Nextflow pipeline to group genes on polyploid phased assemblies that are orthologous and syntelogous based on GENESPACE results. This pipeline is part of the LongPolyASE framework for long-read RNA-seq allele-specific expressin analysis in polyploid organisms.

Requirements:

• nextflow

• conda

The following packages are not in bioconda/pip so need to be installed manually if running with --profile conda (for singularity this is not necessary):

• McxScan (follow instructions here and provide path to installation to --mcscanx_path)
• GENESPACE (instructions)(inside the conda environment genespace-env (syntelogfinder/modules/local/genespace/genespace_run/environment.yml))

• parameter file (params.json)
• genome fasta of phased reference (chromosome names like this: >chr[]01_1, >chr[]01_2 Where the _suffix is the haplotype )
• gff or gtf with CDS corresponding to the reference (same chromosome names!)

The gff file should look like this https://agat.readthedocs.io/en/latest/gff_to_gtf.html#the-gff-file-to-convert with the following features:

• gene
• mRNA/transcript
• exon
• CDS

Or a gtf file with the following features:

• gene
• mRNA/transcript
• exon
• CDS

Mandatory Attributes

• gene_id - must be present on ALL lines
• transcript_id - required for transcript, exon, CDS features
• Parent - links child features to parent

The parms.json should look like this

{
    "reference_fasta": "genome.fa",
    "reference_gff": "annotation.gff",
    "ploidy": 3,
    "outdir": "output_path"
}

Run like this:

git clone https://github.com/NIB-SI/syntelogfinder.git

cd syntelogfinder

nextflow run main.nf -params-file params/params.json \
                     -profile singularity \
                     -resume

or with conda

nextflow run main.nf -params-file params/params.json \
                     -profile singularity \
                     --mcscanx_path [path to McScaX installation]
                     -resume

A test dataset is available for testing and demonstration purposes. This dataset contains a phased genome assembly and annotation for chromosome 1 across all haplotypes of the tetraploid potato cultivar Atlantic.

• fasta
• gtf

Then the params_test.json should look like this:

{
    "reference_fasta": "{download_dir}/ATL_v3.asm.chr01_all_haplotypes.fa",
    "reference_gff": "{download_dir}/ATL_unitato_liftoff.chr01_all_haplotypes.gtf",
    "ploidy": 4,
    "outdir": "output_path"
}

After downloading the fasta and gtf file and preperation of the parameter file the pipeline can be run like this:

git clone https://github.com/NIB-SI/syntelogfinder.git --branch v1.0.0
cd syntelogfinder
conda create -n nextflow -c bioconda nextflow 
conda activate nextflow
nextflow run main.nf \
  -params-file params/params_test.json \
  -profile singularity \
  --run_blast \
  -resume 

Expected runtime: 10 minutes (if all singularity images are already pulled)

• Running syntelogfinder on phased reference of diplpid rice
• Running syntelogfinder on phased reference of haxaploid wheat

Here's the formatted sample output for your README:

The pipeline generates a tab-separated file with the following columns:

gene_id	transcript_id	Synt_id	synteny_category	syntenic_genes	haplotype	CDS_length_category	CDS_haplotype_with_longest_annotation
TraesAK58CH7A01G122800	TraesAK58CH7A01G122800.1	Synt_id_0	1hapA_3hapB_1hapD_no_s	TraesAK58CH7A01G122800.1,TraesAK58CH1B01G017800.1,TraesAK58CH4B01G024800.1,TraesAK58CH2B01G118200.1,TraesAK58CH2D01G119400.1	hapA
TraesAK58CH1A01G005100	TraesAK58CH1A01G005100.1	Synt_id_1	2hapA_1hapB_2hapD_no_s	TraesAK58CH1A01G005100.1,TraesAK58CH3A01G490400.1,TraesAK58CH1B01G017500.1,TraesAK58CH1D01G000500.1,TraesAK58CH7D01G525700.1	hapA
TraesAK58CH3A01G490400	TraesAK58CH3A01G490400.1	Synt_id_1	2hapA_1hapB_2hapD_no_s	TraesAK58CH1A01G005100.1,TraesAK58CH3A01G490400.1,TraesAK58CH1B01G017500.1,TraesAK58CH1D01G000500.1,TraesAK58CH7D01G525700.1	hapA
TraesAK58CH3A01G236000	TraesAK58CH3A01G236000.1	Synt_id_2	1hapA_1hapB_2hapD_no_s	TraesAK58CH3A01G236000.1,TraesAK58CH1B01G017200.1,TraesAK58CH1D01G000900.1,TraesAK58CH5D01G521100.1	hapA
TraesAK58CH1A01G006000	TraesAK58CH1A01G006000.1	Synt_id_3	3hapA_0hapB_1hapD_no_s	TraesAK58CH1A01G006000.1,TraesAK58CH3A01G436000.1,TraesAK58CH5A01G002300.1,<NA>,TraesAK58CH1D01G365400.1	hapA
TraesAK58CH3A01G436000	TraesAK58CH3A01G436000.1	Synt_id_3	3hapA_0hapB_1hapD_no_s	TraesAK58CH1A01G006000.1,TraesAK58CH3A01G436000.1,TraesAK58CH5A01G002300.1,<NA>,TraesAK58CH1D01G365400.1	hapA

Key Features:

• Each gene is assigned to a synteny group (Synt_id)
• The synteny_category shows the distribution pattern (e.g., 2hapA_1hapB_2hapD_no_s means 2 genes in hapA, 1 in hapB, 2 in hapD, with no specific pattern)
• Missing syntenic genes are indicated with <NA>
• All syntenic gene members are listed in the syntenic_genes column

• if GENESPACE process is interuped, running with -resume flag will fail. To chace the other processes, delete the genespace work dir before resuming