Prepare and configure the pipeline

Creating the configuration file: inputs and other parameters

The configuration file config.yaml will contain all the input files required for the pipeline and some other parameters.

• Prepare the query genome data

Parameter in config.yaml	format	description	Example
annotationQuery	GFF3	The gff file of the annotation we want to transfert onto the new Target genome	annotationQuery: “/path/to/IWGSC_RefSeqv1_annotation.gff3”
featureType	[STRING]	The feature we want to use to anchore the annotation. Here we use the gene feature of the GFF.	featureType: ‘gene
queryFasta	FASTA	Fasta file of the genome reference sequence. Must match the GFF file used in annotationQuery parameter	queryFasta: ‘/path/to/IWGSC_RefSeqv1_annotation.fasta’
blastdb	[blast database]	blast db of all mRNAs (all isoforms) of the query annotation. This will be used to rescue genes which have failed in the transfert	blastdb: ‘data/IWGSCv1.1_all_mrna’
chromosomes	python list	list of all the chromosomes in the query reference genome. This will be used to split all the data per chromosome and speed up the analysis	chromosomes: [‘1A’, ‘2A’, ‘3A’, ‘4A’, ‘5A’, ‘6A’, ‘7A’, ‘1B’, ‘2B’, ‘3B’, ‘4B’, ‘5B’, ‘6B’, ‘7B’, ‘1D’, ‘2D’, ‘3D’, ‘4D’, ‘5D’, ‘6D’, ‘7D’, ‘U’]

• Prepare the markers/ISBPs input data

The pipeline uses markers/ISBPs as anchores to target a restricted part of the target genome on which our genes are suspected to be located.

Parameter in config.yaml	format	description	Example
isbpBed	BED	Initial coordinates of the markers/ISBPs on the query genome	isbpBed: ‘data/ISBP_refseqv1.bed’
mapq	[INT]	Minimum mapping quality of the marker/ISBP to be kept in the anchoring process	mapq: 30
mismatches	[INT]	Max missmatches allowed for a marker to be kept in the analysis	mismatches: 0

• Prepare the target genome data

Here, we only need the fasta of the new genome assembly

Parameter in config.yaml	format	description	Example
targetFasta	FASTA	fasta file of the target genome assembly on which we transfert the annotation	targetFasta: ‘data/CS_pesudo_v2.1.fa’
targetBwaIdx	BWA index	Prefix for the BWA index files	targetBwaIdx: ‘/data/db/triticum_aestivum/julius/current/bwa/all’
targetGmapIndex	PATH	Name of the GMAP index directory. This will be used with the -d option of gmapl	targetGmapIndex: ‘ensembl_Triticum_aestivum_julius_2022-9-16’
targetGmapIndexPath	PATH	Full path to the directory in which the GMAPindex is found. This will be used with the -D option of gmapl	targetGmapIndexPath: ‘/data/db/triticum_aestivum/julius/gmapdb/all/’

• Output parameters/settings

Parameter in config.yaml	format	description	Example
results	[STRING]	directory in which all the Snakemake rules will be executed	results: ‘results’
finalPrefix	[STRING]	Prefix for all the final output files (annotaion, mrna/pep fasta sequences ect)	finalPrefix: ‘IWGSC_refseqv2.0_annotv2.0’
chromMapID	CSV	Mapping file which sets the correspondance between the chromosome names in the GFF and the chromosome ID in the newlygenerated gene IDs	chromMapID: ‘data/chromosomeMappingID.csv’
transferType	[STRING]	transfert all isoforms or only the representative transcript (.1) for each gene in the reference genome	transferType: ‘all’ ; transferType: ‘first’

Example of chromosomeMappingID.csv file :

$ cat data/chromosomeMappingID.csv
Chr1A   1A
Chr1B   1B
Chr1D   1D
Chr2A   2A
Chr2B   2B
Chr2D   2D
Chr3A   3A
Chr3B   3B
Chr3D   3D
Chr4A   4A
Chr4B   4B
Chr4D   4D
Chr5A   5A
Chr5B   5B
Chr5D   5D
Chr6A   6A
Chr6B   6B
Chr6D   6D
Chr7A   7A
Chr7B   7B
Chr7D   7D

Once all those parameters has been set up, the final configuration file may look like this:

##### QUERY related files/parameters (refseqv1.0)
# GFF annotatin to transfert
annotationQuery: 'data/IWGSC_v1.1_20170706.gff3'
# feature type used for anchoring on target genome
featureType: 'gene'
# FASTA of the query (used to check the sequences after the coordinates are calculated on the target genome)
queryFasta: 'data/161010_Chinese_Spring_v1.0_pseudomolecules.fasta'
# blastdb of all mrnas. used to rescue genes which have failed in the transfert using the targeted approache
blastdb: 'data/IWGSCv1.1_all_mrna'
# map of all chromosome ids --> NEED TO BE UPDATED in another version WITH ONE ARRAY FOR THE QUERY AND ONE ARRAY FOR THE TARGET GENOME ASSEMBLY
chromosomes: ['1A', '2A', '3A', '4A', '5A', '6A', '7A', '1B', '2B', '3B', '4B', '5B', '6B', '7B', '1D', '2D', '3D', '4D', '5D', '6D', '7D', 'U']
refChrom: ['chr1A', 'chr1B', 'chr1D', 'chr2A', 'chr2B', 'chr2D', 'chr3A', 'chr3B', 'chr3D', 'chr4A', 'chr4B', 'chr4D', 'chr5A', 'chr5B', 'chr5D', 'chr6A', 'chr6B', 'chr6D', 'chr7A', 'chr7B', 'chr7D', 'chrUn']

##### TARGET related files/parameters (refseqv2.1)
targetFasta: 'data/CS_pesudo_v2.1.fa'
#GMAP index of the genome for -d option
targetGmapIndex: 'ensembl_Triticum_aestivum_julius_2022-9-16'
#GMAP index: path to the gmapindex directory, for -D option
targetGmapIndexPath: '/data/db/triticum_aestivum/julius/current/gmapdb/all/'


##### ISBP/markers related config and parameters
# BAM file of markers/ISBPs mapped on the target genome
# isbpBam: '/home/masirvent/wheat10plus-pangenome/data/mappingISBP/session2/arina/arina_CS_ISBP.bam'
# BED file of coordinates on the query genome (REFSEQ v2.1)
isbpBed: 'data/Tae.Chinese_Spring.refSeqv2.1.ISBPs.bed'
# BWA threads for mapping
bwaThreads: 16
# FLAG : F flag for samtools
flag_F: 3844
# minimum mapping quality of markers on the target genome
mapq: 30
# max mismatches per ISBP/marker
mismatches: 2


##### OUTPUT directory
results: 'resultsDEV'
finalPrefix: 'IWGSC_refseqv2.0_annotv2.0'
# this file contains two columns: the first is the chromosome name as it appears in the genome.fasta of the new reference,
# and the second the chromosome name as it will appear in the new gene Names
chromMapID: 'data/chromosomeMappingID.csv'