The script get_pangenes.pl computes whole genome alignments (WGA) to define clusters of collinear, orthologous genes/features annotated in GFF files. Such clusters define pangenes across a pangenome. Several WGA algorithms are available and some parameters are customizable. It is designed to process (in a multicore computer or HPC cluster) files contained in a directory (-d), so that new .fna & .gff files can be added while conserving previous results.
This script calls _cut_sequences.pl, _collinear_genes.pl and _cluster_analysis.pl and produces different types of output:
- clusters of CDS (nucl & pep), cDNA sequences and genomic segments (gdna) of collinear genes (FASTA)
- pangene matrices that summarize the genome occupancy of clusters
- matrix of % conserved sequences that summarize shared clusters across genomes
- optionally (-c) matrices with core- and pangene set growth simulations
- Objective
- Definition of pangene
- How it works
- Example 1: default core pangene set analysis
- Example 2: pangene and Presence-Absence Variation (PAV) analysis
- Example 3: splitting genome in chromosomes
- Example 4: using GSAlign instead of minimap2
- Example 5: simulation of pangene set growth
- Example 6: estimation of haplotype diversity
- Explaining pangene matrices and other result files
- Plotting the results
- Sequence alignments of clusters
- Quality metrics of clusters
- Evidence supporting clusters
- Whole genome alignment evidence
- Plotting the genome context of a pangene cluster
- Remediating pangene models with check_evidence-pl
- Pangene analysis with GFF patches
- Dotplots of gene-based whole-genome alignments
- Matching nucleotide sequences to precomputed pangene clusters
- Troubleshooting
- Funding
- Citation
The main task of these scripts is to cluster collinear/orthologous genes across a set of genomes (or pangenome) so that pangenes can be defined:
Figure 1. Graphical summary of pangene set analysis.
| occupancy class | definition |
|---|---|
| core | Genes contained in all considered genomes/taxa. |
| soft-core | Genes contained in 95% of the considered genomes/taxa. |
| cloud | Genes present only in a few genomes/taxa, generally 1 or 2. The cutoff is defined as the class next to the most populated non-core class. |
| shell | Remaining genes, present in several genomes/taxa. |
Table 1. Definitions of occupancy classes for pangenes, taken from GET_HOMOLOGUES. Accessory genes include both shell and cloud genes.
According to our paper https://doi.org/10.1186/s13059-023-03071-z, a pangene can be defined as follows:
A gene model or allele/haplotype found in some or all individuals of a species in a similar genomic location.
A pangene should integrate additional naming schemes, e.g., so that a cluster of gene models can
share a common identifier that links back to their original gene identifiers. A pangene set defines
our current understanding of the total coding potential of a species and can assist in gene model
curation, by providing a pool of possible gene models for assessment.
The next flowchart shows the three main tasks of the pipeline:
Figure 2. Pipeline flowchart.
Note that each of the three scripts called by get_pangenes.pl (cut_sequences.pl, _collinear_genes.pl & _cluster_analysis.pl) create their own logfiles.
By default GET_PANGENES performs the required tasks serially, which equals to option -m local,
but it can also run in parallel on a cluster both with options -m cluster
and -m dryrun, if you prefer to copy and paste your commands in batches directly in the terminal.
This is recommended for large or multiple genomes.
Sample configuration files for LSF and SLURM managers are provided at
(HPC.conf.sample) and
(HPC.conf.sample.slurm).
Please adapt them and rename then HPC.conf for them to work.
The second block of the flow aligns genome sequences (in pairs A & B) and uses the resulting alignments to transform gene coordinates:
Figure 3. Whole Genome Alignment (WGA) and gene mapping.
This is how the overlap of genes is computed (with bedtools intersect) to call collinear pairs:
Figure 4. How gene overlap is computed.
The overlap value is computed from WGA coordinates (subject) and gene coordinates from the source GFF file (query). Note that the WGA alignment might be partial, thus the estimated overlap ratio could be actually smaller if the subject GFF coordinates were used. Note that overlaps can be also computed among gene models annotated in one assembly and matching genomic segments from others.
Collinear pairs are internally stored in Compara-like TSV files, with 1-based coordinates, that look like this:
gene_stable_id protein_stable_id species overlap homology_type homology_gene_stable_id homology_protein_stable_id homology_species overlap dn ds goc_score wga_coverage is_high_confidence coordinates
gene:BGIOSGA002569 gene:BGIOSGA002569 Oryza_indica.ASM465v1.chr1 6223 ortholog_collinear gene:ONIVA01G00100 gene:ONIVA01G00100 Oryza_nivara_v1.chr1 6223 NULL NULL NULL 100.00 1 1:30219-36442(+);1:104920-116326(+)
Oryza_indica.ASM465v1.chr1:1:217360-222398:+ segment Oryza_indica.ASM465v1.chr1 5038 segment_collinear gene:ONIVA01G00180 gene:ONIVA01G00180 Oryza_nivara_v1.chr1 5038 NULL NULL NULL 100.00 1 1:217360-222398(+);1:155040-165322(+)
gene:BGIOSGA002594 gene:BGIOSGA002594 Oryza_indica.ASM465v1.chr1 3838 segment_collinear Oryza_nivara_v1.chr1:1:178848-182686:+ segment Oryza_nivara_v1.chr1 3838 NULL NULL NULL 100.00 1 1:246911-252389(+);1:178848-182686(+)
TSV files are merged and sorted by gene and overlap. The resulting file is used to drive the construction of clusters from pairs of collinear genes as follows:
Figure 5. Clustering sequences from pairs of collinear genes. Option -N controls the max distance (in genes) among sequences of same species in a cluster.
A few parameters are encoded as variables in the scripts and their values printed to log files. Here I list the most important ones, they can be changed by editing the script source if needed:
| script | variable | value | meaning |
|---|---|---|---|
| get_pangenes.pl | MINGFFLEN | 100 | min length of sequences of features (cdna, cds) extracted from input GFF files |
| get_pangenes.pl | NOFSAMPLESREPORT | 20 | number of samples while simulating pangene growth with -c |
| get_pangenes.pl | GFFACCEPTEDFEATS | gene,mRNA,transcript,exon,CDS | accepted features in input GFF files |
| get_pangenes.pl | GFFVALIDGENEFEAT | gene,mRNA,transcript | features used to call valid genes in input GFF files |
| get_pangenes.pl | MINPERCID | 95.0 | min percent identity of aligned overlapping genes |
| get_pangenes.pl | MAXDISTNEIGHBORS | 2 | neighbor genes in a cluster cannot be more than 2 genes away on same strand |
| _collinear_genes.pl | MINIMAPPARS | --secondary=no --cs -x asm20 -r1k,5k | minimap2 settings |
| _collinear_genes.pl | GSALIGNPARS | -sen -no_vcf -fmt 1 | GSAlign settings |
| _collinear_genes.pl | BEDINTSCPAR | -wo -f XXX -F XXX -e | bedtools intersect parameters, XXX replaced with user selected overlap [0-1] |
| _collinear_genes.pl | MINMASKLEN | 1000000 | mask longer (intergenic, repetitive) fragments with -H |
| _collinear_genes.pl | GENEMARGIN | 5000 | do not mask gene margins |
| _collinear_genes.pl | MINALNLEN | 100 | min alignment length when mapping & transforming gene coords on WGA |
| _cluster_analysis.pl | MINEDGESTOMERGE | 0.75 | ratio of edges connecting two clusters so they can be merged |
| check_evidence.pl | GMAPARAMS | -t 1 -2 -z sense_force -n 1 -F | gmap settings |
| check_evidence.pl | MAXSEGMENTSIZE | 100000 | max length of genomic segment containing candidate split genes |
| check_evidence.pl | MINPAIRPECNONOUTLIERS | 0.25 | min %pairs of genes from same species among non-outliers, used to correct long gene models |
| check_evidence.pl | MINLIFTIDENTITY | 95.0 | min % of identity of gmap CDS/cDNA to genome alignments to be considered |
| check_evidence.pl | MINFIXOVERLAP | 0.75 | min overlap [0-1] of mapped genes to fix long/split gene models |
| _dotplot.pl | MINCONTIGSIZE | 100000 | min length of chrs/contigs to be considered for dotplot |
In addition to Perl, these scripts require:
- https://github.com/lh3/minimap2
- https://github.com/gpertea/gffread
- https://bedtools.readthedocs.io/en/latest/
Assuming bedtools are installed in most settings,and that gcc & g++ compilers are available, the remaining dependencies can be installed on Ubuntu/Debian in folder bin/ with:
# does not require root privileges
cd ../..
make install_pangenes
Note this will also download a test rice dataset. You can test everything is in place with:
perl pangenes/get_pangenes.pl -v
make test_pangenes
This should print something like this:
Checking required binaries and data sources, set in pangeneTools.pm or in command line:
EXE_MINIMAP : OK (path:/home/contrera/plant-scripts/pangenes/../lib/minimap2/minimap2)
EXE_BEDTOOLS : OK (path:bedtools)
EXE_GFFREAD : OK (path:/home/contrera/plant-scripts/pangenes/bin/gffread/gffread)
EXE_COLLINEAR : OK (path:/home/contrera/plant-scripts/pangenes/_collinear_genes.pl)
EXE_CUTSEQUENCES : OK (path:/home/contrera/plant-scripts/pangenes/_cut_sequences.pl)
EXE_CLUSTANALYSIS : OK (path:/home/contrera/plant-scripts/pangenes/_cluster_analysis.pl)
EXE_GZIP : OK (path:gzip)
EXE_BZIP2 : OK (path:bzip2)
EXE_SORT : OK (path:sort)
In addition to minimap2, two other genome aligners have been integrated:
| software | flag | source | installation instructions | notes |
|---|---|---|---|---|
| GSAlign (benchmarked) | -g | https://doi.org/10.1186/s12864-020-6569-1 | cd ../.. && make install_gsalign | requires gcc compiler |
| Wfmash (experimental) | -w | https://github.com/ekg/wfmash | cd ../.. && make install_wfmash | requires sudo & g++ compiler |
See all options with:
perl get_pangenes.pl -h
If the installation was succesfull you should have a copy of a test dataset. You can browse it with:
ls ../files/test_rice/
This toy dataset comprises three annotated genome assemblies:
| accession | assembly FASTA file | annotation GFF file |
|---|---|---|
| Oryza_indica.ASM465v1.chr1 | Oryza_indica.ASM465v1.chr1.fa.gz | Oryza_indica.ASM465v1.chr1.gff3.gz |
| Oryza_sativa.IRGSP-1.0 | Oryza_sativa.IRGSP-1.0.chr1.fa.gz | Oryza_sativa.IRGSP-1.0.chr1.gff.gz |
| Oryza_nivara_v1.chr1 | Oryza_nivara_v1.chr1.fa.gz | Oryza_nivara_v1.chr1.gff3.gz |
As you can see there's a FASTA file and a matching GFF file for each genome. Each pair of files has a common prefix or accession, which is the name of each genome.
Accepted extensions for FASTA files are .fna, .fa and .fasta, which can all be GZIP-compressed and have also .gz appended.
Accepted extensions for GFF files are .gff and .gff3. These can also be compressed.
As shown in section Parameters, genes are parsed from GFF files. In order to be called valid,
a gene must include the following GFF features: gene, mRNA and transcript.
Other accepted features include: exon and CDS.
You can see also a file named include.split which shows how a custom subset of accessions can be analyzed with option -I.
To compute a core pangene set out of the test dataset you can start with:
perl get_pangenes.pl -d ../files/test_rice
Note that you can use -m cluster or -m dryrun to run tasks in parallel,
section Runmodes and HPC configuration.
While computing WGA alignments you can tell the script to split each genome
in chromosomes and align only homologous chromosomes. Please use option -s
for this, which requires a regular expression.
For instance, use -s '\d+' to split in chromosomes named with natural numbers.
The output of the test looks like this:
$ perl get_pangenes.pl -d ../files/test_rice
# get_pangenes.pl -d ../files/test_rice -o 0 -r 0 -t all -c 0 -z 0 -I 0 -m local -w 0 -g 0 -O 0.5 -Q 50 -N 5 -s '' -H 0 -W '' -G '' -B '' -S '' -n 4 -R 0
# version ...
# results_directory=/home/contrera/github/plant-scripts/pangenes/test_rice_pangenes
# parameters: MINGFFLEN=100 GFFACCEPTEDFEATS=gene,mRNA,transcript,exon,CDS GFFVALIDGENEFEAT=gene,mRNA,transcript
# checking input files...
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_indica.ASM465v1.chr1.fna
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_indica.ASM465v1.chr1.gff
# ../files/test_rice/Oryza_indica.ASM465v1.chr1.fa.gz 45.84MB genes=5292 non-valid=0
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_nivara_v1.chr1.fna
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_nivara_v1.chr1.gff
# ../files/test_rice/Oryza_nivara_v1.chr1.fa.gz 41.54MB genes=5143 non-valid=0
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_sativa.IRGSP-1.0.chr1.fna
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_sativa.IRGSP-1.0.chr1.gff
# ../files/test_rice/Oryza_sativa.IRGSP-1.0.chr1.fa.gz 42.56MB genes=5271 non-valid=0
# 3 genomes, 15706 genes
# done
# taxa considered = 3 genes = 15706
# mask=Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_split_ (_5neigh_algMmap_split)
# indexing genomes ...
...
# done
# running pairwise genome alignments ...
...
# done
# sorting collinearity results...
# WGA summary (N50, %mapped genes in blocks of 3+)
31792.0 79.7 Oryza_indica.ASM465v1.chr1
33230.0 80.3 Oryza_nivara_v1.chr1
34983.0 83.2 Oryza_sativa.IRGSP-1.0.chr1
# clustering sequences ...
# done
# number of clusters = 7801 (core = 3008)
# cluster_list = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1.cluster_list
# cluster_directory = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1
# percent_conserved_sequences_file = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/POCS.matrix.tab
# pangene_file (occup) = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/pangene_matrix.tab
# pangene_file (occup, transposed) = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/pangene_matrix.tr.tab
# pangene_file (names) = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/pangene_matrix_genes.tab
# pangene_file (names, transposed) = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/pangene_matrix_genes.tr.tab
In this example, the clusters are stored in folder
test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1
and a text file describing the clusters is also produced
test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1.cluster_list
which looks like this:
cluster gene:ONIVA01G52180 size=3 taxa=3 taxa(gdna)=NA cdnafile: gene:ONIVA01G52180.cdna.fna cdsfile: gene:ONIVA01G52180.cds.fna pepfile: gene:ONIVA01G52180.cds.faa gdnafile: void
: Oryza_indica.ASM465v1.chr1
: Oryza_sativa.IRGSP-1.0.chr1
: Oryza_nivara_v1.chr1
...
Note that up to four types of clusters are generated (cdna, cds, pep & gdna), depending on the nature of the gene and also on the existence of WGA alignments supporting the alignment of annotated genes from one assembly to genomic segments on another.
| cluster type | sequence type | definition | notes |
|---|---|---|---|
| cdna | nucleotides | transcript/mRNA features in GFF file | Often several for the same gene. |
| cds | nucleotides | CDS features in GFF file | Often several for the same gene. Might be empty on non-coding genes. |
| pep | amino acids | CDS features in GFF file | Often several for the same gene. Might be empty on non-coding genes. |
| gdna | nucleotides | genomic segment in one genome matching a gene model in another | Provided as obvious candidate regions for lifting over genes. |
Clusters are FASTA files like this, and might include several sequences for the same gene:
$ grep ">" test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1/gene:ONIVA01G52180.cdna.fna
>transcript:ONIVA01G52180.1 gene:ONIVA01G52180 1:42818942-42824598(-) [Oryza_nivara_v1.chr1]
>transcript:ONIVA01G52180.2 gene:ONIVA01G52180 1:42818942-42824598(-) [Oryza_nivara_v1.chr1]
>transcript:ONIVA01G52180.3 gene:ONIVA01G52180 1:42818944-42824598(-) [Oryza_nivara_v1.chr1]
>transcript:Os01t0978100-01 gene:Os01g0978100 1:43232027-43238506(-) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:Os01t0978100-02 gene:Os01g0978100 1:43232034-43238012(-) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:Os01t0978100-03 gene:Os01g0978100 1:43232036-43237974(-) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:BGIOSGA000001-TA gene:BGIOSGA000001 1:47275570-47278635(-) [Oryza_indica.ASM465v1.chr1]
While cdna, cds and pep cluster files contain sequences annotated in the input GFF files, the gdna FASTA files contain genomic segments from assemblies matching gene models annotated in other genomes. The latter files are good starting points for lifting over genes, as explained in section Lifting over gene models in genomic segment clusters.
The gene collinearity evidence supporting the clusters is summarized in a compressed file which is added to the output folder of each run of the script. In this example this is:
test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/mergedpairs.tsv.gz
See section Evidence supporting clusters to learn how to extract the evidence for a selected cluster.
The script also produces % of Conserved Sequence (POCS) and pangene matrices, which look like this:
$ cat test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/POCS.matrix.tab
genomes Oryza_nivara_v1.chr1 Oryza_sativa.IRGSP-1.0.chr1 Oryza_indica.ASM465v1.chr1
Oryza_nivara_v1.chr1 100.00 61.81 60.62
Oryza_sativa.IRGSP-1.0.chr1 61.81 100.00 62.65
Oryza_indica.ASM465v1.chr1 60.62 62.65 100.00
And
$ head test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/pangene_matrix.tr.tab
source:test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5niegh_algMmap_/Oryzanivarav1.chr1 Oryza_nivara_v1.chr1 Oryza_sativa.IRGSP-1.0.chr1 Oryza_indica.ASM465v1.chr1
chrunsorted NA NA NA
gene:ONIVA01G52180 1 1 1
gene:ONIVA01G52140 1 1 1
gene:ONIVA01G52120 1 1 1
gene:ONIVA01G52090 1 1 1
gene:ONIVA01G52080 1 1 1
gene:ONIVA01G52070 1 1 1
gene:ONIVA01G52060 1 1 1
gene:ONIVA01G52030 1 1 1
$ head test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/pangene_matrix_genes.tr.tab
source:test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1 Oryza_nivara_v1.chr1 Oryza_sativa.IRGSP-1.0.chr1 Oryza_indica.ASM465v1.chr1
chr:unsorted NA NA NA
gene:ONIVA01G52180 gene:ONIVA01G52180 gene:Os01g0978100 gene:BGIOSGA000001
gene:ONIVA01G52140 gene:ONIVA01G52140 gene:Os01g0977600 gene:BGIOSGA000002
gene:ONIVA01G52120 gene:ONIVA01G52120 gene:Os01g0977300 gene:BGIOSGA000003
gene:ONIVA01G52090 gene:ONIVA01G52090 gene:Os01g0976900 gene:BGIOSGA000004
gene:ONIVA01G52080 gene:ONIVA01G52080 gene:Os01g0976800 gene:BGIOSGA000005
gene:ONIVA01G52070 gene:ONIVA01G52070 gene:Os01g0976700 gene:BGIOSGA000006
gene:ONIVA01G52060 gene:ONIVA01G52060 gene:Os01g0976600 gene:BGIOSGA000007
gene:ONIVA01G52030 gene:ONIVA01G52030 gene:Os01g0976200 gene:BGIOSGA000008
While POCS matrices summarize the percentage of genes shared by any two annotated genomes, pangenome matrices contain the composition of those clusters, named in the first columns.
As opposed to Example 1, where only core pangenes where produced, in this example we will build a complete pangene set with clusters of all occupancy classes (-t 0, see Table 1):
perl get_pangenes.pl -d ../files/test_rice -t 0
The output is similar to the previous one, but saved in a different folder (Oryza_nivara_v1chr1_0taxa_5neigh_algMmap_). The main differences are shown below, note the larger number of clusters:
# number of clusters = 7801 (core = 3008)
# cluster_list = test_rice_pangenes/Oryza_nivara_v1chr1_0taxa_5neigh_algMmap_/Oryzanivarav1.chr1.cluster_list
# cluster_directory = test_rice_pangenes/Oryza_nivara_v1chr1_0taxa_5neigh_algMmap_/Oryzanivarav1.chr1
...
# pangene_file (occup, transposed) = test_rice_pangenes/Oryza_nivara_v1chr1_0taxa_5neigh_algMmap_/pangene_matrix.tr.tab
...
# pangene_file (names, transposed) = test_rice_pangenes/Oryza_nivara_v1chr1_0taxa_5neigh_algMmap_/pangene_matrix_genes.tr.tab
The last two output files can be easily parsed to discover pangenes with Presence-Absence Variation (PAV). For instance, to find pangenes that contain gene models from Oryza_indica.ASM465v1.chr1 and Oryza_sativa.IRGSP-1.0.chr1 but no models from Oryza_nivara_v1.chr1 you could do:
perl -lane 'print if($F[1] eq "-" && $F[2] ne "-" && $F[3] ne "-")' test_rice_pangenes/Oryza_nivara_v1chr1_0taxa_5neigh_algMmap_/pangene_matrix_genes.tr.tab | wc -l
#508
perl -lane 'print if($F[1] eq "-" && $F[2] ne "-" && $F[3] ne "-")' test_rice_pangenes/Oryza_nivara_v1chr1_0taxa_5neigh_algMmap_/pangene_matrix_genes.tr.tab | head
gene:BGIOSGA000032 - gene:BGIOSGA000032 gene:Os01g0969700
gene:BGIOSGA000039 - gene:BGIOSGA000039 gene:Os01g0968200
gene:BGIOSGA000074 - gene:BGIOSGA000074 gene:Os01g0960300
gene:BGIOSGA000085 - gene:BGIOSGA000085 gene:Os01g0955550
gene:BGIOSGA000116 - gene:BGIOSGA000116 gene:Os01g0947700
gene:BGIOSGA000117 - gene:BGIOSGA000117 gene:Os01g0946800
gene:BGIOSGA000118 - gene:BGIOSGA000118 gene:Os01g0947000
gene:BGIOSGA000123 - gene:BGIOSGA000123 gene:Os01g0946850
gene:BGIOSGA000126 - gene:BGIOSGA000126 gene:Os01g0945600,gene:Os01g0945700,gene:Os01g0945800
gene:BGIOSGA000127 - gene:BGIOSGA000127 gene:Os01g0945300
As explained earlier, the first column shows the actual cluster names, which can be found with different extensions at
test_rice_pangenes/Oryza_nivara_v1chr1_0taxa_5neigh_algMmap_/Oryzanivarav1.chr1
In this example we will split the input genomes in chromosomes and will limit the alignments to homologous chromosomes, which might be what you expect when talking about collinear genes. This has also the beneficial side-effect of reducing the RAM consumption of the software (see also option -H).
In order to identify homologous chromosomes you'll need to pass a regular expression as an argument:
perl get_pangenes.pl -d ../files/test_rice -s '^\d+$'
This particular example tells the script to identify chromosomes named with a natural number, as done for instance in Ensembl Plants. This matches the nuclear chromosomes in the test data, see:
grep "^>" test_rice_pangenes/_Oryza*.fna
test_rice_pangenes/_Oryza_indica.ASM465v1.chr1.fna:>1 dna:chromosome chromosome:ASM465v1:1:1:47283185:1 REF
test_rice_pangenes/_Oryza_nivara_v1.chr1.fna:>1 dna:chromosome chromosome:Oryza_nivara_v1.0:1:1:42845077:1 REF
test_rice_pangenes/_Oryza_sativa.IRGSP-1.0.chr1.fna:>1 dna:chromosome chromosome:IRGSP-1.0:1:1:43270923:1 REF
test_rice_pangenes/_Oryza_sativa.IRGSP-1.0.chr1.fna:>Mt dna:chromosome chromosome:IRGSP-1.0:Mt:1:490520:1 REF
test_rice_pangenes/_Oryza_sativa.IRGSP-1.0.chr1.fna:>Pt dna:chromosome chromosome:IRGSP-1.0:Pt:1:134525:1 REF
Other possibly useful regexes include '^\d+H$' or 'chr\d+$'.
Any chromosome names that don't match the regex are pooled in a dummy 'unplaced' chromosome.
When you run it you'll see a couple differences in the output:
- the number of chrs/contigs parsed in each input FASTA file
- a BED-like pangene matrix
# checking input files...
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_indica.ASM465v1.chr1.fna
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_indica.ASM465v1.chr1.gff
# ../files/test_rice/Oryza_indica.ASM465v1.chr1.fa.gz 45.84MB genes=5292 non-valid=0 chrs/contigs=1
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_nivara_v1.chr1.fna
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_nivara_v1.chr1.gff
# ../files/test_rice/Oryza_nivara_v1.chr1.fa.gz 41.54MB genes=5143 non-valid=0 chrs/contigs=1
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_sativa.IRGSP-1.0.chr1.fna
# re-using /home/contrera/github/plant-scripts/pangenes/test_rice_pangenes/_Oryza_sativa.IRGSP-1.0.chr1.gff
# ../files/test_rice/Oryza_sativa.IRGSP-1.0.chr1.fa.gz 42.56MB genes=5271 non-valid=0 chrs/contigs=1
...
# clusters sorted by position in chr 1 = 7808
...
# pangene_file (BED-like) = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_split_/pangene_matrix.tr.bed
The BED file contents should be like this, with genome occupancy in column 5:
$ head test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_split_/pangene_matrix.tr.bed
#1 NA NA gene:BGIOSGA002568 1 0 NA NA gene:BGIOSGA002568
1 4848 11824 gene:ONIVA01G00010 1 + gene:ONIVA01G00010 NA NA
1 43371 62621 gene:ONIVA01G00020 1 + gene:ONIVA01G00020 NA NA
1 62743 64526 gene:ONIVA01G00030 1 + gene:ONIVA01G00030 NA NA
1 64707 65654 gene:ONIVA01G00040 1 + gene:ONIVA01G00040 NA NA
1 68827 69733 gene:ONIVA01G00050 1 + gene:ONIVA01G00050 NA NA
1 73630 75670 gene:ONIVA01G00060 1 + gene:ONIVA01G00060 NA NA
1 78105 78695 gene:ONIVA01G00070 1 + gene:ONIVA01G00070 NA NA
1 98770 99876 gene:ONIVA01G00080 1 - gene:ONIVA01G00080 NA NA
1 100726 101071 gene:ONIVA01G00090 1 - gene:ONIVA01G00090 NA NA
In our tests GSAlign produces comparable results to minimap2 but using less RAM. You can try it out with:
perl get_pangenes.pl -d ../files/test_rice -g
Note that the output folder is now
test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algGSal_
A unique output produce by GSAlign is an Average Nucleotide identitiy (ANI) matrix, which summarizes the %identity of pairs of aligned genomes:
$ cat test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algGSal_/ANI.matrix.tab
genomes Oryza_indica.ASM465v1.chr1 Oryza_nivara_v1.chr1 Oryza_sativa.IRGSP-1.0.chr1
Oryza_indica.ASM465v1.chr1 100.00 97.88 97.47
Oryza_nivara_v1.chr1 97.88 100.00 96.86
Oryza_sativa.IRGSP-1.0.chr1 97.47 96.86 100.00
A pangene set growth analysis can be performed by adding option -c, which will produce two files with random-sampling simulations on how the core- and pangene set grow as new genomes are added, named core_gene.tab and pan_gene.tab
perl get_pangenes.pl -d ../files/test_rice/ -s '^\d+$' -g -c
# genome composition report (samples=6,seed=12345)
## sample 0 (Oryza_nivara_v1.chr1 | 0,1,2,)
# adding Oryza_nivara_v1.chr1: core=5063 pan=5063
# adding Oryza_sativa.IRGSP-1.0.chr1: core=3155 pan=6801
# adding Oryza_indica.ASM465v1.chr1: core=2975 pan=7840
## sample 1 (Oryza_sativa.IRGSP-1.0.chr1 | 1,2,0,)
# adding Oryza_sativa.IRGSP-1.0.chr1: core=4893 pan=4893
# adding Oryza_indica.ASM465v1.chr1: core=3510 pan=6394
# adding Oryza_nivara_v1.chr1: core=2975 pan=7840
## sample 2 (Oryza_nivara_v1.chr1 | 0,1,2,)
# adding Oryza_nivara_v1.chr1: core=5063 pan=5063
# adding Oryza_sativa.IRGSP-1.0.chr1: core=3155 pan=6801
# adding Oryza_indica.ASM465v1.chr1: core=2975 pan=7840
## sample 3 (Oryza_indica.ASM465v1.chr1 | 2,1,0,)
# adding Oryza_indica.ASM465v1.chr1: core=5011 pan=5011
# adding Oryza_sativa.IRGSP-1.0.chr1: core=3510 pan=6394
# adding Oryza_nivara_v1.chr1: core=2975 pan=7840
## sample 4 (Oryza_sativa.IRGSP-1.0.chr1 | 1,0,2,)
# adding Oryza_sativa.IRGSP-1.0.chr1: core=4893 pan=4893
# adding Oryza_nivara_v1.chr1: core=3155 pan=6801
# adding Oryza_indica.ASM465v1.chr1: core=2975 pan=7840
## sample 5 (Oryza_nivara_v1.chr1 | 0,2,1,)
# adding Oryza_nivara_v1.chr1: core=5063 pan=5063
# adding Oryza_indica.ASM465v1.chr1: core=3437 pan=6637
# adding Oryza_sativa.IRGSP-1.0.chr1: core=2975 pan=7840
# pan-gene (number of clusters) = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algGSal_split_/pan_gene.tab
# core-gene (number of clusters) = test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algGSal_split_/core_gene.tab
The resulting pan and core gene files look like this:
$ cat test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algGSal_split_/pan_gene.tab
g1 g2 g3
5063 6801 7840
4893 6394 7840
5063 6801 7840
5011 6394 7840
4893 6801 7840
5063 6637 7840
Pangene clusters can be used to estimate the diversity of haplotypes or alleles of one or more genes. While this can be done with cDNA FASTA files, haplotypes of coding sequences, particularly those harboring missense and nonsense mutations, are the easiest to interpret in relation to phenotype.
The commands in this section can be used to compute the haplotype diversity of a pangene set.
They include script check_quality.pl, introduced in section
Quality metrics of clusters,
two optional packages are required:
| software | source | installation instructions |
|---|---|---|
| parallel | https://www.gnu.org/software/parallel | sudo apt install parallel |
| trimAl | https://github.com/inab/trimal/releases/tag/v1.5.0 | download and save in appropriate folder |
cd .. && make install_pangenes_quality && cd pangenes
# select protein FASTA files of pangenes with ideally 4+ taxa, 3 in this toy example
perl -lne 'if(/\s+taxa=(\d+) \S+ cdnafile: (\S+) \S+ \S+ pepfile: (\S+)/){ print "$3" if($1 >= 3)}' \
test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1.cluster_list \
> test_rice_pangenes/list.diversity.pep.txt
#wc test_rice_pangenes/list.diversity.pep.txt
# compute multiple alignments of selected FASTA files with 10 parallel threads,
# -o is the name of the output folder
# -I means that only the 1st isoform of each gene model is used,
cat list.diversity.pep.txt | parallel --gnu -j 10 ./check_quality.pl -d \
test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_ -i {} \
-n -I -o test_rice_pangenes/diversity.pep ::: &> test_rice_pangenes/log.diversity.pep.I.tsv
# header of log.diversity.pep.I.tsv
# file 1stisof occup seqs mode_len SE_len mode_exons SE_exons mode_dist max_dist SE_dist sites Ca Cr_max Cr_min Cc_max Cc_min Cij_max Cij_min
# run trimAl to define well aligned blocks, in the example located in
# folder ~/soft/trimAl_Linux_x86-64 , and save results in new folder trimal.out
mkdir test_rice_pangenes/trimal.out
cd test_rice_pangenes/diversity.pep
for file in *.cds.aln.faa; do ~/soft/trimAl_Linux_x86-64/trimal -keepheader -automated1 -terminalonly -in $file -out ../trimal.out/$file; done
cd ..
# count alleles in trimmed blocks
cd trimal.out
for file in *.cds.aln.faa; do
echo -n $file
perl -ne 'chomp; if(/^>/){ print "\n$_\n" }else{print}' $file | \
perl -ne 'next if(/^$/);if(/^>.*\[(\S+)\]/){ $tx=$1 } else { $hp{$_}.="$tx,"; $sq{$_}++ }; END{ print "\t".scalar(keys(%sq))."\t"; foreach $s (keys(%sq)){ print "$sq{$s}:$hp{$s};" } }'
echo
done > ../haplotypes.trimmed.tsv
cd ../..
The resulting file test_rice_pangenes/haplotypes.trimmed.tsv can be used to create a histogram of haplotypes/alleles:
head -6 test_rice_pangenes/haplotypes.trimmed.tsv
gene:ONIVA01G00100.cds.aln.faa 3 1:Oryza_indica.ASM465v1.chr1,;1:Oryza_sativa.IRGSP-1.0.chr1,;1:Oryza_nivara_v1.chr1,;
gene:ONIVA01G00110.cds.aln.faa 3 1:Oryza_sativa.IRGSP-1.0.chr1,;1:Oryza_indica.ASM465v1.chr1,;1:Oryza_nivara_v1.chr1,;
gene:ONIVA01G00120.cds.aln.faa 3 1:Oryza_indica.ASM465v1.chr1,;1:Oryza_sativa.IRGSP-1.0.chr1,;1:Oryza_nivara_v1.chr1,;
gene:ONIVA01G00130.cds.aln.faa 3 1:Oryza_indica.ASM465v1.chr1,;1:Oryza_sativa.IRGSP-1.0.chr1,;1:Oryza_nivara_v1.chr1,;
gene:ONIVA01G00140.cds.aln.faa 3 1:Oryza_nivara_v1.chr1,;1:Oryza_indica.ASM465v1.chr1,;1:Oryza_sativa.IRGSP-1.0.chr1,;
gene:ONIVA01G00150.cds.aln.faa 2 2:Oryza_nivara_v1.chr1,Oryza_sativa.IRGSP-1.0.chr1,;1:Oryza_indica.ASM465v1.chr1,;
# histogram, note that clusters with tandem copies might inflate the diversity
#perl -lane 'print $F[1] if($F[1])' test_rice_pangenes/haplotypes.trimmed.tsv | sort | uniq -c | sort -k2,2n
Figure 6. Histogram of trimmed protein haplotypes encoded in test pangene set.
The main results of the pipeline include a directory (cluster_dir) and a list (cluster_list) of pangene clusters and pangene matrices in several formats, which have already been introduced in Example 1 and Example 3.
The different types of pangene matrices that can be produced are summarized in the following tables, assuming g genomes, n chromosomes and p pangenes with occupancy => numberOfTaxa ([alltaxa] by default). In these matrices an extra, artifical chromosome named 'chrunsorted' is created to accomodate unsorted pangenes. Note that the contents of these matrices correspond to clustered FASTA sequences in cluster_dir.
- If genome was split in chromosomes with an optional regular expression, as in Example 3, pangenes are sorted by position along the reference chromosomes when possible:
| filename | dimensions | contents |
|---|---|---|
| pangene_matrix.tr.tab | p+n+2 X g+1 | (int) number of genes from genome gi in pangene cluster, TSV format, n+1 chromosome blocks |
| pangene_matrix_genes.tr.tab | p+n+2 X g+1 | (string) names of genes from genome gi in pangene cluster, TSV format, n+1 chromosome blocks |
| pangene_matrix.tr.bed | p X g+6 | (string) names of genes from genome gi in pangene cluster, BED-like format, non-reference pangenes lack exact coordinates and are commented out with # |
BED-like pangene_matrix.tr.bed is special for it includes all pangene clusters regardless of their occupancy,
which is useful for plotting the genomic context of a pangene cluster.
Matrices pangene_matrix.tab and pangene_matrix_genes.tab are transposed with respect to pangene_matrix.tr.tab and pangene_matrix_genes.tr.tab; pangene_matrix.fasta is a FASTA compressed version of pangene_matrix.tab.
- If genome was not split in chromosomes all-vs-all chromosome alignments are computed and pangenes are stacked in the chromosome 'chrunsorted', with clusters that include the reference genome first and singletons last. In this case, a pangene cluster might contain genes encoded in different chromosomes from different genomes:
| filename | dimensions | contents |
|---|---|---|
| pangene_matrix.tr.tab | p+2 X g+1 | (int) number of genes from genome gi in pangene cluster, TSV format |
| pangene_matrix_genes.tr.tab | p+2 X g+1 | (string) names of genes from genome gi in pangene cluster, TSV format |
There are different types of intermediate result files produced by the pipeline. Probably the most valuable files are those containing collinear gene models resulting from a pairwise WGA, which are stored in TSV format (see also Dotplots). For instance, let's inspect one such file, in this case produced with the GSal algorithm:
head test_rice_pangenes/_Oryza_nivara_v1.chr1.Oryza_sativa.IRGSP-1.0.chr1.algGSal.overlap0.5.tsv
gene:Os01g0100466 gene:Os01g0100466 Oryza_nivara_v1.chr1 1173 segment_collinear Oryza_sativa.IRGSP-1.0.chr1:1:116866-118039(-) segment Oryza_sativa.IRGSP-1.0.chr1 1173 NULL NULL NULL 100.00 1 NA;1:116866-118039(-)
...
gene:ONIVA01G00100 gene:ONIVA01G00100 Oryza_nivara_v1.chr1 7827 ortholog_collinear gene:Os01g0100100 gene:Os01g0100100 Oryza_sativa.IRGSP-1.0.chr1 7827 NULL NULL NULL 100.00 1 1:104920-116326(+);1:2982-10815(+)
...
Note that there are two types of rows: ortholog_collinear and segment_collinear. The first type describe a pair of collinear genes from two input taxa, their respective genomic coordinates and the length of their overlap in the underlying WGA. The second type indicate cases where a gene model in a taxon overlaps a genomic segment in another. Note also that the strand of each region is indicated, which might be useful to spot genes that are inverted/translocated genomic fragments.
In addition, each of these TSV files have a matching logfile with extension .queue. In our example, that would be:
test_rice_pangenes/_Oryza_nivara_v1.chr1.Oryza_sativa.IRGSP-1.0.chr1.algGSal.overlap0.5.tsv.queue
These files also contain useful bits of information such as:
# WGA blocks: N50 18935 median 3055
# 4557 genes mapped (68.2% in 3+blocks) ... (19 unmapped)
...
# 4243 genes mapped (60.1% in 3+blocks) ... (reverse, 4 unmapped)
# 3596 collinear gene pairs , 2057 collinear segments, 1.121 hits/gene
The first line summarizes the length of the blocks that make up the WGA, which will be shorter for poor assemblies. The 2nd and 3rd line indicate how many of the gene models from the two compared taxa/annotations were actually mapped in the WGA, in both directions (A->B and B->A). The 4th line reports how many collinear gene pairs and segments where found, and how many hits in the WGA were found on average per gene.
These files can be analyzed in bulk in the terminal for quality control, for instance with:
grep "collinear gene pairs" test_rice_pangenes/*Mmap.overlap0.5.tsv.queue | perl -lane 'print $F[1]' | sort -r
This is the location of genome alignments in PAF format and genome indices.
These are temporary files that are re-used by future jobs if possible to save computing time.
As the number of input genomes increases, so will the disk space required by tmp/.
For instance, the analysis of 22 barley genomes resulted in 956GB of temporary data.
It is perfectly safe to remove/compress this folder if you need to save space,
but that will mean that previous temporary files need to be computed again.
Data files produced can be plotted in many ways, for instance in Rstudio, but can also be conveniently done with help from scripts from software GET-HOMOLOGUES, which must first be installed as follows (manual here):
git clone https://github.com/eead-csic-compbio/get_homologues.git
perl install.pl
Here are a few examples:
get_homologues/plot_pancore_matrix.pl -f core_both -i core_gene.tab
get_homologues/plot_pancore_matrix.pl -f pan -i pan_gene.tab
get_homologues/plot_matrix_heatmap.sh -i POCS.matrix.tab -k "Percent Conserved Sequences (POCS)"
get_homologues/plot_matrix_heatmap.sh -i ANI.matrix.tab -k "Average Nucleotide Identity"
get_homologues/parse_pangenome_matrix.pl -m pangene_matrix.tab -s
These will produce figures such as these:
Figure 7. Pangene set growth after pooling 11 Oryza species, generated with get_homologues/plot_pancore_matrix.pl
Figure 8. Core-pangene set growth after pooling 11 Oryza species, generated with get_homologues/plot_pancore_matrix.pl.
Figure 9. Occupancy of pangene clusters of 11 Oryza species, generated with get_homologues/parse_pangenome_matrix.pl
Multiple alignments can be computed for each cluster FASTA file to determine, for instance, if there is a conserved gene structure. For instance, we can align the cDNA cluster gene:ONIVA01G52180.cdna.fna:
>transcript:Os01t0978100-02 gene:Os01g0978100 1:43232034-43238012(-) [Oryza_sativa.IRGSP-1.0.chr1]
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GGGAGGGGATTAGGCAACAAAAGCTCGTCGTCCATCCGCAGATACGGAACTACTCCCCTATCCAACACCTCCGAGTCCGAGCAACGCAAGATGGCGTCGTGGTCGTCGCCCGTCGCCGCCGCCGCCTTGCAGGTCCATTTCGGGTCCTCCTGCTTCTTCTCCGCCCGATCGCCACGACAGACCCTCCTCCTACCACCTCTCGCCCGCAACCCTACACTGACCATCCAGCCCCGGCCCCATCCCTTCCGGAACATCAACTCCTCCTCCTCCTCCAGCTGGATGTGCCACGCCGTCGCCGCCGAGGTCGAGGGCCTCAACATCGCCGACGACGTCACCCAGCTCATCGGCAAGACTCCAATGGTATATCTCAACAACATCGTCAAGGGATGTGTTGCCAATGTCGCTGCTAAGCTCGAGATTATGGAGCCCTGTTGCAGTGTCAAGGACAGGATAGGATACAGTATGATTTCTGATGCGGAAGAGAAAGGCTTGATAACTCCTGGAA------------------------------------------------------AGAGTGTTTTGGTGGAACCAACAAGTGGAAATACAGGCATTGGTCTTGCCTTCATTGCTGCTTCCAGAGGATATAAATTAATATTGACCATGCCTGCATCAATGAGCATGGAGAGAAGAGTTCTACTCAAAGCTTTTGGCGCTGAACTTGTCCTTACTGATGCCGCAAAAGGGATGAAGGGGGCTGTAGATAAGGCTACAGAGATTTTAAATAAGACACCTGATGCCTATATGCTGCAGCAGTTTGACAACCCTGCCAACCCAAAGGTACATTATGAGACTACTGGGCCAGAAATCTGGGAGGATTCTAAAGGGAAGGTGGATGTATTCATTGGTGGAATTGGAACAGGTGGAACAATATCTGGTGCTGGCCGTTTCCTGAAAGAGAAAAATCCTGGAATTAAGGTTATTGGTATTGAGCCTTCTGAGAGTAACATACTCTCTGGTGGAAAACCTGGCCCACATAAGATTCAAGGCATTGGGGCAGGATTTGTTCCAAGGAACTTGGATAGTGAAGTTCTCGATGAAGTGATTGAGATATCTAGTGATGAGGCTGTTGAGACAGCAAAGCAATTGGCTCTTCAGGAAGGATTACTGGTTGGAATTTCATCTGGGGCAGCAGCAGCAGCTGCCATTAAAGTTGCAAAAAGACCAGAAAATGCTGGAAAGTTGGTAGTGGTTGTGTTTCCAAGCTTTGGTGAGAGGTACCTTTCATCTATCCTTTTTCAGTCGATAAGAGAAGAATGTGAGAAGTTGCAACCTGAACCATGAGCCTAACTTCAGTGTTCACAACATCATAATTGTTTCTGAGATTTCTGGCCATTAGTTTTTTTTTTCTGAGAAGTATCATACCACTCCATAGCTGTTTGTTCGATAAATAAAACAGTTACCTTTGCACTTATAATGAGGCTTGTGAGGGTACTGTGAAATTTCTCTGAACATCTTCTACTCTTCTCTTTTATCCTTAAATCAATCTGGGAGCAGTTTGTAATACATACGTAAATTTAAAGCTGGGTGTTTGGTAATTGTAAACAAATGTTTCGAAGAGCCGTGAAACATTATCAATTAGCATGAAGCACTTTAAAAGTGCTTTCCGG-------
>transcript:ONIVA01G52180.1 gene:ONIVA01G52180 1:42818942-42824598(-) [Oryza_nivara_v1.chr1]
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ATGGCGTCGTGGTCGTCGCCCGTCGCCGCCGCCGCCTTGCAGGTCCATTTCGGGTCCTCCTGCTTCTTCTCCGCCCGATCGCCACGACAGACCCTCCTCCTACCACCTCTCGCCCGCAACCCTACACTGACCATCCAGCCCCGGCCCCATCCCTTCCGGAACATCAACTCCTCCTCCTCCTCCAGCTGGATGTGCCACGCCGTCGCCGCCGAGGTCGAGGGCCTCAACATCGCCGACGA---------CCTCATCGGCAAGACTCCAATGGTATATCTCAACAACATCGTCAAGGGATGTGTTGCCAATGTCGCTGCTAAGCTCGAGATTATGGAGCCCTGTTGCAGTGTCAAGGACAGGATAGGATACAGTATGATTTCTGATGCGGAAGAGAAAGGCTTGATAACTCCTGGAA------------------------------------------------------AGAGTGTTTTGGTGGAACCAACAAGTGGAAATACAGGCATTGGTCTTGCCTTCATTGCTGCTTCCAGAGGATATAAATTAATATTGACCATGCCTGCATCAATGAGCATGGAGAGAAGAGTTCTACTCAAAGCTTTTGGCGCTGAACTTGTCCTTACTGATGCCGCAAAAGGGATGAAGGGGGCTGTAGATAAGGCTACAGAGATTTTAAATAAGACACCTGATGCCTATATGCTGCAGCAGTTTGACAACCCTGCCAACCCAAAGGTACATTATGAGACTACTGGGCCAGAAATCTGGGAGGATTCTAAAGGGAAGGTGGATGTATTCATTGGTGGAATTGGAACAGGTGGAACAATATCTGGTGCTGGCCGTTTCCTGAAAGAGAAAAATCCTGGAATTAAGGTTATTGGTATTGAGCCTTCTGAGAGTAACATACTCTCTGGTGGAAAACCTGGCCCACATAAGATTCAAGGCATTGGGGCAGGATTTGTTCCAAGGAACTTGGATAGTGAAGTTCTCGATGAAGTGATTGAGATATCTAGTGATGAGGCTGTTGAGACAGCAAAGCAATTGGCTCTTCAGGAAGGATTACTGGTTGGAATTTCATCTGGGGCAGCAGCAGCAGCTGCCATTAAAGTTGCAAAAAGACCAGAAAATGCTGGAAAGTTGGTAGTGGTTGTGTTTCCAAGCTTTGGTGAGAGGTACCTTTCATCTATCCTTTTTCAGTCGATAAGAGAAGAATGTGAGAAGTTGCAACCTGAACCATGAGC-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
>transcript:BGIOSGA000001-TA gene:BGIOSGA000001 1:47275570-47278635(-) [Oryza_indica.ASM465v1.chr1]
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ATGGAGAGAAGAGTTCTACTCAAAGCTTTTGGCGCTGAACTTGTCCTTACTGATGCCGCAAAAGGGATGAAGGGGGCTGTAGATAAGGCTACAGAGATTTTAAATAAGACACCTGATGCCTATATGCTGCAGCAGTTTGACAACCCTGCCAACCCAAAGGTACATTATGAGACTACTGGGCCAGAAATCTGGGAGGATTCTAAAGGGAAGGTGGATGTATTCATTGGTGGAATTGGAACAGGTGGAACAATATCTGGTGCTGGCCGTTTCCTGAAAGAGAAAAATCCTGGAATTAAGGTTATTGGTATTGAGCCTTCTGAGAGTAACATACTCTCTGGTGGAAAACCTGGCCCACATAAGATTCAAGGCATTGGGGCAGGATTTGTTCCAAGGAACTTGGATAGTGAAGTTCTCGATGAAGTGATTGAGATATCTAGTGATGAGGCTGTTGAGACAGCAAAGCAATTGGCTCTTCAGGAAGGATTACTGGTTGGAATTTCATCTGGGGCAGCAGCAGCAGCTGCCATTAAAGTTGCAAAAAGACCAGAAAATGCTGGAAAGTTGGTAGTGGTTGTGTTTCCAAGCTTTGGTGAGAGGTACCTTTCATCTATCCTTTTTCAGTCGATAAGAGAAGAATGTGAGAAGTTGCAACCTGAACCATGA---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
>transcript:Os01t0978100-01 gene:Os01g0978100 1:43232027-43238506(-) [Oryza_sativa.IRGSP-1.0.chr1]
TGGGCCCCACCTTCAGAGAAAACCGCGTCTCCCCAACTTCCACCGCTAATTTCCGCCGCTGGCTTCGTACTTTCCAACTCCACGGCGGTTCGGCGGCGTACGGCGGCGACCCCCGCCGCTTACTCCTCTTCCCTCCTCTCCTCCCACCGGCGGCGCACAGCCGGCCTTCCACCGCAGCGGAGAGGCCGGCGCGCCAACGCTCGGATCCGGCCTCCGCGCCGTGGCCCAAGCGGCGACGGCGACGAGCGGACGACTTGGAGCGGCGTCTCCCCCTTCCTCGTCCGGCCTCCGCGCCGTGGCCCGAGCGGCGACGGCGTCGGGCGGTGCTTTTCAGCTGTGCGCCTGCGCCTGAGCTTCGCTTTTCAGCCAAGAACGGGTGACCAAGTTTGGCCACTCGGTCTTCACTAGGCTACACATGTGGATGAGGACGTGGCACCGAATATGCCGAACATGTGGAGGAGCATTGTAAACGCCTCACAAAAGACCGTACATTAGGGAGGGGATTAGGCAACAAAAGCTCGTCGTCCATCCGCAGATACGGAACTACTCCCCTATCCAACACCTCCGAGTCCGAGCAACGCAAGATGGCGTCGTGGTCGTCGCCCGTCGCCGCCGCCGCCTTGCAGGTCCATTTCGGGTCCTCCTGCTTCTTCTCCGCCCGATCGCCACGACAGACCCTCCTCCTACCACCTCTCGCCCGCAACCCTACACTGACCATCCAGCCCCGGCCCCATCCCTTCCGGAACATCAACTCCTCCTCCTCCTCCAGCTGGATGTGCCACGCCGTCGCCGCCGAGGTCGAGGGCCTCAACATCGCCGACGACGTCACCCAGCTCATCGGCAAGACTCCAATGGTATATCTCAACAACATCGTCAAGGGATGTGTTGCCAATGTCGCTGCTAAGCTCGAGATTATGGAGCCCTGTTGCAGTGTCAAGGACAGGATAGGATACAGTATGATTTCTGATGCGGAAGAGAAAGGCTTGATAACTCCTGGAA------------------------------------------------------AGAGTGTTTTGGTGGAACCAACAAGTGGAAATACAGGCATTGGTCTTGCCTTCATTGCTGCTTCCAGAGGATATAAATTAATATTGACCATGCCTGCATCAATGAGCATGGAGAGAAGAGTTCTACTCAAAGCTTTTGGCGCTGAACTTGTCCTTACTGATGCCGCAAAAGGGATGAAGGGGGCTGTAGATAAGGCTACAGAGATTTTAAATAAGACACCTGATGCCTATATGCTGCAGCAGTTTGACAACCCTGCCAACCCAAAGGTACATTATGAGACTACTGGGCCAGAAATCTGGGAGGATTCTAAAGGGAAGGTGGATGTATTCATTGGTGGAATTGGAACAGGTGGAACAATATCTGGTGCTGGCCGTTTCCTGAAAGAGAAAAATCCTGGAATTAAGGTTATTGGTATTGAGCCTTCTGAGAGTAACATACTCTCTGGTGGAAAACCTGGCCCACATAAGATTCAAGGCATTGGGGCAGGATTTGTTCCAAGGAACTTGGATAGTGAAGTTCTCGATGAAGTGATTGAGATATCTAGTGATGAGGCTGTTGAGACAGCAAAGCAATTGGCTCTTCAGGAAGGATTACTGGTTGGAATTTCATCTGGGGCAGCAGCAGCAGCTGCCATTAAAGTTGCAAAAAGACCAGAAAATGCTGGAAAGTTGGTAGTGGTTGTGTTTCCAAGCTTTGGTGAGAGGTACCTTTCATCTATCCTTTTTCAGTCGATAAGAGAAGAATGTGAGAAGTTGCAACCTGAACCATGAGCCTAACTTCAGTGTTCACAACATCATAATTGTTTCTGAGATTTCTGGCCATTAGTTTTTTTTTTCTGAGAAGTATCATACCACTCCATAGCTGTTTGTTCGATAAATAAAACAGTTACCTTTGCACTTATAATGAGGCTTGTGAGGGTACTGTGAAATTTCTCTGAACATCTTCTACTCTTCTCTTTTATCCTTAAATCAATCTGGGAGCAGTTTGTAATACATACGTAAATTTAAAGCTGGGTGTTTGGTAATTGTAAACAAATGTTTCGAAGAGCCGTGAAACATTATCAATTAGCATGAAGCACTTTAAAAGTGCTTTCCGGATGCTGC
>transcript:ONIVA01G52180.3 gene:ONIVA01G52180 1:42818944-42824598(-) [Oryza_nivara_v1.chr1]
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ATGGCGTCGTGGTCGTCGCCCGTCGCCGCCGCCGCCTTGCAGGTCCATTTCGGGTCCTCCTGCTTCTTCTCCGCCCGATCGCCACGACAGACCCTCCTCCTACCACCTCTCGCCCGCAACCCTACACTGACCATCCAGCCCCGGCCCCATCCCTTCCGGAACATCAACTCCTCCTCCTCCTCCAGCTGGATGTGCCACGCCGTCGCCGCCGAGGTCGAGGGCCTCAACATCGCCGACGA---------CCTCATCGGCAAGACTCCAATGGTATATCTCAACAACATCGTCAAGGGATGTGTTGCCAATGTCGCTGCTAAGCTCGAGATTATGGAGCCCTGTTGCAGTGTCAAGGACAGGATAGGATACAGTATGATTTCTGATGCGGAAGAGAAAGGCTTGATAACTCCTGGAAAGCTCTCACCCAGTGACTTACCATGCATGACATATTACTTTATGCTCTATGCTCAGAGTGTTTTGGTGGAACCAACAAGTGGAAATACAGGCATTGGTCTTGCCTTCATTGCTGCTTCCAGAGGATATAAATTAATATTGACCATGCCTGCATCAATGAGCATGGAGAGAAGAGTTCTACTCAAAGCTTTTGGCGCTGAACTTGTCCTTACTGATGCCGCAAAAGGGATGAAGGGGGCTGTAGATAAGGCTACAGAGATTTTAAATAAGACACCTGATGCCTATATGCTGCAGCAGTTTGACAACCCTGCCAACCCAAAGGTACATTATGAGACTACTGGGCCAGAAATCTGGGAGGATTCTAAAGGGAAGGTGGATGTATTCATTGGTGGAATTGGAACAGGTGGAACAATATCTGGTGCTGGCCGTTTCCTGAAAGAGAAAAATCCTGGAATTAAGGTTATTGGTATTGAGCCTTCTGAGAGTAACATACTCTCTGGTGGAAAACCTGGCCCACATAAGATTCAAGGCATTGGGGCAGGATTTGTTCCAAGGAACTTGGATAGTGAAGTTCTCGATGAAGTGATTGAGATATCTAGTGATGAGGCTGTTGAGACAGCAAAGCAATTGGCTCTTCAGGAAGGATTACTGGTTGGAATTTCATCTGGGGCAGCAGCAGCAGCTGCCATTAAAGTTGCAAAAAGACCAGAAAATGCTGGAAAGTTGGTAGTGGTTGTGTTTCCAAGCTTTGGTGAGAGGTACCTTTCATCTATCCTTTTTCAGTCGATAAGAGAAGAATGTGAGAAGTTGCAACCTGAACCATGA---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
>transcript:Os01t0978100-03 gene:Os01g0978100 1:43232036-43237974(-) [Oryza_sativa.IRGSP-1.0.chr1]
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CAGATACGGAACTACTCCCCTATCCAACACCTCCGAGTCCGAGCAACGCAAGATGGCGTCGTGGTCGTCGCCCGTCGCCGCCGCCGCCTTGCAGGTCCATTTCGGGTCCTCCTGCTTCTTCTCCGCCCGATCGCCACGACAGACCCTCCTCCTACCACCTCTCGCCCGCAACCCTACACTGACCATCCAGCCCCGGCCCCATCCCTTCCGGAACATCAACTCCTCCTCCTCCTCCAGCTGGATGTGCCACGCCGTCGCCGCCGAGGTCGAGGGCCTCAACATCGCCGACGACGTCACCCAGCTCATCGGCAAGACTCCAATGGTATATCTCAACAACATCGTCAAGGGATGTGTTGCCAATGTCGCTGCTAAGCTCGAGATTATGGAGCCCTGTTGCAGTGTCAAGGACAGGATAGGATACAGTATGATTTCTGATGCGGAAGAGAAAGGCTTGATAACTCCTGGAA------------------------------------------------------AGAGTGTTTTGGTGGAACCAACAAGTGGAAATACAGGCATTGGTCTTGCCTTCATTGCTGCTTCCAGAGGATATAAATTAATATTGACCATGCCTGCATCAATGAGCATGGAGAGAAGAGTTCTACTCAAAGCTTTTGGCGCTGAACTTGTCCTTACTGATGCCGCAAAAGGGATGAAGGGGGCTGTAGATAAGGCTACAGAGATTTTAAATAAGACACCTGATGCCTATATGCTGCAGCAGTTTGACAACCCTGCCAACCCAAAGGTACATTATGAGACTACTGGGCCAGAAATCTGGGAGGATTCTAAAGGGAAGGTGGATGTATTCATTGGTGGAATTGGAACAGGTGGAACAATATCTGGTGCTGGCCGTTTCCTGAAAGAGAAAAATCCTGGAATTAAGGTTATTGGTATTGAGCCTTCTGAGAGTAACATACTCTCTGGTGGAAAACCTGGCCCACATAAGATTCAAGGCATTGGGGCAGGATTTGTTCCAAGGAACTTGGATAGTGAAGTTCTCGATGAAGTGATTGAGATATCTAGTGATGAGGCTGTTGAGACAGCAAAGCAATTGGCTCTTCAGGAAGGATTACTGGTTGGAATTTCATCTGGGGCAGCAGCAGCAGCTGCCATTAAAGTTGCAAAAAGACCAGAAAATGCTGGAAAGTTGGTAGTGGTTGTGTTTCCAAGCTTTGGTGAGAGGTACCTTTCATCTATCCTTTTTCAGTCGATAAGAGAAGAATGTGAGAAGTTGCAACCTGAACCATGAGCCTAACTTCAGTGTTCACAACATCATAATTGTTTCTGAGATTTCTGGCCATTAGTTTTTTTTTTCTGAGAAGTATCATACCACTCCATAGCTGTTTGTTCGATAAATAAAACAGTTACCTTTGCACTTATAATGAGGCTTGTGAGGGTACTGTGAAATTTCTCTGAACATCTTCTACTCTTCTCTTTTATCCTTAAATCAATCTGGGAGCAGTTTGTAATACATACGTAAATTTAAAGCTGGGTGTTTGGTAATTGTAAACAAATGTTTCGAAGAGCCGTGAAACATTATCAATTAGCATGAAGCACTTTAAAAGTGCTTTCC---------
>transcript:ONIVA01G52180.2 gene:ONIVA01G52180 1:42818942-42824598(-) [Oryza_nivara_v1.chr1]
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ATGGCGTCGTGGTCGTCGCCCGTCGCCGCCGCCGCCTTGCAGGTCCATTTCGGGTCCTCCTGCTTCTTCTCCGCCCGATCGCCACGACAGACCCTCCTCCTACCACCTCTCGCCCGCAACCCTACACTGACCATCCAGCCCCGGCCCCATCCCTTCCGGAACATCAACTCCTCCTCCTCCTCCAGCTGGATGTGCCACGCCGTCGCCGCCGAGGTCGAGGGCCTCAACATCGCCGACGA---------CCTCATCGGCAAGACTCCAATGGTATATCTCAACAACATCGTCAAGGGATGTGTTGCCAATGTCGCTGCTAAGCTCGAGATTATGGAGCCCTGTTGCAGTGTCAAGGACAGGATAGGATACAGTATGATTTCTGATGCGGAAGAGAAAGGCTTGATAACTCCTGGAAAGCTCTCACCCAGTGACTTACCATGCATGACATATTACTTTATGCTCTATGCTCAGAGTGTTTTGGTGGAACCAACAAGTGGAAATACAGGCATTGGTCTTGCCTTCATTGCTGCTTCCAGAGGATATAAATTAATATTGACCATGCCTGCATCAATGAGCATGGAGAGAAGAGTTCTACTCAAAGCTTTTGGCGCTGAACTTGTCCTTACTGATGCCGCAAAAGGGATGAAGGGGGCTGTAGATAAGGCTACAGAGATTTTAAATAAGACACCTGATGCCTATATGCTGCAGCAGTTTGACAACCCTGCCAACCCAAAGGTACATTATGAGACTACTGGGCCAGAAATCTGGGAGGATTCTAAAGGGAAGGTGGATGTATTCATTGGTGGAATTGGAACAGGTGGAACAATATCTGGTGCTGGCCGTTTCCTGAAAGAGAAAAATCCTGGAATTAAGGTTATTGGTATTGAGCCTTCTGAGAGTAACATACTCTCTGGTGGAAAACCTGGCCCACATAAGATTCAAGGCATTGGGGCAGGATTTGTTCCAAGGAACTTGGATAGTGAAGTTCTCGATGAAGTGATTGAGATATCTAGTGATGAGGCTGTTGAGACAGCAAAGCAATTGGCTCTTCAGGAAGGATTACTGGTTGGAATTTCATCTGGGGCAGCAGCAGCAGCTGCCATTAAAGTTGCAAAAAGACCAGAAAATGCTGGAAAGTTGGTAGTGGTTGTGTTTCCAAGCTTTGGTGAGAGGTACCTTTCATCTATCCTTTTTCAGTCGATAAGAGAAGAATGTGAGAAGTTGCAACCTGAACCATGAGC-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
The script check_evidence.pl, introduced in section
Quality metrics of clusters,
calls clustal-omega to perform such alignments.
If you installed GET-HOMOLOGUES earlier, you can also compute local BLAST alignments, coverage and sequence identity to the longest sequence in a cluster as follows:
get_homologues/annotate_cluster.pl -f test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1/gene:ONIVA01G52180.cdna.fna
get_homologues/annotate_cluster.pl -P -f test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1/gene:ONIVA01G52180.cds.faa
In addition to running and inspecting sequence alignments of pangene clusters, script check_quality.pl can produce quality reports of pangene clusters. Note it requires the installation of optional software, clustal-omega and AliStat, which can be done as follows:
cd .. && make install_pangenes_quality
The automatic report includes metrics based on multiple sequence alignment and the corresponding distance matrix. It can be produced as follows:
$ perl check_evidence.pl -d test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_ -i gene:ONIVA01G50850.cdna.fna
file 1stisof occup seqs mode_len SE_len mode_exons SE_exons mode_dist max_dist SE_dist sites Ca Cr_max Cr_min Cc_max Cc_min Cij_max Cij_min
gene:ONIVA01G50850.cdna.fna 0 3 6 2067 267.9 10 1.5 0.000000 0.776042 0.000000 2069 0.609554 0.999033 0.092799 0.833333 0.166667 0.880135 0.000000
The metrics Ca, Cr_max, Cr_min, Cc_max, Cc_min, Cij_max, Cij_min constitute the minimum reporting standard proposed by AliStat, where:
Ca - Completeness score for the alignment
Cr - Completeness score for individual sequences
Cc - Completeness score for individual sites
Cij - Completeness score for pairs of sequences
By default all isoforms are analyzed, but this can be changed with option -I, which will take the first isoform found in the GFF file. This choice is recorded in the second column of the output. Also, option -o can be used to save the multiple sequence alignment and distance matrix files in a folder.
In sections Pairwise genome comparisons and Example 1 we saw that collinear gene pairs are stored in TSV files. These files summarize the collinearity evidence supporting the produced gene clusters. For each get_pangenes.pl run these files are merged and sorted in a compressed TSV file such as
test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/mergedpairs.tsv.gz
It is possible to extract the collinearity evidence supporting selected clusters as follows:
$ perl check_evidence.pl -d test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_ -i gene:ONIVA01G50850.cdna.fna
# sequence-level stats
# isoform length in cluster: median=1239 mode(s): 1823,1239,1179,192
# short isoform: transcript:ONIVA01G50850.2 gene:ONIVA01G50850 [Oryza_nivara_v1.chr1] length=1067
# short isoform: transcript:ONIVA01G50860.1 gene:ONIVA01G50860 [Oryza_nivara_v1.chr1] length=192
# long isoform: transcript:Os01t0961600-01 gene:Os01g0961600 [Oryza_sativa.IRGSP-1.0.chr1] length=2067
# long isoform: transcript:Os01t0961600-02 gene:Os01g0961600 [Oryza_sativa.IRGSP-1.0.chr1] length=1823
# cluster gene:ONIVA01G50850.cdna.fna genes = 4 (3 taxa)
# re-using database
#gene_stable_id protein_stable_id species overlap homology_type homology_gene_stable_id homology_protein_stable_id homology_species overlap dn ds goc_score wga_coverage is_high_confidence coordinates
gene:ONIVA01G50850 gene:ONIVA01G50850 Oryza_nivara_v1.chr1 4539 ortholog_collinear gene:Os01g0961600 gene:Os01g0961600 Oryza_sativa.IRGSP-1.0.chr1 4539 NULL NULL NULL 100.00 1 1:42029287-42033826(-);1:42393585-42401178(-)
gene:ONIVA01G50860 gene:ONIVA01G50860 Oryza_nivara_v1.chr1 548 segment_collinear Oryza_indica.ASM465v1.chr1:1:46475865-46476413(-) segment Oryza_indica.ASM465v1.chr1 548 NULL NULL NULL 100.00 1 1:42033863-42034305(-);1:46475865-46476413(-)
gene:ONIVA01G50860 gene:ONIVA01G50860 Oryza_nivara_v1.chr1 442 ortholog_collinear gene:Os01g0961600 gene:Os01g0961600 Oryza_sativa.IRGSP-1.0.chr1 442 NULL NULL NULL 100.00 1 1:42033863-42034305(-);1:42393585-42401178(-)
gene:BGIOSGA000064 gene:BGIOSGA000064 Oryza_indica.ASM465v1.chr1 4598 ortholog_collinear gene:Os01g0961600 gene:Os01g0961600 Oryza_sativa.IRGSP-1.0.chr1 4598 NULL NULL NULL 100.00 1 1:46471290-46475888(-);1:42393585-42401178(-)
gene:BGIOSGA000064 gene:BGIOSGA000064 Oryza_indica.ASM465v1.chr1 4538 ortholog_collinear gene:ONIVA01G50850 gene:ONIVA01G50850 Oryza_nivara_v1.chr1 4538 NULL NULL NULL 100.00 1 1:46471290-46475888(-);1:42029287-42033826(-)
# gene-level stats
#len pairs overlap gene_name species
7594 3 9579 gene:Os01g0961600 Oryza_sativa.IRGSP-1.0.chr1
4599 2 9136 gene:BGIOSGA000064 Oryza_indica.ASM465v1.chr1
4540 2 9077 gene:ONIVA01G50850 Oryza_nivara_v1.chr1
443 1 442 gene:ONIVA01G50860 Oryza_nivara_v1.chr1
4569 2 9106 median values
Note this script builds a local BerkeleyDB database the first time is run, which takes a minute, so that subsequent calls run efficiently.
While in the previous section we described the evidence for pairs of overlapping gene models, the primary evidence of this algorithm are actually pairs of aligned genomic segments, which are stored in PAF format.
The following line, taken from file tmp/_Oryza_sativa.IRGSP-1.0.chr1.Oryza_indica.ASM465v1.chr1.minimap2.paf, shows a segment from chr1 in Oryza_sativa.IRGSP-1.0 aligned to a collinear segment in Oryza_indica.ASM465v1. The segments have coordinates 1:5902373-6068137 and 1:6345936-6511263, respectively. The last column is a CIGAR string that summarizes the actual alignment:
1 43270923 5902373 6068137 + 1 47283185 6345936 6511263 164108 166011 60 NM:i:2054 ms:i:157821 AS:i:158450 nn:i:151 tp:A:P cm:i:27339 s1:i:157194 s2:i:1845 de:f:0.0050 zd:i:2 rl:i:279032 cs:Z::5*ca:...
As depicted on Figure 3, gene models are placed within aligned collinear genomic segments to check whether they overlap across. During this process some genes might fail to be mapped. It is possible to see exactly which ones failed and the actual reason by inspecting the logs. The following lines, taken from log file _Oryza_nivara_v1.chr1.Oryza_sativa.IRGSP-1.0.chr1.algMmap.overlap0.5.id95.tsv.queue, indicate that 165 genes could not be confidently mapped, and the list below shows some examples:
# 4569 genes mapped (85.7% in 3+blocks) in tmp/_Oryza_sativa.IRGSP-1.0.chr1.Oryza_nivara_v1.chr1.minimap2.gene.mapped.bed (165 unmapped)
# unmapped: [sequence identity 91.949685534591195 < 95] 1 26030520 26031331 gene:Os01g0554400 888 + 91.95
# unmapped: [quality 37 < 50] 1 34167935 34171573 gene:Os01g0805900 9999 + 1 34170717 34171348 - 111145515 11146146 560 37 631
# unmapped: [overlap 54 < 100] 1 30059934 30059987 gene:Os01g0742150 53 + 98.89
This happens in function query2ref_coords within _collinear_genes.pl. Note that gene models might fail to map for having less sequence identity than $MINPERCID = 95 or less than $MINALNLEN = 100 aligned nucleotides or for mapping genomic regions aligned with poor quality (parameter -q).
It is often useful to check visually the genomic context of the genes in a pangene cluster. The script introduced in the previous section can be used for that. Note that it requires the BED-like pangene matrix obtained as explained in Example 3 and also the installation of pyGenomeViz. This is how you can run it:
perl check_evidence.pl -d Otest_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_split_ -i gene:ONIVA01G52020.cds.fna -P -n
After adding option -P the output should now include:
# write code for plotting cluster genomic context
# WARN: Cannot get strand of gene:ONIVA01G52010 , will plot as box, re-run get_pangenes.pl with options -t 0 -s
# log file: gene:ONIVA01G52020.cds.fna.plot.log.tsv
# plotting script file: gene:ONIVA01G52020.cds.fna.plot.py
# install if required: pip install pygenomeviz
# see other installation options at https://pypi.org/project/pygenomeviz
# run it as: python3 gene:ONIVA01G52020.cds.fna.plot.py
# will produce: gene:ONIVA01G52020.cds.fna.plot.png
Please follow the instructions to produce the plot. Note that you can change the last line of the generated Python script to change the default graphic format (PNG) to others such as PDF o SVG by simply changing the extension of the target file from .png to .pdf or .svg .
Figure 10. Genomic context of cluster gene:ONIVA01G52020.cds.fna, generated with script check_evidence.pl. Note that several genes might fit in the same slot, due to split gene models or to tandem copies.
The check_evidence.pl script can also be used to try and fix individual gene models based on the evidence supporting a pangene cluster. This requires the software GMAP, which is installed by default as explained in section Dependencies and installation. Currently, the following fixes have been tested:
| problem | hypothesis | proposed solution |
|---|---|---|
| long gene model | long model actually merges two single genes by mistake/overlapping RNAseq reads | liftover individual consensus models against genomic segment containing long gene |
| split gene model | the real gene is long and was split in 2+ partial genes | liftover consensus (longer) models on genomic segment containing both split models |
| missing gene model | gene model exists but failed to be annotated | liftover consensus models over matching genomic segment, used precomputed clusters with genomic segments, with extension .gdna.fna |
Figure 11. Flowchart of script check_evidence.pl -f
The following call shows an example cluster analyzed with argument -f (option -n avoids the TSV evidence to be printed):
perl check_evidence.pl -d test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_ -f -i gene:ONIVA01G25360.cds.fna
# sequence-level stats
# isoform length in cluster: median=297 mode(s): 297
# long isoform: transcript:ONIVA01G25360.2 gene:ONIVA01G25360 [Oryza_nivara_v1.chr1] length=408
# cluster gene:ONIVA01G25360.cds.fna genes = 5 (3 taxa)
# gene-level stats
#len pairs overlap gene_name species
7935 4 1678 gene:ONIVA01G25360 Oryza_nivara_v1.chr1
412 2 832 gene:Os01g0606200 Oryza_sativa.IRGSP-1.0.chr1
422 2 842 gene:BGIOSGA001248 Oryza_indica.ASM465v1.chr1
436 1 435 gene:BGIOSGA001246 Oryza_indica.ASM465v1.chr1
412 1 411 gene:BGIOSGA001247 Oryza_indica.ASM465v1.chr1
422 2 832 median values
# FIX PARAMETERS:
# -p 0 $MINPAIRPECNONOUTLIERS=0.25 $MINLIFTIDENTITY=95 $MINFIXOVERLAP=0.75 $MAXSEGMENTSIZE=100000
# long gene model: corrected gene:ONIVA01G25360 [Oryza_nivara_v1.chr1]
## replaces gene:ONIVA01G25360 [Oryza_nivara_v1.chr1] source=Oryza_indica.ASM465v1.chr1 matches=855 mismatches=18 indels=9
1 gmap gene 22440345 22440755 . - . ID=gene:BGIOSGA001247.path1;Name=gene:BGIOSGA001247;Dir=sense;old_locus_tag=gene:ONIVA01G25360;
1 gmap mRNA 22440345 22440755 . - . ID=gene:BGIOSGA001247.mrna1;Name=gene:BGIOSGA001247;Parent=gene:BGIOSGA001247.path1;Dir=sense;coverage=100.0;identity=99.0;matches=285;mismatches=3;indels=0;unknowns=0
1 gmap exon 22440692 22440755 98 - . ID=gene:BGIOSGA001247.mrna1.exon1;Name=gene:BGIOSGA001247;Parent=gene:BGIOSGA001247.mrna1;Target=gene:BGIOSGA001247 1 64 +
1 gmap exon 22440345 22440568 99 - . ID=gene:BGIOSGA001247.mrna1.exon2;Name=gene:BGIOSGA001247;Parent=gene:BGIOSGA001247.mrna1;Target=gene:BGIOSGA001247 65 288 +
1 gmap CDS 22440692 22440755 98 - 0 ID=gene:BGIOSGA001247.mrna1.cds1;Name=gene:BGIOSGA001247;Parent=gene:BGIOSGA001247.mrna1;Target=gene:BGIOSGA001247 1 64 +
1 gmap CDS 22440345 22440568 99 - 1 ID=gene:BGIOSGA001247.mrna1.cds2;Name=gene:BGIOSGA001247;Parent=gene:BGIOSGA001247.mrna1;Target=gene:BGIOSGA001247 65 288 +
1 gmap gene 22447844 22448278 . - . ID=gene:BGIOSGA001246.path1;Name=gene:BGIOSGA001246;Dir=sense;old_locus_tag=gene:ONIVA01G25360;
1 gmap mRNA 22447844 22448278 . - . ID=gene:BGIOSGA001246.mrna1;Name=gene:BGIOSGA001246;Parent=gene:BGIOSGA001246.path1;Dir=sense;coverage=100.0;identity=98.3;matches=292;mismatches=5;indels=0;unknowns=0
1 gmap exon 22448215 22448278 100 - . ID=gene:BGIOSGA001246.mrna1.exon1;Name=gene:BGIOSGA001246;Parent=gene:BGIOSGA001246.mrna1;Target=gene:BGIOSGA001246 1 64 +
1 gmap exon 22447844 22448076 97 - . ID=gene:BGIOSGA001246.mrna1.exon2;Name=gene:BGIOSGA001246;Parent=gene:BGIOSGA001246.mrna1;Target=gene:BGIOSGA001246 65 297 +
1 gmap CDS 22448215 22448278 100 - 0 ID=gene:BGIOSGA001246.mrna1.cds1;Name=gene:BGIOSGA001246;Parent=gene:BGIOSGA001246.mrna1;Target=gene:BGIOSGA001246 1 64 +
1 gmap CDS 22447844 22448076 97 - 1 ID=gene:BGIOSGA001246.mrna1.cds2;Name=gene:BGIOSGA001246;Parent=gene:BGIOSGA001246.mrna1;Target=gene:BGIOSGA001246 65 297 +
Arguments -o and -a can be used to append any GFF output to a patch GFF file which can be used downstream (see next section). Argument -v can be added to increase verbosity and see the raw GMAP alignments used while lifting-over features. Argument -p might be used to allow partial (not multiple of 3) CDS lifted-over by GMAP.
# locate cluster of interest
grep "^>" test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1/gene:ONIVA01G50620.cdna.fna
>transcript:ONIVA01G50620.6 gene:ONIVA01G50620 1:41880970-41884290(+) [Oryza_nivara_v1.chr1]
>transcript:ONIVA01G50620.1 gene:ONIVA01G50620 1:41880970-41888135(+) [Oryza_nivara_v1.chr1]
>transcript:ONIVA01G50620.2 gene:ONIVA01G50620 1:41880970-41888135(+) [Oryza_nivara_v1.chr1]
>transcript:ONIVA01G50620.3 gene:ONIVA01G50620 1:41880970-41888135(+) [Oryza_nivara_v1.chr1]
>transcript:ONIVA01G50620.4 gene:ONIVA01G50620 1:41882945-41888135(+) [Oryza_nivara_v1.chr1]
>transcript:ONIVA01G50620.5 gene:ONIVA01G50620 1:41882945-41888135(+) [Oryza_nivara_v1.chr1]
>transcript:Os01t0958200-01 gene:Os01g0958200 1:42238811-42240420(+) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:Os01t0958400-01 gene:Os01g0958400 1:42240825-42244043(+) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:Os01t0958400-02 gene:Os01g0958400 1:42240837-42244651(+) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:Os01t0958400-03 gene:Os01g0958400 1:42240858-42245248(+) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:BGIOSGA005213-TA gene:BGIOSGA005213 1:46293601-46294992(+) [Oryza_indica.ASM465v1.chr1]
>transcript:BGIOSGA005214-TA gene:BGIOSGA005214 1:46296594-46299549(+) [Oryza_indica.ASM465v1.chr1]
# fix a gene model and save GFF patch in folder patch/
perl check_evidence.pl -d test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_/ -i gene:ONIVA01G50620.cdna.fna -f -o patch/
# copy patch GFF files to same input dir used in original run (Example 1)
cp patch/*.patch.gff ../files/test_rice/
# re-run pan-gene analysis using GFF patches
perl get_pangenes.pl -d ../files/test_rice/ -p -m
# check how the updated clusters look like
grep "^>" test_rice_pangenes/Oryza_nivara_v1chr1_patch_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1/gene:BGIOSGA005213.path1.cdna.fna
>gene:BGIOSGA005213.mrna1 gene:BGIOSGA005213.path1 1:41880970-41882361(+) [Oryza_nivara_v1.chr1]
>transcript:Os01t0958200-01 gene:Os01g0958200 1:42238811-42240420(+) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:BGIOSGA005213-TA gene:BGIOSGA005213 1:46293601-46294992(+) [Oryza_indica.ASM465v1.chr1]
grep "^>" test_rice_pangenes/Oryza_nivara_v1chr1_patch_alltaxa_5neigh_algMmap_/Oryzanivarav1.chr1/gene:BGIOSGA005214.path1.cdna.fna
>gene:BGIOSGA005214.mrna1 gene:BGIOSGA005214.path1 1:41883977-41886935(+) [Oryza_nivara_v1.chr1]
>transcript:Os01t0958400-01 gene:Os01g0958400 1:42240825-42244043(+) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:Os01t0958400-02 gene:Os01g0958400 1:42240837-42244651(+) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:Os01t0958400-03 gene:Os01g0958400 1:42240858-42245248(+) [Oryza_sativa.IRGSP-1.0.chr1]
>transcript:BGIOSGA005214-TA gene:BGIOSGA005214 1:46296594-46299549(+) [Oryza_indica.ASM465v1.chr1]
The _dotplot.pl script can be used to make a genome-wide dotplot of collinear gene models resulting from a pairwise WGA, which are stored in TSV format (see relevant section). This can be done in two steps- First, the TSV file must be converted to a PAF file as follows:
perl _dotplot.pl test_rice_pangenes/_Oryza_nivara_v1.chr1.Oryza_sativa.IRGSP-1.0.chr1.algGSal.overlap0.5.tsv
The second step involves executing a few output R lines in order to actually produce the plot below; note that this requires installing the pafr R package:
# $MINCONTIGSIZE = 100000
# PAF file: test_rice_pangenes/_Oryza_nivara_v1.chr1.Oryza_sativa.IRGSP-1.0.chr1.algGSal.overlap0.5.genes.paf
# Make a dotplot of aligned models coords with the following R script:
#https://dwinter.github.io/pafr/articles/Introduction_to_pafr.html
#install.packages(devtools)
#devtools::install_github("dwinter/pafr")
library(pafr, quietly=TRUE)
pafile = "test_rice_pangenes/_Oryza_nivara_v1.chr1.Oryza_sativa.IRGSP-1.0.chr1.algGSal.overlap0.5.genes.paf"
ali <- read_paf(pafile)
dotplot(ali, label_seqs = TRUE, xlab='Oryza_nivara_v1.chr1', ylab='Oryza_sativa.IRGSP-1.0.chr1')
Figure 11. Dotplot of collinear genes in chr1 of Oryza_nivara_v1 and Oryza_sativa.IRGSP-1.0
check_evidence.pl check_evidence.pl check_evidence.pl
If you have a set of precomputed pangene clusters you might want to assign arbitrary sequences to them without recomputing new pangenes. For instance, these sequences could be transcripts from a new cultivar/genotype not considered before. You can do this with script match_cluster.pl, which creates a sequence index with nucleotide sequences from clusters and uses GMAP to scan them. By default it uses cDNA sequences to build the index, but can also use CDS (-C). The next example shows how to do this:
perl match_cluster.pl -d test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_5neigh_algMmap_ -s ../files/test_transcripts.fna -o test_transcripts.gmap.tsv
This will produce TAB-separated (TSV) output similar to this:
#query qlength pangene length matches perc_qcover perc_identity coords
TR2 NA NA NA NA NA NA NA
TR1 NA NA NA NA NA NA NA
TR4 1768 gene:ONIVA01G40940.cdna.fna 1820 1 100 97.1 1:35501996-35505052(-)
TR3 890 gene:ONIVA01G42530.cdna.fna 617 2 69 87.4 1:35899105-35900529(+)
The column 'matches' indicates how many individual alignments support the assignment of the input sequence to the same cluster, which in the last example is 2. Note that potentially a sequence could match more than one cluster.
Before you run this script it might be handy to add genomic coordinates to the headers of the input FASTA file, so that you can quickly check whether they match clusters in the same chromosome.
The standard output of get_pangenes.pl can inform you about potential errors. For instance, please pay attention to the number of genes parsed from each input GFF file. If one of them yields 0 genes, it might be due to a lack of 'gene' records. This can be fixed with
plant-scripts/pangenes/bin/gffread/gffread --keep-genes geneless.gff > genes.gff
If you encounter an error, or the program stops, it is useful to look for error messages in the logfiles. As get_pangenes.pl includes 3 other scripts, logs are split in independent files:
| script | example logfile |
|---|---|
| _cut_sequences.pl | test_rice_pangenes/_Oryza_nivara_v1.chr1.fa.gz.queue |
| _collinear_genes.pl | test_rice_pangenes/_Oryza_indica.ASM465v1.chr1.Oryza_nivara_v1.chr1.algMmap.overlap0.5.tsv.queue |
| _cluster_analysis.pl | test_rice_pangenes/Oryza_nivara_v1chr1_alltaxa_algMmap_.queue |
The main log of get_pangenes.pl might contain error messages such as: 1125 /homes/bcontreras/panoryza/plant-scripts/pangenes/bin/gffread/gffread --keep-genes BarkeBaRT2v18.gff |less
-
EXIT, folder_pangenes/_oryza_sativa_arc.oryza_sativa_chaomeo.algMmap.overlap0.5.patch.tsv does not exist, WGA might have failed or hard drive is still writing it (please re-run). This can happen in HPC cluster jobs due to drive latency issues. The fix is to open the relevant specific log (_collinear_genes.pl in this case) and look for the failing command, which in this example looks like:
plant-scripts/pangenes/_collinear_genes.pl -sp1 oryza_sativa_arc -fa1 folder_pangenes/_oryza_sativa_arc.fna -gf1 folder_pangenes/_oryza_sativa_arc.patched.gff -sp2 oryza_sativa_chaomeo -fa2 folder_pangenes/_oryza_sativa_chaomeo.fna -gf2 folder_pangenes/_oryza_sativa_chaomeo.patched.gff -out folder_pangenes/_oryza_sativa_arc.oryza_sativa_chaomeo.algMmap.overlap0.5.patch.tsv -p 1 -a 1 -ovl 0.5 -q 50 -wf 0 -gs 0 -A -c 0 -s '' -M plant-scripts/pangenes/../lib/minimap2/minimap2 -W plant-scripts/pangenes/bin/wfmash/build/bin/wfmash -G ./ -B bedtools -T folder_pangenes/tmp/ -t 4 -i 0 -r 1 -H 0
Then, the failing command should be run locally as follows:
$ plant-scripts/pangenes/_collinear_genes.pl -sp1 oryza_sativa_arc -fa1 folder_pangenes/_oryza_sativa_arc.fna -gf1 folder_pangenes/_oryza_sativa_arc.patched.gff -sp2 oryza_sativa_chaomeo -fa2 folder_pangenes/_oryza_sativa_chaomeo.fna -gf2 folder_pangenes/_oryza_sativa_chaomeo.patched.gff -out folder_pangenes/_oryza_sativa_arc.oryza_sativa_chaomeo.algMmap.overlap0.5.patch.tsv -p -a -M plant-scripts/pangenes/../lib/minimap2/minimap2 -B bedtools -T folder_pangenes/tmp/ -r
The log of _cluster_analysis.pl might contain warnings like these:
-
WARN: skip gene model XXX as it lacks cDNA (no coordinates)
-
WARN: merged clusters XXX & YYY (40,10,4). Explanation: two clusters containing 10 and 4 sequences from different species (disjoint) were merged. A total of 40 collinear gene pairs are supported by WGA evidence. See global variable $MINEDGESTOMERGE above.
-
WARN: partially overlapping clusters XXX & YYY (2,3,2): Explanation: two clusters containing 3 and 2 sequences from different species (disjoint) were not merged as only 2 collinear gene pairs are supported by WGA evidence. See global variable $MINEDGESTOMERGE above.
-
WARN: conflicting clusters ZZZ & YYY (Oryza_indica). Explanation: two individual clusters have WGA evidence connecting their genes but each have 1+ sequences from the same species, preventing the merge.
-
WARN: remove ZZZ from cluster XXX (46). This happens when a non-neighbor gene is removed from a cluster for having too many intervening genes in between.
This prototype was produced as part of the project "PanOryza: globally coordinated genomes, proteomes and pathways for rice", funded by BBSRC.NSF/BIO.
The paper describing this protocol is:
Contreras-Moreira B, Saraf S, Naamati G, Casas AM, Amberkar SS, Flicek P, Jones AR & Dyer S (2023) GET_PANGENES: calling pangenes from plant genome alignments confirms presence-absence variation. Genome Biol 24, 223. https://doi.org/10.1186/s13059-023-03071-z
Check all the references you need to cite in each script by running:
perl get_pangenes.pl -v
perl check_evidence.pl -c
perl check_quality.pl -c
perl match_cluster.pl -c