ResynPy is a time-efficient pipeline written in Python that interrogates the genotyping data of a segregating population of plants, aiming to detect individuals with complementary genotyping profiles. This tool has been created with the aim to automatize, in a time-efficient way, the selection process of individuals as part of the Resynthesis method, as explained by Eduardo et al. (2020). The aim of Resynthesis is to obtain a variety that is a slightly modified variant of an elite commercial line but with new traits of agronomic importance. This method can greatly reduce the required time of variety production in plant species with high intergeneration periods, such as fruit trees.
Apart from python, all the other packages are optional for the user to install. These packages are required for the graphical representation of data generated by the pipeline.
- python (version 3.8.10 or higher)
- pandas (version 0.25.3 or higher)
- matplotlib (version 3.1.2 or higher)
- seaborn (0.11.2 or higher)
Note for Windows users: instead of using the Command Prompt window, a user can also use the "Terminal" tab in the lower left area of the Rstudio environment.
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Python installation
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Pandas, matplotlib and seaborn installation (Linux/Windows)
install pandas:
pip install pandasinstall matplotlib:
pip install matplotlibinstall seaborn:
pip install seaborn
ResynPy performs a pairwise individual comparison of the genotyping data of a segregating population for detecting complementary pairs of individuals.
To see all the available options for running the pipeline, type python (or python3) ResynPy.py -h:
usage: ResynPy.py [-h] [--genos TAB FILE] [--markers TAB FILE] [--results_dir STR] [--not_phased] [--scores_file FILE] [--invariable FLOAT] [--hetero FLOAT] [--out_prefix STR]
Analysis of F2 genotyping data for detection of highly complementary individuals.
optional arguments:
-h, --help show this help message and exit
--genos TAB FILE Tab-delimited file (.tab) containing the genotyping data of the F2 population. Markers should be in columns and individuals in rows. Genotypes should be in the format "A,B,H,-", where "-" represents missing data. This argument has to be used together with
--markers. [Required]
--markers TAB FILE A 2-column tab-delimited file with the markers used for the F2 genotyping, in the format of "chromosome<tab>marker name". Marker order per chromosome should match that of markers in the genotyping file. [Required]
--scores_file FILE A tab-delimited file containing user-defined scores for the different combinations of genotypes, assigned during the comparison of the individuals. If not one available, the user can use the scores_default.tab file found in the downloaded github directory.
[Default: ResynPy/scores_default.tab]
--results_dir STR Name of the results directory [Default: ResynPy_results]
--not_phased Use this argument if your genotyping data are not phased. [Default: FALSE]
--invariable FLOAT Keep individual pairs that have a ratio of AA or BB combinations that are lower than FLOAT. [Default: 0.01]
--score_threshold FLOAT
Keep individual pairs with total score ratio higher than FLOAT. [Default: 0.8]
--hetero FLOAT Keep individuals that have heterozygosity ratio lower than FLOAT. [Default: 0.5]
--out_prefix STR Prefix for the output files.[Default: resynpyOut]
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Example data provided in the repository:
SDmarkers.tab: markers fileSD_F2data.tab: genotyping file with data of 62 markers from 418 individuals, originating from Eduardo et al., 2020.
During this process, the genotypic profile of each individual is compared with the corresponding profile of the rest of the individuals in the F2 population. Each pair of genotypes in a specific marker has a score assigned to it. All scores assigned at each genotype pair are summed up and the total score is tagged to the respective pair of individuals. The file scores_default.tab provides scores for each combination of genotypes. The user can also provide a tab-delimited file with different scores (see below). For the pairwise comparison analysis, the user should provide a file with the genotyping data (--genos) and a file with markers information (--markers) (see below). The pipeline considers that genotyping data are phased. If data are not phased then the user should declare it by using the argument --not_phased.
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Example command:
python ResynPy.py \ --genos SD_F2data.tab \ --markers SDmarkers.tab \ [--results_dir ResynPy_results] \ [--scores_file scores_default.tab] \ [--filter_invariable 0.01] \ [--hetero 0.5] \ [--not_phased]
--genos: A tab-delimited file with individuals in rows and markers in columns. Markers should be ordered first by chromosome and then by their physical or genetic position. The individual column should have a title. Data should be in A, B, H format; i.e. "A" represents the homogyzous genotype for one allele, "B" is the homozygous genotype for the other allele and "H" is the heterozygous genotype. Missing data should be represented as "-".
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example of genotyping data.
indv SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 SNP8 SNP9 SNP10 ... indv_1 A - A - B A A H B B ... indv_2 A - B - B H B H H H ... indv_3 A - H - B A B A H B ... indv_4 B - H - A H H A H B ... ... .... indv_n A - H - H H A H A A ...
--markers: A 2-column tab-delimited file. Column 1: marker name used for genotyping the population. Column 2: chromosome the marker belongs to. The marker order should match that of the genotyping file.
--not_phased: Should be used if genotyping data are not phased. The use of this argument will cause the exclusion of recombination information from the output file (see Output data Section).
--scores_file: A 2-column tab-delimited file containing scores assigned to each possible genotype combination, when two individuals are compared. Column 1: genotype pair, Column 2: score. By default, the pipeline will use the score information present inside the file scores_default.tab, provided in the repository's main directory. However if the user wants to apply a different scoring, a tab-delimited file can be provided by using this argument.
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contents of
scores_default.tab:AA 0 BB 0 -- 0.5 HH 0.5 H- 0.625 A- 0.75 B- 0.75 AH 0.75 BH 0.75 AB 1
--invariable: We also offer the possibility to filter out pairs of individuals that have a number of genotype identities (A/A or B/B) higher than the threshold float value set by this argument. The use of this argument can speed up the whole comparison process since the pipeline does not proceed to score assignment if the pair is discarded. In theory, there shouldn't be any genotype identity between the two individuals, since the presence of genotype identity implies that the corresponding region of the genome will not contribute to obtaining the near-identical variety when the two individuals are crossed. Therefore, the value of this argument should be as low as possible. Default value: 0.01
--hetero: A value defining the maximum level of heterozygosity for any individual in the pair. Low heterozygosity of an individual increases the probability to find a matching individual with a complementary genotype in the following generation. Default value: 0.5
--out_prefix: Prefix to be used for all the output files. Default value: resynpyOut
All the output files are saved into the --results_dir directory (Default results directory: ResynPy_results).
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selected_pairs.tabA file containing all the accepted pairs of individuals. This file can contain up to seven columns.
column 1: selected pair of individuals
column 2: total score of genotyping
column 3: total number of invariable pairs (AA or BB)
column 4: (heterozygosity in individual 1)|(heterozygosity in individual 2)
column 5: Range of similarity of the hybrid, generated by the selected pair, to the initial hybrid genotype
#### If data are phased, the following three columns are included in the output:
column 6: number of recombinations present in the first individual
column 7: number of recombinations present in the second individual
column 8: total number of recombinations in the pair
Example of the output file for unphased data:
#indiv_pair score invariable_pairs hetero1|hetero2 similarity_to_hybrid SDF2-343|SDF2-386 55.0 0 0.23|0.20 66.13-100.00 SDF2-37|SDF2-217 53.75 0 0.26|0.27 56.45-100.00 SDF2-131|SDF2-180 52.875 0 0.31|0.27 43.55-100.00 SDF2-28|SDF2-365 52.25 0 0.24|0.37 50.00-100.00 SDF2-200|SDF2-217 52.0 0 0.36|0.27 45.16-100.00Example of the output file for phased data:
#indiv_pair score invariable_pairs hetero1|hetero2 similarity_to_hybrid recomb_no1 recomb_no2 sum_recomb SDF2-343|SDF2-386 55.0 0 0.23|0.20 66.13-100.00 0 0 0 SDF2-37|SDF2-217 53.75 0 0.26|0.27 56.45-100.00 0 0 0 SDF2-131|SDF2-180 52.875 0 0.31|0.27 3.55-100.00 1 0 1 SDF2-28|SDF2-365 52.20 0 0.24|0.37 50.00-100.00 2 0 2 SDF2-200|SDF2-217 52.0 0 0.36|0.27 45.16-100.00 1 0 1 -
discarded_individuals_and_pairs.tabA file containing all individuals and/or pairs that have not met the filters set by the user, supplemented with the parameter that did not meet the criteria and its value.
Example:
PR20CG15-1840-3988 heterozygosity: 0.43
PR20CG15-1840-3995 heterozygosity: 0.46
PR20CG15-1840-002|PR20CG15-1840-003 invariable genotype ratio: 0.27
PR20CG15-1840-002|PR20CG15-1840-004 invariable genotype ratio: 0.23
PR20CG15-1840-002|PR20CG15-1840-204 pair score: 19.75
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<prefix>.png,<prefix>.pdfA figure showing the haplotypes of the individuals in the first 10 selected pairs, in a colour-coded mode.
<prefix>corresponds to argument--out_prefix. -
PairComparison.logLog file with the arguments used by the user, the different steps implemented by the pipeline and counts of accepted and discarded individuals and pairs. When the user runs again the pipeline the file is appended with the new information. This can help the user to evaluate different parameters of the pipeline.