nPhase is a command line ploidy agnostic phasing pipeline and algorithm which phases samples of any ploidy with sequence alignment of long and short read data to a reference sequence.
Project description
nPhase
Ploidy agnostic phasing pipeline and algorithm
nPhase is a ploidy agnostic tool developed in python which predicts the haplotypes of a sample that was sequenced by both long and short reads by aligning them to a reference. It should work with any ploidy.
Quick-start
If you have bioconda you can install nPhase by running the following commands in your terminal:
conda create -n polyploidPhasing python=3.8
conda activate polyploidPhasing
conda install -c oakheart nphase
Then you can phase your data with the following command:
nphase pipeline --sampleName Individual_1 --reference /path/to/Individual_referenceGenome.fasta
--R1 /path/to/Individual_1_shortReads_R1.fastq.gz --R2 /path/to/Individual_1_shortReads_R2.fastq.gz
--longReads /path/to/Individual_1_longReads.fastq.gz --longReadPlatform [ont|pacbio]
--output /path/to/outputFolder
Installation
With bioconda
Install bioconda and set the correct channels by following the first two steps here: https://bioconda.github.io/user/install.html
Then you can create a new environment and install nPhase with the following commands:
conda create -n polyploidPhasing python=3.8
conda activate polyploidPhasing
conda install -c oakheart nphase
Without bioconda
Pre-requisites
You will have to install the following software before nPhase:
*Currently, installing samtools v1.10 or higher will cause an error to occur due to a bug in the way ngmlr handles MAPQ scores.
Installation via PyPI
You can now install nPhase via
pip install -U nPhase
Usage
There are two ways to run nPhase:
nphase pipeline
will run the entire pipeline from start to finish and requires the following inputs:
nphase pipeline --sampleName SAMPLE_NAME --reference REFERENCE --output OUTPUT_FOLDER --longReads LONG_READ_FILE
--longReadPlatform {ont,pacbio} --R1 SHORT_READ_FILE_R1 --R2 SHORT_READ_FILE_R2
Optional parameters are described further down.
nphase algorithm
will only run the phasing algorithm, it requires inputs generated by nphase pipeline
. This is useful if you want to test different paramaters on your dataset after having generated the pre-processed files once. Here are the inputs required by nphase algorithm
:
nphase algorithm --sampleName SAMPLE_NAME --reference REFERENCE --output OUTPUT_FOLDER --longReads LONG_READ_FILE
--contextDepth CONTEXT_DEPTHS_FILE --processedLongReads VALIDATED_SNP_ASSIGNMENTS_FILE
Optional parameters are described further down.
Parameters
nphase pipeline [-h] [--threads [THREADS]] [--maxID [MAXID]] [--minOvl [MINOVL]] [--minSim [MINSIM]] [--minLen [MINLEN]] --sampleName SAMPLE_NAME
--reference REFERENCE --output OUTPUT_FOLDER --longReads LONG_READ_FILE --longReadPlatform {ont,pacbio} --R1 SHORT_READ_FILE_R1 --R2
SHORT_READ_FILE_R2
or
nphase algorithm [-h] [--threads [THREADS]] [--maxID [MAXID]] [--minOvl [MINOVL]] [--minSim [MINSIM]] [--minLen [MINLEN]] --sampleName SAMPLE_NAME
--reference REFERENCE --output OUTPUT_FOLDER --longReads LONG_READ_FILE --contextDepth CONTEXT_DEPTHS_FILE --processedLongReads
VALIDATED_SNP_ASSIGNMENTS_FILE
positional arguments:
pipeline Run the entire nPhase pipeline on your sample
algorithm Only run the nPhase algorithm. NOTE: This will require files generated by running the pipeline mode
arguments always required:
--sampleName STRAINNAME
Name of your sample, ex: "Individual_1"
--reference REFERENCE
Path to fasta file of reference genome to align to, ex: /home/reference/Individual_reference.fasta
--output OUTPUTFOLDER
Path to output folder, ex: /home/phased/
--longReads LONGREADFILE
Path to long read FastQ file, ex: /home/longReads/Individual_1.fastq.gz
additional arguments required by nphase pipeline:
--longReadPlatform {ont,pacbio}
Long read platform, must be 'ont' or 'pacbio'
--R1 SHORTREADFILE_R1
Path to paired end short read FastQ file #1, ex: /home/shortReads/Individual_1_R1.fastq.gz
--R2 SHORTREADFILE_R2
Path to paired end short read FastQ file #2, ex: /home/shortReads/Individual_1_R2.fastq.gz
additional arguments required by nphase algorithm:
--contextDepth CONTEXTDEPTHSFILE
Path to context depths file, ex: /home/phased/Individual_1/Overlaps/Individual_1.contextDepths.tsv
--processedLongReads VALIDATEDSNPASSIGNMENTSFILE
Path to validated long read SNPs, ex:
/home/phased/Individual_1/VariantCalls/longReads/Individual_1.hetPositions.SNPxLongReads.validated.tsv
optional arguments:
-h, --help show this help message and exit
--threads [THREADS] Number of threads to use on some steps, default 8
--maxID [MAXID] MaxID parameter, determines how different two clusters must be to prevent them from merging. Default 0.05
--minOvl [MINOVL] minOvl parameter, determines the minimal percentage of overlap required to allow a merge between two clusters that have fewer than
100 heterozygous SNPs in common. Default 0.1
--minSim [MINSIM] minSim parameter, determines the minimal percentage of similarity required to allow a merge between two clusters. Default 0.01
--minLen [MINLEN] minLen parameter, any cluster based on fewer than N reads will not be output. Default 0
Paper
nPhase: An accurate and contiguous phasing method for polyploids
Media
Online lightning talk I gave about nPhase for an Oxford Nanopore event [5:44]: link
Misc
Current recommendations for default parameters are at least 20X coverage per haplotype (so 3*20=60X for a triploid) and a heterozygosity level of at least 0.4% (average of 1 heterozygous SNP every 250 bp).
It is currently untested on pacbio data so if you have a pacbio dataset (with a known ground truth) please contact me (raise an issue on github or email me), especially if you have errors.
If you have a hybrid sample that has an acquired genomic copy which is genetically distant from the rest of the genome, nPhase will struggle to predict accurate results as the "distant" haplotype will make the other haplotypes look incredibly similar to each other. The solution is to separate the long reads based on their genetic distance to a reference genome. More details in this PDF file.
Contact me
email: oabousaada@unistra.fr
discord: Peaceful#6956
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