫 | 🚫 |
| bwasw | 🚫 | 🚫 | 🚫 |
| graphmap2 | 🚫 | 🚫 | 🚫 |
| hisat2 | --rna-strandness [ F / R / FR / RF ] | SF / SR / ISF OSF MSF / ISR OSR MSR | strand information |
| hisat2 | --fr / --rf / --ff | I / O / M | read orientation |
| minimap2 | 🚫 | 🚫 | 🚫 |
| ngmlr | 🚫 | 🚫 | 🚫 |
| novoalign | 🚫 | 🚫 | 🚫 |
| nucmer | 🚫 | 🚫 | 🚫 |
| star | 🚫 | 🚫 | 🚫 |
| star 2pass mode | 🚫 | 🚫 | 🚫 |
| subread | -S fr / -S rf / -S ff | ISF ISR IU / OSF OSR OU / MSF MSR MU | read orientation |
| sublong | 🚫 | 🚫 | 🚫 |
| tophat2 | fr-unstranded / fr-firststrand / fr-secondstrand | U / SR / SF | strand information |
Legend
U unstranded; SR stranded reverse; SF stranded forward; IU inward unstranded; OU outward unstranded; MU matching unstranded; ISF inward stranded forward; ISR inward stranded reverse; OSF outward stranded forward; OSR outward stranded reverse; MSF matching stranded forward; MSR matching stranded reverse (see herefor morde details)
🚫 Not applicable
By default the --library_type is in auto mode and the pipeline will automatically detect the library type.
You can also specify manually the library type to use via the --library_type parameter.
If the skip_libray_usage paramater is set, the information about the library type—either provided by the user or inferred by the pipeline using the --library_type parameter—will be ignored.
Note: If you explicitly specify the library type via the aligner parameter (e.g. hisat2_options for hisat2), that value will take precedence over any information provided or inferred using --library_type.
If you provide an annotation file the pipeline will pass automatically the file to the following aligner:
| Tool | accept |
|---|---|
| bbmap | 🚫 |
| bowtie2 | 🚫 |
| bwaaln | 🚫 |
| bwamem | 🚫 |
| bwasw | 🚫 |
| graphmap2 | GTF (--gtf) |
| hisat2 | 🚫 |
| minimap2 | 🚫 |
| ngmlr | 🚫 |
| novoalign | 🚫 |
| nucmer | 🚫 |
| star | GTF / GFF ( --sjdbGTFfile + --sjdbGTFtagExonParentTranscript Parent in case of GFF ) |
| star 2pass mode | GTF / GFF (--sjdbGTFfile + --sjdbGTFtagExonParentTranscript Parent in case of GFF ) |
| subread | GTF or compatible GFF format (-a) |
| sublong | 🚫 |
| tophat | GTF/GFF3 (-G) |
Legend
🚫 Not applicable
---
config:
theme: neutral
---
graph TD;
Genome-->Index;
Index-->Aligner1;
Index-->Aligner2;
Annotation[Annotation - optional]--> Aligner1;
Annotation--> Aligner2;
Reads --> QCraw[QC raw];
Reads --> StandardizeScore[Standardize score]
StandardizeScore --> Trim;
Trim[Trim - optional] --> LibraryGuessing[Library guessing<br>strandedness and orientation];
Trim --> QCtrim;
LibraryGuessing --> Aligner1;
LibraryGuessing --> Aligner2;
Trim --> Aligner1;
Aligner1 --> QCaligner1[QC aligner1];
Trim --> Aligner2;
Aligner2 --> QCaligner2[QC aligner2];
QCraw[QC raw] --> MultiQC;
QCtrim[QC trim] --> MultiQC;
QCaligner1 --> MultiQC;
QCaligner2 --> MultiQC;
The prerequisites to run the pipeline are:
- Nextflow >= 22.04.0
- Docker or Singularity
-
Via conda
See here
``` conda create -n nextflow conda activate nextflow conda install nextflow ``` -
Manually
See here
Nextflow runs on most POSIX systems (Linux, macOS, etc) and can typically be installed by running these commands:# Make sure 11 or later is installed on your computer by using the command: java -version # Install Nextflow by entering this command in your terminal(it creates a file nextflow in the current dir): curl -s https://get.nextflow.io | bash # Add Nextflow binary to your user's PATH: mv nextflow ~/bin/ # OR system-wide installation: # sudo mv nextflow /usr/local/bin
To run the workflow you will need a container platform: docker or singularity.
Please follow the instructions at the Docker website
Please follow the instructions at the Singularity website
You can first check the available options and parameters by running:
nextflow run Juke34/AliNe -r v1.0.0 --help
To run the workflow you must select a profile according to the container platform you want to use:
singularity, a profile using Singularity to run the containersdocker, a profile using Docker to run the containers
The command will look like that:
nextflow run Juke34/AliNe -r v1.0.0 -profile docker <rest of paramaters>
Another profile is available (/!\ actually not yet implemented):
slurm, to add if your system has a slurm executor (local by default)
The use of the slurm profile will give a command like this one:
nextflow run Juke34/AliNe -r v1.0.0 -profile singularity,slurm <rest of paramaters>
Test data are included in the AliNe repository in the test folder.
Test with short single reads:
nextflow run -profile docker,test_illumina_single Juke34/AliNe -r v1.0.0
Test with short paired reads:
nextflow run -profile docker,test_illumina_paired Juke34/AliNe -r v1.0.0
Test with ont reads:
nextflow run -profile docker,test_ont Juke34/AliNe -r v1.0.0
Test with pacbio reads:
nextflow run -profile docker,test_pacbio Juke34/AliNe -r v1.0.0
On success you should get a message looking like this:
AliNe Pipeline execution summary
--------------------------------------
Completed at : 2024-03-07T21:40:23.180547+01:00
UUID : e2a131e3-3652-4c90-b3ad-78f758c06070
Duration : 8.4s
Success : true
Exit Status : 0
Error report : -
--help prints the help section
General Parameters
--reads path to the reads file or folder
--reads_extension extension of the reads files (default: .fastq.gz)
--genome path to the genome file
--aligner aligner(s) to use among this list (comma or space separated) [bbmap, bowtie2, bwaaln, bwamem, bwasw, graphmap2, hisat2, minimap2, novoalign, nucmer, ngmlr, star, subread, sublong, tophat2]
--outdir path to the output directory (default: alignment_results)
--annotation [Optional][used by STAR, Tophat2] Absolute path to the annotation file (gtf or gff3)
Type of input reads
--read_type type of reads among this list [short_paired, short_single, pacbio, ont] (default: short_paired)
--paired_reads_pattern pattern to detect paired reads (default: {1,2})
--library_type Set the library_type of your reads (default: auto). In auto mode salmon will guess the library type for each sample.
If you know the library type you can set it to one of the following: [U, IU, MU, OU, ISF, ISR, MSF, MSR, OSF, OSR]. See https://salmon.readthedocs.io/en/latest/library_type.html for more information.
In such case the sample library type will be used for all the samples.
--skip_libray_usage Skip the usage of library type provided by the user or guessed by salmon.
Extra steps
--trimming_fastp run fastp for trimming (default: false)
--fastqc run fastqc on raw and aligned reads (default: false)
--multiqc_config path to the multiqc config file (default: config/multiqc_conf.yml)
Aligner specific options
--bbmap_options additional options for bbmap
--bowtie2_options additional options for bowtie2
--bwaaln_options additional options for bwaaln
--bwamem_options additional options for bwamem
--bwasw_options additional options for bwasw
--graphmap2_options additional options for graphmap2
--hisat2_options additional options for hisat2
--minimap2_options additional options for minimap2 (default: -a (to get sam output))
--minimap2_index_options additional options for minimap2 index
--ngmlr_options additional options for ngmlr
--novoalign_options additional options for novoalign
--novoalign_license license for novoalign. You can ask for one month free trial license at http://www.novocraft.com/products/novoalign/
--nucmer_options additional options for nucmer
--star_options additional options for star
--star_2pass set to true to run STAR in 2pass mode (default: false)
--read_length [Optional][used by STAR] length of the reads, if none provided it is automatically deduced
--subread_options additional options for subread
--sublong_options additional options for sublong
--tophat2_options additional options for tophat