Full parameters
This page provides the full options and parameters for each subcommand, along with additional explanations and links where necessary. The available subcommands can be viewed using the command:
hybsuite -h/--help
or:
bash <the path to HybSuite.sh> -h/--help
Parameters for running hybsuite stage1
Stage 1 Manual
--------------------------------------------------------------------------------
Usage: hybsuite stage1 ...
Mandatory arguments: -input_list -input_data (required when including user-provided data) -output_dir
Essential arguments: -sra_maxsize -NGS_dir -nt -process
Arguments for inputs:
-input_list <FILE> The file listing input sample names and corresponding data types. (Default: None)
-input_data <DIR> The directory containing all input data (required when the inputs include your own data / pre-assembled data). (Default: None).
Arguments for outputs:
-output_dir <DIR> The output directory for all pipeline results (better to be consistent across all stages). (Default: None)
-NGS_dir <DIR> The output directory containing raw and cleaned reads files (Default: <output_dir>/NGS_dataset).
Notes: Pre-existing cleaned reads will skip reads trimming steps.
General arguments:
=== Threads control ===
-nt <INT|AUTO> Global thread setting. (Default: 1)
-nt_fasterq_dump <INT>
fasterq-dump threads. (Default: 1)
-nt_pigz <INT> pigz compression threads. (Default: 1)
-nt_trimmomatic <INT> Trimmomatic threads. (Default: 1)
=== Parallel control ===
-process <INT|all> Number of public data downloading and raw reads trimming to run concurrently. (Default: 1)
"all" means running all samples concurrently. (be cautious to set this option)
=== Public raw reads doenloading control ===
-rm_sra <TRUE/FALSE> Whether to remove SRA files after conversion. (Default: TRUE)
-download_format <fastq|fastq_gz>
Downloaded data format. (Default: fastq_gz)
=== Logfile Control ===
-log_mode <simple|cmd|full>
The output mode of hybsuite logfile. (Default: cmd)
Arguments for integrated tools:
=== SRAToolkit ===
-sra_maxsize <NUM> The maximum size of sra files to download. (Default: 20GB)
=== Trimmomatic ===
-trimmomatic_leading_quality <3-40>
Leading base quality cutoff. (Default: 3)
-trimmomatic_trailing_quality <3-40>
Trailing base quality cutoff. (Default: 3)
-trimmomatic_min_length <36-100>
Minimum read length. (Default: 36)
-trimmomatic_sliding_window_s <4-10>
Sliding window size. (Default: 4)
-trimmomatic_sliding_window_q <15-30>
Window average quality. (Default: 15)
Command example:
# Run HybSuite stage1 with 1 thread and 1 parallel processing
$ hybsuite stage1 -input_list ./input_list.txt -input_data ./Input_data -NGS_dir ./NGS_dir -output_dir ./
# Run HybSuite stage1 with 5 threads and 5 parallel processing
$ hybsuite stage1 -input_list ./input_list.txt -input_data ./Input_data -NGS_dir ./NGS_dir -output_dir ./ -nt 5 -process 5
Parameters for running hybsuite stage2
Stage 2 Manual
--------------------------------------------------------------------------------
Usage: hybsuite stage2 ...
Mandatory arguments: -input_list -NGS_dir -t -output_dir
Essential arguments: -eas_dir -seqs_min_length -seqs_min_sample_coverage -nt -process
Arguments for inputs:
-input_list <FILE> The file listing input sample names and corresponding data types used in stage 1. (Default: None)
-input_data <DIR> The directory containing all input data (in this stage, only required when the inputs include pre-assembled data). (Default: None).
-NGS_dir <DIR> The directory containing NGS raw and cleaned reads files (generated in stage 1). (Default: ./NGS_dir)
-t <FILE> Target file for data assembly. (follows the format required in HybPiper)
Arguments for outputs:
-output_dir <DIR> The Output directory for all pipeline results (better to be consistent across all stages). (Default: None)
-eas_dir <DIR> The output directory containing HybPiper assembly sequences. (Default: <output_dir>/01-Assembled_data)
Note: Pre-existing data in this directory will skip redundant assembly steps.
General arguments:
=== Putative paralogs filtering control ===
-seqs_min_length <INT>
Minimum sequence length for filtered paralogs. (Default: 0)
Putative paralogs shorter than this value will be filtered.
-seqs_mean_length_ratio <0-1>
Minimum sequence length ratio relative to the mean value per locus for putative paralogs. (Default: 0)
Putative paralogs shorter than this percentage of the maximum length will be filtered.
-seqs_max_length_ratio <0-1>
Minimum length ratio relative to the longest value per locus for putative paralogs. (Default: 0)
Putative paralogs shorter than this percentage of the maximum length will be filtered.
-seqs_min_sample_coverage <0-1>
Minimum sample coverage for putative paralogs. (Default: 0)
For all putative paralogs in stage 2, HRS and RLWP sequences in stage 3, loci lower than this sample coverage will be filtered.
-seqs_min_locus_coverage <0-1>
Minimum locus coverage for putative paralogs. (Default: 0)
For all putative paralogs in stage 2, taxa (samples) with lower than this locus coverage will be filtered.
=== Heatmap control ===
-heatmap_color {black,blue,red,green,purple,orange,yellow,brown,pink}
Color scheme for heatmap gradient. (Default: black)
=== Threads control ===
-nt <INT|AUTO> Global thread setting (Default: 1)
-nt_hybpiper <INT> HybPiper threads (Default: 1)
=== Parallel control ===
-process <INT|all> Number of data assembly ('hybpiper assemble') to run concurrently (Default: 1)
"all" means running all samples concurrently (be cautious to set this option)
=== Logfile control ===
-log_mode <simple|cmd|full>
The output mode of hybsuite logfile. (Default: cmd)
Arguments for integrated tools:
=== HybPiper ===
-hybpiper_mapping_tool <blast|diamond>
The tool used for mapping reads to targets in HybPiper (only for protein targets) (Default: blast)
-hybpiper_check_chimeric_contigs <FALSE|TRUE>
Check whether a stitched contig is a potential chimera of contigs from multiple paralogs when running "hybpiper assemble". (Default: TRUE)
-hybpiper_cov_cutoff <INT>
Specify the value of "-cov_cutoff" when running "hybpiper assemble" in Stage 2. (Default: 8)
Increasing this value may increase the loci recovery efficiency but potentially introducing errors.
Command example:
# Run HybSuite stage2 with filtering paralog sequences
$ hybsuite stage2 -NGS_dir ./NGS_dir -t ./Angiosperms353.fasta -output_dir ./ -nt 5 -process 5 -seqs_min_length 100 -seqs_min_sample_coverage 0.1
# Run HybSuite stage2 without filtering paralog sequences
$ hybsuite stage2 -NGS_dir ./NGS_dir -t ./Angiosperms353.fasta -output_dir ./ -nt 5 -process 5
Parameters for running hybsuite stage3
Stage 3 Manual
--------------------------------------------------------------------------------
Usage: hybsuite stage3 ...
Mandatory arguments: -input_list -eas_dir -paralogs_dir -t -output_dir
Essential arguments: -PH -prefix -run_phyparts -aln_min_sample -nt -process
Arguments for inputs:
-input_list <FILE> The file listing input sample names and corresponding data types used in stage 1&2. (Default: None)
-input_data <DIR> The directory containing all input data (in this stage, only required when the inputs include pre-assembled data). (Default: None).
-eas_dir <DIR> The output directory containing HybPiper assembly sequences (generated in stage 3). (Default: <output_dir>/01-Assembled_data)
-paralogs_dir <DIR> The directory containing all paralog sequences generated in stage 2 or by users themselves. (Default: None)
It's advisable to set this parameter as '<output_dir>/02-All_paralogs/03-Filtered_paralogs'.
-t <FILE> Target file for data assembly. (follows the format required in HybPiper)
Arguments for outputs:
-output_dir <DIR> Output directory for all pipeline results (better to be consistent across all stages). (Default: None)
-prefix <STRING> Prefix for output files. (Default: HybSuite)
General arguments:
=== Paralog handling control ===
-PH <1-7|a|b|all> Paralog handling methods to execute: (one or more of them can be chosen)
1: HRS, 2: RLWP, 3: LS, 4: MI, 5: MO, 6: RT, 7: 1to1
a: PhyloPyPruner, b: ParaGone (Default: 1a)
=== Sequences and alignments filtering control ===
-seqs_min_length <INT>
Minimum sequence bp length for filtering HRS and RLWP sequences. (Default: 0)
HRS and RLWP sequences shorter than this value will be removed.
-aln_min_length <INT>
Minimum sequence bp length for filtering HRS and RLWP final alignments. (Default: 4)
-aln_min_sample <INT>
Minimum sample number for final alignments. (Default: 0)
Final alignments (aligned and trimmed) with sample number below this threshold will be removed.
=== Gene tree builder control ===
-gene_tree <1/2> Choose the software to construct paralogs gene trees. (1: IQ-TREE; 2: FastTree) (Default: 1)
-gene_tree_bb <INT> Choose the bootstrap value for paralogs gene trees inference. (Default: 1000)
=== Alignments trimming tool control ===
-trim_tool <1/2> Choose the software to trim/clean alignments. (1: trimAl; 2: HMMCleaner) (Default: 1)
=== Nucleotide ambiguity character replacement ===
-replace_n <TRUE|FALSE>
Replace ambiguous characters ('n', 'N', '?') with gaps ('-') in alignment files. (Default: FALSE)
Note: Recommended for phylogenetic software compatibility (e.g., IQ-TREE, trimAl).
=== Threads control ===
-nt <INT|AUTO> Global thread setting. (Default: 1)
-nt_paragone <INT> ParaGone threads. (Default: 1)
-nt_phylopypruner <INT>
PhyloPyPruner threads. (Default: 1)
-nt_mafft <INT> MAFFT threads. (Default: 1)
-nt_amas <INT> AMAS.py threads. (Default: 1)
-nt_modeltest_ng <INT>
ModelTest-NG threads. (Default: 1)
-nt_iqtree <INT> IQ-TREE threads. (Default: 1)
-nt_fasttree <INT> FastTree threads. (Default: 1)
=== Parallel control ===
-process <INT|all> Number of multiple sequences aligning, alignments trimming, and gene trees inference to run concurrently. (Default: 1)
"all" means running all samples concurrently. (be cautious to set this option)
=== Heatmap control ===
-heatmap_color {black,blue,red,green,purple,orange,yellow,brown,pink}
Color scheme for heatmap gradient. (Default: black)
Arguments for integrated tools :
=== PhyloPyPruner ===
-pp_min_taxa <INT> Minimum taxa per cluster. (Default: 4)
-pp_min_support <0-1> Minimum support value. (Default: 0=auto)
-pp_trim_lb <INT> Trim long branches. (Default: 5)
=== ParaGone ===
-paragone_pool <INT> Parallel alignment tasks. (Default: 1, same as the option '-process')
-treeshrink_q_value <0-1>
TreeShrink quantile threshold (Default: 0.05)
-paragone_cutoff_value <FLOAT>
Branch length cutoff (Default: 0.3)
-paragone_minimum_taxa <INT>
Minimum taxa per alignment (Default: 4)
-paragone_min_tips <INT>
Minimum tips per tree (Default: 4)
=== HybPiper ===
-hybpiper_skip_chimeric_genes <FALSE|TRUE>
Whether to skip recovering sequences for putative chimeric genes when running "hybpiper retrieve_sequences" (HRS method) in Stage 3. (Default: FALSE)
-hybpiper_retrieved_seqs_type <dna|intron|supercontig>
The type of sequence to extract when running "hybpiper retrieve_sequences" in Stage 3. (default:dna, which means extracting coding sequences)
=== MAFFT ===
-mafft_algorithm <str>
MAFFT algorithm [auto|linsi] (Default: auto)
-mafft_adjustdirection <TRUE/FALSE>
Whether to adjust sequence directions (Default: TRUE)
-mafft_maxiterate <INT>
Maximum number of iterations for MAFFT (Default: auto)
Specifies the maximum number of iterations MAFFT will perform during multiple sequence alignment. Higher iteration counts may improve alignment accuracy but will increase computation time.
-mafft_pair <str>
Pairing strategy for MAFFT (Default: auto)
Specifies the pairing strategy used by MAFFT during multiple sequence alignment. Options include auto, localpair, globalpair, etc. Choosing the appropriate strategy can affect the alignment results and efficiency.
=== trimAl ===
-trimal_mode <str>
trimAl mode [automated1|strict|strictplus|gappyout|nogaps|noallgaps] (Default: automated1)
-trimal_gapthreshold <0-1>
Gap threshold (Default: 0.12)
-trimal_simthreshold <0-1>
Similarity threshold (Default: auto)
-trimal_cons <0-100>
Consensus threshold (Default: auto)
-trimal_block <INT>
Minimum block size (Default: auto)
-trimal_w <INT>
Window size (Default: auto)
-trimal_gw <INT>
Gap window size (Default: auto)
-trimal_sw <INT>
Similarity window size (Default: auto)
-trimal_resoverlap <0-1>
Minimum overlap of a positions with other positions in the column. (Default: auto)
-trimal_seqoverlap <0-100>
Minimum percentage of sequences without gaps in a column. (Default: auto)
=== HMMCleaner ===
-hmmcleaner_cost <NUM1_NUM2_NUM3_NUM4>
Cost parameters that defines the low similarity segments detected by HmmCleaner. (Default: -0.15_-0.08_0.15_0.45)
Users can change each value but they have to be in increasing order. (NUM1 < NUM2 < 0 < NUM3 < NUM4)
Command example :
# Run HybSuite stage3 without alignments filtering
$ hybsuite stage3 -eas_dir ./01-Assembled_data -paralogs_dir ./02-All_paralogs/03-Filtered_paralogs -t ./Angiosperms353 -PH 1234567a -output_dir ./ -nt -process 5
# Run HybSuite stage3 with alignments filtering
$ hybsuite stage3 -eas_dir ./01-Assembled_data -paralogs_dir ./02-All_paralogs/03-Filtered_paralogs -t ./Angiosperms353 -PH 124567b -output_dir ./ -nt -process 5 -aln_min_length 100 -aln_min_sample 0.1
Parameters for running hybsuite stage4
Stage 4 Manual
--------------------------------------------------------------------------------
Usage: hybsuite stage4 ...
Mandatory arguments: -input_list -aln_dir -output_dir
Essential arguments: -PH -sp_tree -prefix -run_phyparts -nt -process
Arguments for inputs:
-input_list <FILE> The file listing input sample names and corresponding data types used in stage 1&2. (Default: None)
-aln_dir The directory containing different orthogroups alignments generated in stage 3. (Default: <output_dir>/06-Final_alignments)
It's advisable to set this parameter as '<output_dir>/06-Final_alignments'.
-PH <1-7|a|b|all> Choose alignments generated via paralog handling methods as input:
1: HRS, 2: RLWP, 3: LS, 4: MI, 5: MO, 6: RT, 7: 1to1 (one or more of them can be chosen)
a: PhyloPyPruner, b: ParaGone (Default: 1a)
Arguments for outputs:
-output_dir <DIR> Output directory for all pipeline results (better to be consistent across all stages). (Default: None)
-prefix <STRING> Prefix for output files. (Default: HybSuite)
General arguments:
=== Species tree builder control ===
-sp_tree <1-5|all> Species tree inference method:
1: IQ-TREE, 2: RAxML, 3: RAxML-NG, 4: ASTRAL-IV, 5: wASTRAL
=== Steps control ===
-run_coalescent_step <INT>
Control which coalescent analysis steps to run:
1: Construct single gene trees, 2: Combine and collapse gene trees, 3: Infer species tree, 4: Reroot gene trees, 5: PhyParts concordance analysis
(Default: 1234)
-run_concatenated_step <INT>
Control which concatenated analysis steps to run:
1: Construct concatenated alignment, 2: Infer species tree
(Default: 12)
=== Gene tree builder control ===
-gene_tree <1/2> Choose the software to construct paralogs gene trees. (1: IQ-TREE; 2: FastTree) (Default: 1)
-gene_tree_bb <INT> Choose the bootstrap value for paralogs gene trees inference. (Default: 1000)
=== Gene trees collapse threshold ===
-collapse_threshold <VALUE>
Specify the minimum support value threshold for internal nodes in gene trees. (Default: 0)
Nodes with support values ≤ this threshold will be collapsed into polytomies.
=== Nucleotide ambiguity character replacement ===
-replace_n <TRUE|FALSE>
Replace ambiguous characters ('n', 'N', '?') with gaps ('-') in alignment files. (Default: FALSE)
Note: Recommended for phylogenetic software compatibility (e.g., IQ-TREE, trimAl).
=== Threads control ===
-nt <INT|AUTO> Global thread setting. (Default: 1)
-nt_amas <INT> AMAS.py threads (Default: 1)
-nt_modeltest_ng <INT>
ModelTest-NG threads (Default: 1)
-nt_iqtree <INT> IQ-TREE threads (Default: 1)
-nt_fasttree <INT> FastTree threads (Default: 1)
-nt_raxml_ng <INT> RAxML-NG threads (Default: 1)
-nt_raxml <INT> RAxML threads (Default: 1)
-nt_astral4 <INT> ASTRAL-IV threads (Default: 1)
-nt_wastral <INT> wASTRAL threads (Default: 1)
-nt_astral_pro <INT> ASTRAL-Pro3 threads (Default: 1)
=== Parallel control ===
-process <INT|all> Number of gene trees inference in coalescent analysis to run concurrently. (Default: 1)
"all" means running all samples concurrently. (be cautious to set this option)
Arguments for integrated tools :
=== IQ-TREE (cancatenated analysis)===
-iqtree_bb <INT> IQ-TREE bootstrap replicates (Default: 1000)
-iqtree_alrt <INT> SH-aLRT replicates (Default: 1000)
-iqtree_run_option <str>
IQ-TREE run mode [standard|undo] (Default: undo)
-iqtree_partition <TRUE/FALSE>
Whether to use partition models in IQ-TREE (Default: TRUE)
-iqtree_constraint_tree <Treefile>
The pathway to the constraint tree for running IQ-TREE (Default: none)
=== ModelTest-NG ===
-run_modeltest_ng <TRUE/FALSE>
Whether to run ModelTest-NG (Default: TRUE)
=== RAxML ===
-raxml_m <str> RAxML model [GTRGAMMA|PROTGAMMA] (Default: GTRGAMMA)
-raxml_bb <INT> RAxML bootstrap replicates (Default: 1000)
-raxml_constraint_tree <Treefile>
The pathway to the constraint tree for running RAxML (Default: no constraint tree)
=== RAxML-NG ===
-rng_bs_trees <INT> RAxML-NG bootstrap replicates (Default: 1000)
-rng_force <TRUE/FALSE>
Ignore thread warnings (Default: FALSE)
-rng_constraint_tree <Treefile>
The pathway to the constraint tree for running RAxML-NG (Default: no constraint tree)
=== ASTRAL-IV ===
-astral4_root Outermost (most distant) outgroup taxon name for ASTRAL-IV branch length calculation. (Default: none)
(Strongly recommended for accurate branch length estimation. Specify only the single outermost outgroup.)
-astral_r <INT> ASTRAL-IV rounds of search. (Default: 4)
-astral_s <INT> ASTRAL-IV rounds of subsampling. (Default: 4)
=== wASTRAL ===
-wastral_mode <1-4> wASTRAL mode [1|2|3|4] (Default: 1)
1: hybrid weighting, 2: support only, 3: length only, 4: unweighted
-wastral_r <INT> wASTRAL rounds of search. (Default: 4)
-wastral_s <INT> wASTRAL rounds of subsampling. (Default: 4)
=== ASTRAL-Pro ===
-astral_pro_r <INT> ASTRAL-Pro rounds of search. (Default: 4)
-astral_pro_s <INT> ASTRAL-Pro rounds of subsampling. (Default: 4)
=== MAFFT (only for paralogs inclusion method -> ASTRAL-Pro) ===
-mafft_algorithm <str>
MAFFT algorithm [auto|linsi] (Default: auto)
-mafft_adjustdirection <TRUE/FALSE>
Whether to adjust sequence directions (Default: TRUE)
-mafft_maxiterate <INT>
Maximum number of iterations for MAFFT (Default: auto)
Specifies the maximum number of iterations MAFFT will perform during multiple sequence alignment. Higher iteration counts may improve alignment accuracy but will increase computation time.
-mafft_pair <str>
Pairing strategy for MAFFT (Default: auto)
Specifies the pairing strategy used by MAFFT during multiple sequence alignment. Options include auto, localpair, globalpair, etc. Choosing the appropriate strategy can affect the alignment results and efficiency.
=== trimAl (only for paralogs inclusion method -> ASTRAL-Pro) ===
-trimal_mode <str>
trimAl mode [automated1|strict|strictplus|gappyout|nogaps|noallgaps] (Default: automated1)
-trimal_gapthreshold <0-1>
Gap threshold (Default: 0.12)
-trimal_simthreshold <0-1>
Similarity threshold (Default: auto)
-trimal_cons <0-100>
Consensus threshold (Default: auto)
-trimal_block <INT>
Minimum block size (Default: auto)
-trimal_w <INT>
Window size (Default: auto)
-trimal_gw <INT>
Gap window size (Default: auto)
-trimal_sw <INT>
Similarity window size (Default: auto)
-trimal_resoverlap <0-1>
Minimum overlap of a positions with other positions in the column. (Default: auto)
-trimal_seqoverlap <0-100>
Minimum percentage of sequences without gaps in a column. (Default: auto)
=== HMMCleaner (only for paralogs inclusion method -> ASTRAL-Pro) ===
-hmmcleaner_cost <NUM1_NUM2_NUM3_NUM4>
Cost parameters that defines the low similarity segments detected by HmmCleaner. (Default: -0.15_-0.08_0.15_0.45)
Users can change each value but they have to be in increasing order. (NUM1 < NUM2 < 0 < NUM3 < NUM4)
=== PhyPartsPieCharts & modified_phypartspiecharts ===
-run_phyparts <TRUE|FALSE>
Enable/disable PhyParts concordance analysis and modified pie chart visualization. (Default: TRUE)
Note: Requires successful completion of previous coalescent analysis.
-phypartspiecharts_tree_type <cladogram/circle>
The tree type of displaying when running modified_phypartspiecharts.py (Default: cladogram)
-phypartspiecharts_num_mode <num>
Control what numbers to show on branches (specify 0-2 digits) (Default: 12)
0: Hide all numbers
1: Number of genes supporting species tree (blue)
2: Number of genes conflicting with species tree (red+green)
3: Number of genes with no signal (gray)
4: Proportion of supporting genes (blue/total)
5: Proportion of conflicting genes ((red+green)/total)
6: Proportion of no signal genes (gray/total)
7: Ratio of supporting to all signal genes (blue/(blue+red+green))
8: Ratio of conflicting to all signal genes ((red+green)/(blue+red+green))
9: Original node support values from the input tree
Command example :
# Run HybSuite stage4 with IQ-TREE
$ hybsuite stage4 -aln_dir ./06-Final_alignments -t ./Angiosperms353 -PH 1234567a -output_dir ./ -nt -process 5 -sp_tree 1
# Run HybSuite stage4 with ASTRAL-IV
$ hybsuite stage4 -aln_dir ./06-Final_alignments -t ./Angiosperms353 -PH 1234567a -output_dir ./ -nt -process 5 -sp_tree 4
# Run HybSuite stage4 with ASTRAL-IV and PhyParts
$ hybsuite stage4 -aln_dir ./06-Final_alignments -t ./Angiosperms353 -PH 1234567a -output_dir ./ -nt -process 5 -sp_tree 4 -run_phyparts TRUE
Parameters for running hybsuite full_pipeline
HybSuite full pipeline Manual
--------------------------------------------------------------------------------
Usage: hybsuite full_pipeline ...
Mandatory arguments: -input_list -input_data (required when including user-provided data) -t -output_dir
Essential arguments: -PH -sp_tree -seqs_min_length -aln_min_sample -prefix -nt -process
Arguments for inputs:
-input_list <FILE> The file listing input sample names and corresponding data types. (Default: None)
-input_data <DIR> The directory containing all input data (required when the inputs include your own data / pre-assembled data). (Default: None).
-t <FILE> Target file for data assembly. (follows the format required in HybPiper)
Arguments for outputs:
-output_dir <DIR> The output directory for all pipeline results. (Default: None)
-NGS_dir <DIR> The output directory containing raw and cleaned reads files (see GitHub documentation).
Notes: Pre-existing cleaned reads will skip reads trimming steps.
-eas_dir <DIR> The output directory containing HybPiper assembly sequences. (Default: <output_dir>/01-Assembled_data)
Note: Pre-existing data in this directory will skip redundant assembly steps.
-prefix <STRING> Prefix for output files. (Default: HybSuite)
General arguments:
=== Stages running control ===
-skip_stage <1|2|3|12|123|>
Specify pipeline stages to skip during execution. (Default: None, running all stages)
Note: Particularly useful for re-running specific HybSuite pipeline stages.
(e.g., '-skip_stage 1' for skipping stage 1)
-run_to_stage <1|2|3> Specify pipeline stages to run up to (Default: None, running all stages)
(e.g., '-run_to_stage 3' for stopping before stage 4)
=== Public raw reads downloading control (Stage 1) ===
-rm_sra <TRUE/FALSE> Whether to remove SRA files after conversion. (Default: TRUE)
-download_format <fastq|fastq_gz>
Downloaded data format. (Default: fastq_gz)
=== Putative paralogs filtering control (Stage 2) ===
-seqs_min_length <INT>
Minimum sequence length for filtered paralogs. (Default: 0)
Putative paralogs shorter than this value will be filtered.
-seqs_mean_length_ratio <0-1>
Minimum sequence length ratio relative to the mean value per locus for putative paralogs. (Default: 0)
Putative paralogs shorter than this percentage of the maximum length will be filtered.
-seqs_max_length_ratio <0-1>
Minimum length ratio relative to the longest value per locus for putative paralogs. (Default: 0)
Putative paralogs shorter than this percentage of the maximum length will be filtered.
-seqs_min_sample_coverage <0-1>
Minimum sample coverage for putative paralogs. (Default: 0)
For all putative paralogs in stage 2, HRS and RLWP sequences in stage 3, loci lower than this sample coverage will be filtered.
-seqs_min_locus_coverage <0-1>
Minimum locus coverage for putative paralogs. (Default: 0)
For all putative paralogs in stage 2, taxa (samples) with lower than this locus coverage will be filtered.
=== Heatmap control (Stage 2&3) ===
-heatmap_color {black,blue,red,green,purple,orange,yellow,brown,pink}
Color scheme for heatmap gradient. (Default: black)
=== Paralog handling control (Stage 3) ===
-PH <1-7|a|b|all> Paralog handling methods to execute: (one or more of them can be chosen)
1: HRS, 2: RLWP, 3: LS, 4: MI, 5: MO, 6: RT, 7: 1to1
a: PhyloPyPruner, b: ParaGone (Default: 1a)
=== Sequences and alignments filtering control (Stage 3) ===
-seqs_min_length <INT>
Minimum sequence bp length for filtering HRS and RLWP sequences. (Default: 0)
HRS and RLWP sequences shorter than this value will be removed.
-aln_min_length <INT>
Minimum sequence bp length for filtering HRS and RLWP final alignments. (Default: 4)
-aln_min_sample <INT>
Minimum sample number for final alignments. (Default: 5)
Final alignments (aligned and trimmed) with sample number below this threshold will be removed.
=== Alignments trimming tool control (Stage 3) ===
-trim_tool <1/2> Choose the software to trim/clean alignments. (1: trimAl; 2: HMMCleaner) (Default: 1)
=== Gene trees builder control (Stage 3&4) ===
-gene_tree <1/2> Choose the software to construct paralogs gene trees. (1: IQ-TREE; 2: FastTree) (Default: 1)
-gene_tree_bb <INT> Choose the bootstrap value for paralogs gene trees inference. (Default: 1000)
=== Species tree builder control (Stage 4) ===
-sp_tree <1-5|all> Species tree inference method: (Default: 1)
1: IQ-TREE, 2: RAxML, 3: RAxML-NG, 4: ASTRAL-IV, 5: wASTRAL, 6: ASTRAL-Pro
=== Steps control in stage 4 ===
-run_coalescent_step <INT>
Control which coalescent analysis steps to run:
1: Construct single gene trees, 2: Combine and collapse gene trees, 3: Infer species tree, 4: Reroot gene trees, 5: PhyParts concordance analysis
(Default: 1234)
-run_concatenated_step <INT>
Control which concatenated analysis steps to run:
1: Construct concatenated alignment, 2: Infer species tree
(Default: 12)
=== Nucleotide ambiguity character replacement (Stage 3&4) ===
-replace_n <TRUE|FALSE>
Replace ambiguous characters ('n', 'N', '?') with gaps ('-') in alignment files. (Default: FALSE)
Note: Recommended for phylogenetic software compatibility (e.g., IQ-TREE, trimAl).
=== Gene trees collapse threshold ===
-collapse_threshold <VALUE>
Specify the minimum support value threshold for internal nodes in gene trees. (Default: 0)
Nodes with support values ≤ this threshold will be collapsed into polytomies.
=== Threads Control ===
-nt <INT|AUTO> Global thread setting. (Default: 1)
-nt_fasterq_dump <INT>
fasterq-dump threads. (Default: 1)
-nt_pigz <INT> pigz compression threads. (Default: 1)
-nt_trimmomatic <INT> Trimmomatic threads. (Default: 1)
-nt_hybpiper <INT> HybPiper threads (Default: 1)
-nt_paragone <INT> ParaGone threads. (Default: 1)
-nt_phylopypruner <INT>
PhyloPyPruner threads. (Default: 1)
-nt_mafft <INT> MAFFT threads. (Default: 1)
-nt_amas <INT> AMAS.py threads. (Default: 1)
-nt_modeltest_ng <INT>
ModelTest-NG threads. (Default: 1)
-nt_iqtree <INT> IQ-TREE threads. (Default: 1)
-nt_fasttree <INT> FastTree threads. (Default: 1)
-nt_modeltest_ng <INT>
ModelTest-NG threads (Default: 1)
-nt_raxml_ng <INT> RAxML-NG threads (Default: 1)
-nt_raxml <INT> RAxML threads (Default: 1)
-nt_astral4 <INT> ASTRAL-IV threads (Default: 1)
-nt_wastral <INT> wASTRAL threads (Default: 1)
-nt_astral_pro <INT> ASTRAL-Pro3 threads (Default: 1)
=== Parallel Control ===
-process <INT|all> Number of subprocess to run concurrently. (Default: 1)
"all" means running all subprocesses concurrently. (be cautious to set this option)
The related steps are:
Stage 1: public data downloading and raw reads trimming;
Stage 2: data assembly ('hybpiper assemble');
Stage 3: multiple sequences aligning, alignments trimming, and gene trees inference;
Stage 4: gene trees inference in coalescent analysis.
=== Logfile Control ===
-log_mode <simple|cmd|full>
The output mode of hybsuite logfile. (Default: cmd)
Arguments for integrated tools :
=== SRAToolkit (Stage 1) ===
-sra_maxsize <NUM> The maximum size of sra files to download. (Default: 20GB)
=== Trimmomatic (Stage 1) ===
-trimmomatic_leading_quality <3-40>
Leading base quality cutoff. (Default: 3)
-trimmomatic_trailing_quality <3-40>
Trailing base quality cutoff. (Default: 3)
-trimmomatic_min_length <36-100>
Minimum read length. (Default: 36)
-trimmomatic_sliding_window_s <4-10>
Sliding window size. (Default: 4)
-trimmomatic_sliding_window_q <15-30>
Window average quality. (Default: 15)
=== HybPiper (Stage 2 & 3) ===
-hybpiper_mapping_tool <blast|diamond>
The tool used for mapping reads to targets in HybPiper (only for protein targets) (Default: blast)
-hybpiper_check_chimeric_contigs <FALSE|TRUE>
Check whether a stitched contig is a potential chimera of contigs from multiple paralogs when running "hybpiper assemble". (Default: FALSE)
-hybpiper_cov_cutoff <INT>
Specify the value of "-cov_cutoff" when running "hybpiper assemble" in Stage 2. (Default: 8)
Increasing this value may increase the loci recovery efficiency but potentially introducing errors.
-hybpiper_skip_chimeric_genes <FALSE|TRUE>
Whether to recover sequences for putative chimeric genes when running "hybpiper retrieve_sequences" (HRS method) in Stage 3. (Default: FALSE)
-hybpiper_retrieved_seqs_type <dna|intron|supercontig>
The type of sequence to extract when running "hybpiper retrieve_sequences" in Stage 3.
=== PhyloPyPruner (Stage 3) ===
-pp_min_taxa <INT> Minimum taxa per cluster. (Default: 4)
-pp_min_support <0-1> Minimum support value. (Default: 0=auto)
-pp_trim_lb <INT> Trim long branches. (Default: 5)
=== ParaGone (Stage 3) ===
-paragone_pool <INT> Parallel alignment tasks. (Default: 1, same as the option '-process')
-treeshrink_q_value <0-1>
TreeShrink quantile threshold (Default: 0.05)
-paragone_cutoff_value <FLOAT>
Branch length cutoff (Default: 0.3)
-paragone_minimum_taxa <INT>
Minimum taxa per alignment (Default: 4)
-paragone_min_tips <INT>
Minimum tips per tree (Default: 4)
=== TreeShrink (Stage 3) ===
-treeshrink_q_value <0-1>
TreeShrink quantile threshold (Default: 0.05)
=== MAFFT (Stage 3) ===
-mafft_algorithm <str>
MAFFT algorithm [auto|linsi] (Default: auto)
-mafft_adjustdirection <TRUE/FALSE>
Whether to adjust sequence directions (Default: TRUE)
-mafft_maxiterate <INT>
Maximum number of iterations for MAFFT (Default: auto)
Specifies the maximum number of iterations MAFFT will perform during multiple sequence alignment. Higher iteration counts may improve alignment accuracy but will increase computation time.
-mafft_pair <str>
Pairing strategy for MAFFT (Default: auto)
Specifies the pairing strategy used by MAFFT during multiple sequence alignment. Options include auto, localpair, globalpair, etc. Choosing the appropriate strategy can affect the alignment results and efficiency.
=== trimAl (Stage 3) ===
-trimal_mode <str>
trimAl mode [automated1|strict|strictplus|gappyout|nogaps|noallgaps] (Default: automated1)
-trimal_gapthreshold <0-1>
Gap threshold (Default: 0.12)
-trimal_simthreshold <0-1>
Similarity threshold (Default: auto)
-trimal_cons <0-100>
Consensus threshold (Default: auto)
-trimal_block <INT>
Minimum block size (Default: auto)
-trimal_w <INT>
Window size (Default: auto)
-trimal_gw <INT>
Gap window size (Default: auto)
-trimal_sw <INT>
Similarity window size (Default: auto)
-trimal_resoverlap <0-1>
Minimum overlap of a positions with other positions in the column. (Default: auto)
-trimal_seqoverlap <0-100>
Minimum percentage of sequences without gaps in a column. (Default: auto)
=== HMMCleaner (Stage 3) ===
-hmmcleaner_cost <NUM1_NUM2_NUM3_NUM4>
Cost parameters that defines the low similarity segments detected by HmmCleaner. (Default: -0.15_-0.08_0.15_0.45)
Users can change each value but they have to be in increasing order. (NUM1 < NUM2 < 0 < NUM3 < NUM4)
=== IQ-TREE (Stage 4) ===
-iqtree_bb <INT> IQ-TREE bootstrap replicates (Default: 1000)
-iqtree_alrt <INT> SH-aLRT replicates (Default: 1000)
-iqtree_run_option <str>
IQ-TREE run mode [standard|undo] (Default: undo)
-iqtree_partition <TRUE/FALSE>
Whether to use partition models in IQ-TREE (Default: TRUE)
-iqtree_constraint_tree <Treefile>
The pathway to the constraint tree for running IQ-TREE (Default: none)
=== ModelTest-NG (Stage 4) ===
-run_modeltest_ng <TRUE/FALSE>
Whether to run ModelTest-NG (Default: TRUE)
=== RAxML (Stage 4) ===
-raxml_m <str> RAxML model [GTRGAMMA|PROTGAMMA] (Default: GTRGAMMA)
-raxml_bb <INT> RAxML bootstrap replicates (Default: 1000)
-raxml_constraint_tree <Treefile>
The pathway to the constraint tree for running RAxML (Default: no constraint tree)
=== RAxML-NG (Stage 4) ===
-rng_bs_trees <INT> RAxML-NG bootstrap replicates (Default: 1000)
-rng_force <TRUE/FALSE>
Ignore thread warnings (Default: FALSE)
-rng_constraint_tree <Treefile>
The pathway to the constraint tree for running RAxML-NG (Default: no constraint tree)
=== ASTRAL-IV (Stage 4) ===
-astral4_root Outermost (most distant) outgroup taxon name for ASTRAL-IV branch length calculation. (Default: none)
(Strongly recommended for accurate branch length estimation. Specify only the single outermost outgroup.)
-astral_r <INT> ASTRAL-IV rounds of search. (Default: 4)
-astral_s <INT> ASTRAL-IV rounds of subsampling. (Default: 4)
=== wASTRAL (Stage 4) ===
-wastral_mode <1-4> wASTRAL mode [1|2|3|4] (Default: 1)
1: hybrid weighting, 2: support only, 3: length only, 4: unweighted
-wastral_r <INT> wASTRAL rounds of search. (Default: 4)
-wastral_s <INT> wASTRAL rounds of subsampling. (Default: 4)
=== ASTRAL-Pro ===
-astral_pro_r <INT> ASTRAL-Pro rounds of search. (Default: 4)
-astral_pro_s <INT> ASTRAL-Pro rounds of subsampling. (Default: 4)
=== PhyPartsPieCharts & modified_phypartspiecharts (Stage 4) ===
-run_phyparts <TRUE|FALSE>
Enable/disable PhyParts concordance analysis and modified pie chart visualization. (Default: TRUE)
Note: Requires successful completion of previous coalescent analysis.
-phypartspiecharts_tree_type <cladogram/circle>
The tree type of displaying when running modified_phypartspiecharts.py (Default: cladogram)
-phypartspiecharts_num_mode <num>
Control what numbers to show on branches (specify 0-2 digits) (Default: 12)
0: Hide all numbers
1: Number of genes supporting species tree (blue)
2: Number of genes conflicting with species tree (red+green)
3: Number of genes with no signal (gray)
4: Proportion of supporting genes (blue/total)
5: Proportion of conflicting genes ((red+green)/total)
6: Proportion of no signal genes (gray/total)
7: Ratio of supporting to all signal genes (blue/(blue+red+green))
8: Ratio of conflicting to all signal genes ((red+green)/(blue+red+green))
9: Original node support values from the input tree
Command example :
=== Run the full pipeline with all paralog-handling methods and all species trees inference approaches ===
hybsuite full_pipeline \
-input_list ./Input_list.txt \
-input_data ./Input_data \
-t Angiosperms353.fasta \
-PH 1234567 \
-sp_tree 12345 \
-output_dir ./ \
-nt 5 -process 5
=== Run the full pipeline with only tree-based orthology inference methods (MO/MI/RT/1to1) in ParaGone and ASTRAL-IV ===
hybsuite full_pipeline \
-input_list ./Input_list.txt \
-input_data ./Input_data \
-t Angiosperms353.fasta \
-PH 4567b \
-sp_tree 4 \
-output_dir ./ \
-nt 5 -process 5
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