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Full pipeline parameters
Documentation current for HybPiper version 2.3.0
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 hybpiper --help :
usage: hybpiper [-h] [--version] {assemble,stats,retrieve_sequences,paralog_retriever,recovery_heatmap,check_dependencies,check_targetfile,fix_targetfile} ...
optional arguments:
-h, --help show this help message and exit
--version, -v Print the HybPiper version number.
Subcommands for HybPiper:
assemble Assemble gene, intron, and supercontig sequences for a sample
stats Gather statistics about the HybPiper run(s)
retrieve_sequences
Retrieve sequences generated by the HybPiper run(s)
paralog_retriever
Retrieve paralog sequences generated by the HybPiper run(s)
recovery_heatmap Create a gene recovery heatmap for the HybPiper run(s)
check_dependencies
Run a check for all pipeline dependencies and exit
check_targetfile Check the target file for issues, then exit
fix_targetfile Fix and filter the target file, then exit
filter_by_length Filter the sequences output by command "hybpiper retrieve_sequences" by length (absolute and relative to mean)
To view parameters and help for a subcommand, use e.g. "assemble --help"
To view the full parameters for a particular subcommand, use hybpiper <subcommand> --help.
usage: hybpiper assemble [-h] --readfiles READFILES [READFILES ...]
(--targetfile_dna TARGETFILE_DNA | --targetfile_aa TARGETFILE_AA)
[--unpaired UNPAIRED] [--bwa | --diamond]
[--diamond_sensitivity {mid-sensitive,sensitive,more-sensitive,very-sensitive,ultra-sensitive}]
[--evalue FLOAT] [--max_target_seqs INTEGER]
[--distribute_low_mem] [--cov_cutoff INTEGER]
[--single_cell_assembly]
[--kvals INTEGER [INTEGER ...]] [--merged]
[--timeout_assemble_reads INTEGER] [--no_spades_eta]
[--not_protein_coding]
[--extract_contigs_blast_task {blastn,blastn-short,megablast,dc-megablast}]
[--extract_contigs_blast_evalue FLOAT]
[--extract_contigs_blast_word_size INTEGER]
[--extract_contigs_blast_gapopen INTEGER]
[--extract_contigs_blast_gapextend INTEGER]
[--extract_contigs_blast_penalty INTEGER]
[--extract_contigs_blast_reward INTEGER]
[--extract_contigs_blast_perc_identity INTEGER]
[--extract_contigs_blast_max_target_seqs INTEGER]
[--thresh INTEGER]
[--paralog_min_length_percentage FLOAT]
[--depth_multiplier INTEGER] [--target TARGET]
[--exclude EXCLUDE]
[--timeout_extract_contigs INTEGER]
[--no_stitched_contig]
[--no_pad_stitched_contig_gaps_with_n]
[--chimeric_stitched_contig_check]
[--bbmap_memory INTEGER] [--bbmap_subfilter INTEGER]
[--bbmap_threads INTEGER]
[--chimeric_stitched_contig_edit_distance INTEGER]
[--chimeric_stitched_contig_discordant_reads_cutoff INTEGER]
[--trim_hit_sliding_window_size INTEGER]
[--trim_hit_sliding_window_thresh INTEGER]
[--exonerate_skip_hits_with_frameshifts]
[--exonerate_skip_hits_with_internal_stop_codons]
[--exonerate_skip_hits_with_terminal_stop_codons]
[--exonerate_refine_full] [--no_intronerate]
[--no_padding_supercontigs] [--prefix PREFIX]
[--start_from {map_reads,distribute_reads,assemble_reads,extract_contigs}]
[--end_with {map_reads,distribute_reads,assemble_reads,extract_contigs}]
[--force_overwrite] [--cpu INTEGER]
[--compress_sample_folder] [--skip_targetfile_checks]
[--keep_intermediate_files] [--verbose_logging]
[--hybpiper_output OUTPUT_FOLDER] [--run_profiler]
optional arguments:
-h, --help show this help message and exit
Required input:
--readfiles READFILES [READFILES ...], -r READFILES [READFILES ...]
One or more read files to start the pipeline. If
exactly two are specified, HybPiper will assume it is
paired Illumina reads.
--targetfile_dna TARGETFILE_DNA, -t_dna TARGETFILE_DNA
FASTA file containing DNA target sequences for each
gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--targetfile_aa TARGETFILE_AA, -t_aa TARGETFILE_AA
FASTA file containing amino-acid target sequences for
each gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
Optional input:
--unpaired UNPAIRED Include a single FASTQ file with unpaired reads along
with two paired read files
Options for step: map_reads:
--bwa Use BWA to map reads to targets. Requires BWA and a
target file that is nucleotides! Default is: False
--diamond Use DIAMOND instead of BLASTx for read mapping.
Default is: False
--diamond_sensitivity {mid-sensitive,sensitive,more-sensitive,very-sensitive,ultra-sensitive}
Use the provided sensitivity for DIAMOND read-mapping
searches.
--evalue FLOAT e-value threshold for mapping blastx hits. Default is:
0.0001
--max_target_seqs INTEGER
Max target seqs to save in BLASTx search. Default is:
10
Options for step: distribute_reads:
--distribute_low_mem Distributing and writing reads to individual gene
directories will be 40-50 percent slower, but can use
less memory/RAM with large input files (see wiki).
Default is: False
Options for step: assemble_reads:
--cov_cutoff INTEGER Coverage cutoff for SPAdes. Default is: 8
--single_cell_assembly
Run SPAdes assemblies in MDA (single-cell) mode.
Default is: False
--kvals INTEGER [INTEGER ...]
Values of k for SPAdes assemblies. SPAdes needs to be
compiled to handle larger k-values! Default is auto-
detection by SPAdes.
--merged For assembly with both merged and unmerged
(interleaved) reads. Default is: False.
--timeout_assemble_reads INTEGER
Kill long-running gene assemblies if they take longer
than X percent of average.
--no_spades_eta When SPAdes is run concurrently using GNU parallel,
the "--eta" flag can result in many "sh: /dev/tty:
Device not configured" errors written to stderr. Using
this option removes the "--eta" flag to GNU parallel.
Default is False.
Options for step: extract_contigs:
--not_protein_coding If provided, extract sequences from SPAdes contigs
using BLASTn rather than Exonerate (step:
extract_contigs)
--extract_contigs_blast_task {blastn,blastn-short,megablast,dc-megablast}
Task to use for BLASTn searches during the
extract_contigs step of the assembly pipeline. See
https://www.ncbi.nlm.nih.gov/books/NBK569839/ for a
description of tasks. Default is: blastn
--extract_contigs_blast_evalue FLOAT
Expectation value (E) threshold for saving hits.
Default is: 10
--extract_contigs_blast_word_size INTEGER
Word size for wordfinder algorithm (length of best
perfect match).
--extract_contigs_blast_gapopen INTEGER
Cost to open a gap.
--extract_contigs_blast_gapextend INTEGER
Cost to extend a gap.
--extract_contigs_blast_penalty INTEGER
Penalty for a nucleotide mismatch.
--extract_contigs_blast_reward INTEGER
Reward for a nucleotide match.
--extract_contigs_blast_perc_identity INTEGER
Percent identity. Can be used as a pre-filter at the
BLASTn stage, followed by --thresh (see below).
--extract_contigs_blast_max_target_seqs INTEGER
Maximum number of aligned sequences to keep (value of
5 or more is recommended). Default is: 500
--thresh INTEGER Percent identity threshold for retaining
Exonerate/BLASTn hits. Default is 55, but increase
this if you are worried about contaminant sequences.
Exonerate hit identity is calculated using amino-
acids, BLASTn hit identity is calculated using
nucleotides.
--paralog_min_length_percentage FLOAT
Minimum length percentage of a contig Exonerate hit vs
reference protein length for a paralog warning and
sequence to be generated. Default is: 0.75
--depth_multiplier INTEGER
Assign a long paralog as the "main" sequence if it has
a coverage depth <depth_multiplier> times all other
long paralogs. Set to zero to not use depth. Default
is: 10
--target TARGET Use the target file sequence with this taxon name in
Exonerate/BLASTn searches for each gene. Other targets
for that gene will be used only for read sorting. Can
be a tab-delimited file (one <gene>\t<taxon_name> per
line) or a single taxon name.
--exclude EXCLUDE Do not use any sequence with the specified taxon name
string in Exonerate/BLASTn searches. Sequences from
this taxon will still be used for read sorting.
--timeout_extract_contigs INTEGER
Kill long-running processes if they take longer than X
seconds. Default is: 120
--no_stitched_contig Do not create any stitched contigs. The longest single
Exonerate/BLASTn hit will be used. Default is: False.
--no_pad_stitched_contig_gaps_with_n
When constructing stitched contigs, do not pad any
gaps between hits (with respect to the "best" protein
reference) with a number of Ns corresponding to the
reference gap multiplied by 3 (Exonerate) or reference
gap (BLASTn). Default is: True.
--chimeric_stitched_contig_check
Attempt to determine whether a stitched contig is a
potential chimera of contigs from multiple paralogs.
Default is: False.
--bbmap_memory INTEGER
MB memory (RAM) to use for bbmap.sh if a chimera check
is performed during step extract_contigs. Default: is
1000.
--bbmap_subfilter INTEGER
Ban alignments with more than this many substitutions.
Default is: 7.
--bbmap_threads INTEGER
Number of threads to use for BBmap when searching for
chimeric stitched contig. Default is: 1.
--chimeric_stitched_contig_edit_distance INTEGER
Minimum number of differences between one read of a
read pair vs the stitched contig reference for a read
pair to be flagged as discordant. Default is: 5.
--chimeric_stitched_contig_discordant_reads_cutoff INTEGER
Minimum number of discordant reads pairs required to
flag a stitched contig as a potential chimera of
contigs from multiple paralogs. Default is 5.
--trim_hit_sliding_window_size INTEGER
Size of the sliding window (amino acids for Exonerate,
nucleotides for BLASTn) when trimming hit termini.
Default is: 5 (Exonerate) or 15 (BLASTn).
--trim_hit_sliding_window_thresh INTEGER
Percentage similarity threshold for the sliding window
(amino acids for Exonerate, nucleotides for BLASTn)
when trimming hit termini. Default is: 75 (Exonerate)
or 65 (BLASTn).
--exonerate_skip_hits_with_frameshifts
Skip Exonerate hits where the SPAdes sequence contains
a frameshift. See: https://github.com/mossmatters/HybP
iper/wiki/Troubleshooting-common-issues,-and-
recommendations#42-hits-where-the-spades-contig-
contains-frameshifts. Default is: False.
--exonerate_skip_hits_with_internal_stop_codons
Skip Exonerate hits where the SPAdes sequence contains
an internal in-frame stop codon. See: https://github.c
om/mossmatters/HybPiper/wiki/Troubleshooting,-common-
issues,-and-recommendations#31-sequences-containing-
stop-codons. A single terminal stop codon is allowed,
but see option "--
exonerate_skip_hits_with_terminal_stop_codons" below.
Default is: False.
--exonerate_skip_hits_with_terminal_stop_codons
Skip Exonerate hits where the SPAdes sequence contains
a single terminal stop codon. Only applies when option
"--exonerate_skip_hits_with_internal_stop_codons" is
also provided. Only use this flag if your target file
exclusively contains protein-coding genes with no stop
codons included, and you would like to prevent any in-
frame stop codons in the output sequences. Default is:
False.
--exonerate_refine_full
Run Exonerate searches using the parameter "--refine
full". Default is: False.
--no_intronerate Do not run intronerate to recover fasta files for
supercontigs with introns (if present), and introns-
only. Default is: False.
--no_padding_supercontigs
If Intronerate is run, and a supercontig is created by
concatenating multiple SPAdes contigs, do not add 10
"N" characters between contig joins. By default, Ns
will be added. Default is: False.
General pipeline options:
--prefix PREFIX Directory name for pipeline output, default is to use
the FASTQ file name.
--start_from {map_reads,distribute_reads,assemble_reads,extract_contigs}
Start the pipeline from the given step. Note that this
relies on the presence of output files for previous
steps, produced by a previous run attempt. Default is:
map_reads
--end_with {map_reads,distribute_reads,assemble_reads,extract_contigs}
End the pipeline at the given step. Default is:
extract_contigs
--force_overwrite Overwrite any output from a previous run for pipeline
steps >= --start_from and <= --end_with. Default is:
False
--cpu INTEGER Limit the number of CPUs. Default is to use all cores
available minus one.
--compress_sample_folder
Tarball and compress the sample folder after assembly
has completed (<sample_name>.tar.gz). Default is:
False.
--skip_targetfile_checks
Skip the target file checks. Can be used if you are
confident that your target file has no issues (e.g. if
you have previously run "hybpiper check_targetfile".
Default is: False
--keep_intermediate_files
Keep all intermediate files and logs, which can be
useful for debugging. Default action is to delete
them, which greatly reduces the total file number.
Default is: False.
--verbose_logging If supplied, enable verbose logging. NOTE: this can
increase the size of the log files by an order of
magnitude. Default is: False.
--hybpiper_output OUTPUT_FOLDER, -o OUTPUT_FOLDER
Folder for HybPiper output. Default is None.
--run_profiler If supplied, run the subcommand using cProfile. Saves
a *.csv file of results. Default is: False.
Additional information:
--diamond
By default (i.e. when not providing the flag --bwa to the hybpiper assemble command), HybPiper will use BLASTx to map reads against a target file of amino-acid sequences. While BLASTx can be very sensitive, it can also be quite slow, and the read-mapping step of the pipeline can take some time if the input read files are large. An alternative is to use DIAMOND in place of BLASTx. DIAMOND offers a faster alternative to BLASTx that with results that can be equally sensitive. The sensitivity of the DIAMOND algorithm can be adjusted using the parameter --diamond_sensitivity.
--cov_cutoff
By default, HybPiper performs per-sample/gene assemblies using SPAdes with the parameter --cov-cutoff 8. As noted here, if the --cov-cutoff option is manually provided to SPAdes, it is interpreted as an "average nucleotide coverage". This value can be changed using the HybPiper pipeline parameter --cov_cutoff <int>. Reducing this value to 1, for example, results in a) longer contigs for many samples/genes, and/or b) contigs being generated for some samples/genes that previously had none. However, there is an obvious tradeoff between more/increased length contigs, and confidence in base-level accuracy of contigs - you might want to lower this value to only 4 or 5 and accept some reduced contig lengths/presences for some genes.
--single_cell_assembly
This parameter can be used to run per-sample/gene assemblies using SPAdes in MDA (single-cell) mode, i.e. supplying SPAdes with the flag --sc. This setting provides uneven coverage optimisation implemented for single-cell data, which can also assist with the uneven coverage that can result from target-catpure sequencing approaches. As for the --cov_cutoff setting above, using the --single_cell_assembly flag can result in a) longer contigs for many samples/genes, and/or b) contigs being generated for some samples/genes that previously had none. However, --sc mode tends to produce many more contigs that standard mode for each gene, and this can sometimes result in the introduction of incorrect sequence in the gene sequences output by HybPiper. Therefore, we currently only reccommend running HybPiper with the --single_cell_assembly flag if essential genes are not recovered using the default SPAdes assembly, and that users manually examine the sequence output.
--distribute_low_mem
Using this flag can reduce memory/RAM usage when distributing mapped reads to per-gene fasta files, but takes ~40-50% longer.
--timeout_assemble
In some situations SPAdes assembly for some genes can take a very long time; this is often due to the presence of many (i.e. millions) of low-complexity reads mapping to a given gene. While this situation should be avoided (see here for discussion and recommendations), the --timeout_assemble parameter can also be used to provide a time limit for SPAdes assembly. Any SPAdes assembly that is taking longer than a percentage value of the average gene assembly time (e.g. --timeout_assemble 200) will be stopped, and no sequences will be recovered for that gene.
``
--timeout_exonerate_contigs
In some situations (see --timeout_assemble above) the SPAdes assembly for some genes will comprise hundreds of low-complexity contigs. This causes the next stage of the pipeline - extraction of coding sequences from the SPAdes contigs - to take a very long time, and can produce enormous log files if --verbose_logging is used (see below). By default, sequence extraction for any gene that takes longer than 120 seconds will be cancelled, and no final sequence will be produced NOTE logged and printed to screen. This value can be altered using the --timeout_exonerate_contigs parameter. Note that HybPiper logs and prints a list of genes if they were cancelled due to exceeding the timeout.
--exonerate_refine_full
In HybPiper version <2.3.0, extraction of coding sequences from SPAdes contigs using Exonerate used the Exonerate option --refine full. Although this should improve output alignments, in some cases it can result in spurious alignment regions (e.g. an intron/non-coding region being included as an "exon" alignment) that can get incorporated in to the HybPiper output sequence. Therefore, from HybPiper version 2.3.0 onwards, Exonerate is run without this flag by default. It can be enabled using the option --exonerate_refine_full.
--target
This parameter can be used to provide a single target file taxon name, e.g. Artocarpus. The target file sequence from this taxon will be used during Exonerate searches of SPAdes contigs for each gene. Alternatively, a tab-delimited file can be provided, e.g.:
| Gene name | Target file taxon to use |
|---|---|
| gene001 | Artocarpus |
| gene002 | Morus |
NOTE: do not include the column names row in your file! If a file is provided, it must specify a gene name and a target file taxon name for each gene in your target file.
By default, HybPiper will select the 'best' target file sequence to be used in Exonerate searches for each gene via the highest cumalative MAPQ score (if using BWA) or the highest cumulative bit score (if using BLASTx or DIAMOND).
--exclude
This parameter can be used to provide a single target file taxon name, e.g. Artocarpus. No target file sequence from this taxon will be used during Exonerate searches of SPAdes contigs, for any gene.
--no_stitched_contig
When reads are assembled in to contigs using SPAdes for a given gene/sample, multiple contigs can be produced. These contigs can contain exons corresponding to different regions of the target sequence. By default, HybPiper will attempt to recover as much gene sequence as possible by extracting these exons from different contigs and "stitching" them together to create a contiguous sequence. HybPiper calls these multi-contig sequences stitched_contigs.
This approach is not without its downsides, particularly for samples that have high levels of paralogy. In such cases, creating stitched_contigs can result in contigs from different paralogs being stitched together, resulting in 'chimeric' locus sequences. This potentially results in reduced phylogenetic resolution or misleading results. HybPiper therefore provides the optional flag --no_stitched_contig. When used, stitched_contigs will not be constructed and only single longest sequence originating from a single contig will be returned for each sample/gene.
Note: In cases where your target file corresponds to multi-exon genes with long introns, running the pipeline with the --no_stitched_contig flag can dramatically reduce the total loci sequence length recovered.
--chimeric_stitched_contig_edit_distance
As described above for the flag --no_stitched_contig, HybPiper can potentially create 'chimeric' gene sequences comprising regions from multiple paralogs.
HybPiper implements a work-in-progress method to detect such putative chimeras. Details, including the effect of the related pipeline parameters:
--chimeric_stitched_contig_edit_distance, --chimeric_stitched_contig_discordant_reads_cutoff, --bbmap_memory, --bbmap_subfilter and --bbmap_threads
...are described here.
Note: The HybPiper command hybpiper paralog_retriever will create two folders, one with all "main" and paralog sequences, and one without any putative chimeric sequences. The default folder names are paralogs_all and paralogs_no_chimeras, respectively; these name can be user-specified.
--depth_multiplier
When HybPiper detects 'long' paralogs for a given gene/sample, it assigns one of them as the "main" sequence, and adds the suffix .main to the sequence FASTA header. All other paralogs are arbitrarily assigned an incremented number as a suffix, starting at zero (<sequence_name>.0, <sequence_name>.1, etc.). The .main paralog is assigned first by the read depth of the corresponding SPAdes contig, i.e. if a contig has a coverage depth greater than <depth_multiplier> times that of all other long paralogs. The default value for --depth_multiplier is 10. Note that if no paralog contig has a depth that fulfills this criterion, the .main paralog is assigned via the greatest percentage sequence similarity to the reference target file protein sequence.
--merged
When paired-end sequencing is generated using libraries with short insert sizes, the forwards and reverse reads of a given pair can overlap. In such cases, target locus recovery can be improved when SPAdes assemblies are performed with merged reads*, that is, when a single contig is produced from overlapping pairs when possible. This can be done using the flag --merged. If provided, paired-end reads will be merged using bbmerge.sh with default settings, and SPAdes assemblies will be carried out with both merged and any remaining unmerged output.
*CJJ: benchmarking is required here; this is currently word-of-mouth although it makes intuitive sense...
--keep_intermediate_files
By default, HybPiper will delete intermediate files and folders for each gene after processing. This greatly helps to reduce the total number of files output by the HybPiper pipeline. For example, running the hybpiper assemble commmand on the tutorial test dataset produces 1,924 files by default, or 19,935 files when using the --keep_intermediate_files flag. Total file number can be a limiting factor when running the pipeline on some HPC systems. Intermediate files and folders are:
- The SPAdes assembly folder for each gene.
- A
*.logfile within each gene subfolder, containing details from running theexonerate_hits.pymodule for the given gene. This log file is usually re-logged to the main sample*.logfile and deleted. - Fasta files containing the extracted and trimmed (if necessary) Exonerate hits used to create stitched contigs. One
*.FNAfile contains nucleotide sequences, and one*.FAAcontains amino-acid sequences. These files are found in each<gene_name>/<sample_name>directory. -
chimera_test_stitched_contig.sam. A Sequence Alignment Map file containing mapping details for paired-end reads mapped against a stitched contig reference; used in chimera tests. Found in each<gene_name>/<sample_name>directory.
--no_padding_supercontigs
Unless the flag --no_intronerate is provided, HybPiper will attempt to recover intron sequences (if present for a given gene/sample) and a supercontig sequence. The supercontig sequence comprises both exons and introns; in cases where it has been constructed from more than one SPAdes contig, HybPiper will add a stretch of 10 'N' characters between abutting contigs. This can be turned off using the flag --no_padding_supercontigs
--paralog_min_length_percentage
HybPiper tests for the presence of paralogs for a given gene/sample in two different ways:
-
From a set of exon-only sequences extracted from a set of SPAdes contigs (and where each exon-only sequence originates from a single contig only), it searches for more than one sequence matching a given target file reference, where each sequence is greater than 75% of the reference length. If such sequences are found, they are written to
*.fastafile and can be recovered using thehybpiper paralog_retrievercommand. -
From a set of exon-only sequences extracted from a set of SPAdes contigs (and where each exon-only sequence originates from a single contig only), calculate sequence coverage across the length of the query protein. If coverage is >1 for a given percentage threshold of the query length, produce a paralog warning. This warning is useful as it provides an indication that paralogs might be present for the given gene/sample, without the requirement that SPAdes has assembled each paralog into an almost full-length contig. Note that these sequences are not written to file, as there is currently no way to definitively group contigs containing partial gene sequences into single-paralog clusters. Please let us know if you can think of a way!
HybPiper allows the length percentage cut-off used by each paralog detection approach to be specified using the --paralog_min_length_percentage parameter. The default value is 0.75.
--verbose_logging
When this flag is supplied, HybPiper will capture and log additional data from the pipeline run. This can be useful if you run in to problems/debugging, but note that it can increase the size of the log file by an order of magnitude or more!
--run_profiler
When this flag is supplied, the hybpiper assemble command will be run using the Python profiler cProfile (see https://docs.python.org/3/library/profile.html). A *.csv file of results will be written. This can be useful when e.g. debugging I/O bottlenecks on different computers/HPCs.
usage: hybpiper stats [-h]
(--targetfile_dna TARGETFILE_DNA | --targetfile_aa TARGETFILE_AA)
[--seq_lengths_filename SEQ_LENGTHS_FILENAME]
[--stats_filename STATS_FILENAME]
[--hybpiper_dir HYBPIPER_DIR] [--run_profiler]
{gene,GENE,supercontig,SUPERCONTIG} namelist
positional arguments:
{gene,GENE,supercontig,SUPERCONTIG}
Sequence type (gene or supercontig) to recover lengths
for.
namelist Text file with names of HybPiper output directories,
one per line.
optional arguments:
-h, --help show this help message and exit
--targetfile_dna TARGETFILE_DNA, -t_dna TARGETFILE_DNA
FASTA file containing DNA target sequences for each
gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--targetfile_aa TARGETFILE_AA, -t_aa TARGETFILE_AA
FASTA file containing amino-acid target sequences for
each gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--seq_lengths_filename SEQ_LENGTHS_FILENAME
File name for the sequence lengths *.tsv file. Default
is <seq_lengths.tsv>.
--stats_filename STATS_FILENAME
File name for the stats *.tsv file. Default is:
<hybpiper_stats.tsv>
--hybpiper_dir HYBPIPER_DIR
Specify directory containing HybPiper output sample
folders. Default is the current working directory.
--run_profiler If supplied, run the subcommand using cProfile. Saves
a *.csv file of results
usage: hybpiper recovery_heatmap [-h] [--heatmap_filename HEATMAP_FILENAME]
[--figure_length INTEGER]
[--figure_height INTEGER]
[--sample_text_size INTEGER]
[--gene_text_size INTEGER]
[--heatmap_filetype {png,pdf,eps,tiff,svg}]
[--heatmap_dpi INTEGER] [--no_xlabels]
[--no_ylabels] [--run_profiler]
seq_lengths_file
positional arguments:
seq_lengths_file Filename for the seq_lengths file (output of the
'hybpiper stats' command)
optional arguments:
-h, --help show this help message and exit
--heatmap_filename HEATMAP_FILENAME
Filename for the output heatmap, saved by default as a
*.png file. Defaults to "recovery_heatmap"
--figure_length INTEGER
Length dimension (in inches) for the output heatmap
file. Default is automatically calculated based on the
number of genes
--figure_height INTEGER
Height dimension (in inches) for the output heatmap
file. Default is automatically calculated based on the
number of samples
--sample_text_size INTEGER
Size (in points) for the sample text labels in the
output heatmap file. Default is automatically
calculated based on the number of samples
--gene_text_size INTEGER
Size (in points) for the gene text labels in the
output heatmap file. Default is automatically
calculated based on the number of genes
--heatmap_filetype {png,pdf,eps,tiff,svg}
File type to save the output heatmap image as. Default
is *.png
--heatmap_dpi INTEGER
Dot per inch (DPI) for the output heatmap image.
Default is 100
--no_xlabels If supplied, do not render labels for x-axis (loci) in
the saved heatmap figure
--no_ylabels If supplied, do not render labels for y-axis (samples)
in the saved heatmap figure
--run_profiler If supplied, run the subcommand using cProfile. Saves
a *.csv file of results
usage: hybpiper retrieve_sequences [-h]
(--targetfile_dna TARGETFILE_DNA | --targetfile_aa TARGETFILE_AA)
[--sample_names SAMPLE_NAMES]
[--single_sample_name SINGLE_SAMPLE_NAME]
[--hybpiper_dir HYBPIPER_DIR]
[--fasta_dir FASTA_DIR]
[--skip_chimeric_genes]
[--stats_file STATS_FILE]
[--filter_by column comparison threshold]
[--run_profiler]
{dna,aa,intron,supercontig}
positional arguments:
{dna,aa,intron,supercontig}
Type of sequence to extract.
optional arguments:
-h, --help show this help message and exit
--targetfile_dna TARGETFILE_DNA, -t_dna TARGETFILE_DNA
FASTA file containing DNA target sequences for each
gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--targetfile_aa TARGETFILE_AA, -t_aa TARGETFILE_AA
FASTA file containing amino-acid target sequences for
each gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--sample_names SAMPLE_NAMES
Text file with names of HybPiper output directories,
one per line.
--single_sample_name SINGLE_SAMPLE_NAME
A single sample name to recover sequences for.
--hybpiper_dir HYBPIPER_DIR
Specify directory containing HybPiper output sample
folders. Default is the current working directory.
--fasta_dir FASTA_DIR
Specify directory for output FASTA files.
--skip_chimeric_genes
Do not recover sequences for putative chimeric genes.
This only has an effect for a given sample if the
option "--chimeric_stitched_contig_check" was provided
to command "hybpiper assemble".
--stats_file STATS_FILE
Stats file produced by "hybpiper stats", required for
selective filtering of retrieved sequences.
--filter_by column comparison threshold
Provide three space-separated arguments: 1) column of
the stats_file to filter by, 2) "greater" or
"smaller", 3) a threshold - either an integer (raw
number of genes) or float (percentage of genes in
analysis). This parameter can be supplied more than
once to filter by multiple criteria.
--run_profiler If supplied, run the subcommand using cProfile. Saves
a *.csv file of results.
usage: hybpiper paralog_retriever [-h]
(--targetfile_dna TARGETFILE_DNA | --targetfile_aa TARGETFILE_AA)
[--hybpiper_dir HYBPIPER_DIR]
[--fasta_dir_all FASTA_DIR_ALL]
[--fasta_dir_no_chimeras FASTA_DIR_NO_CHIMERAS]
[--paralog_report_filename PARALOG_REPORT_FILENAME]
[--paralogs_above_threshold_report_filename PARALOGS_ABOVE_THRESHOLD_REPORT_FILENAME]
[--paralogs_list_threshold_percentage FLOAT]
[--no_heatmap]
[--heatmap_filename HEATMAP_FILENAME]
[--figure_length INTEGER]
[--figure_height INTEGER]
[--sample_text_size INTEGER]
[--gene_text_size INTEGER]
[--heatmap_filetype {png,pdf,eps,tiff,svg}]
[--heatmap_dpi INTEGER] [--no_xlabels]
[--no_ylabels] [--run_profiler]
namelist
positional arguments:
namelist Text file containing list of HybPiper output
directories, one per line.
optional arguments:
-h, --help show this help message and exit
--targetfile_dna TARGETFILE_DNA, -t_dna TARGETFILE_DNA
FASTA file containing DNA target sequences for each
gene. Used to extract unique gene names for paralog
recovery. The fasta headers must follow the naming
convention: >TaxonID-geneName
--targetfile_aa TARGETFILE_AA, -t_aa TARGETFILE_AA
FASTA file containing amino-acid target sequences for
each gene. Used to extract unique gene names for
paralog recovery. The fasta headers must follow the
naming convention: >TaxonID-geneName
--hybpiper_dir HYBPIPER_DIR
Specify directory containing HybPiper output sample
folders. Default is the current working directory.
--fasta_dir_all FASTA_DIR_ALL
Specify directory for output FASTA files (ALL).
Default is: paralogs_all.
--fasta_dir_no_chimeras FASTA_DIR_NO_CHIMERAS
Specify directory for output FASTA files (no putative
chimeric sequences). Default is: paralogs_no_chimeras.
--paralog_report_filename PARALOG_REPORT_FILENAME
Specify the filename for the paralog *.tsv report
table. Default is: paralog_report.
--paralogs_above_threshold_report_filename PARALOGS_ABOVE_THRESHOLD_REPORT_FILENAME
Specify the filename for the *.txt list of genes with
paralogs in <paralogs_list_threshold_percentage>
number of samples. Default is:
paralogs_above_threshold_report.
--paralogs_list_threshold_percentage FLOAT
Percent of total number of samples and genes that must
have paralog warnings to be reported in the
<genes_with_paralogs.txt> report file. The default is
0.0, meaning that all genes and samples with at least
one paralog warning will be reported
--no_heatmap If supplied, do not create a heatmap figure. Default
is: False.
--heatmap_filename HEATMAP_FILENAME
Filename for the output heatmap, saved by default as a
*.png file. Default is: paralog_heatmap.
--figure_length INTEGER
Length dimension (in inches) for the output heatmap
file. Default is automatically calculated based on the
number of genes.
--figure_height INTEGER
Height dimension (in inches) for the output heatmap
file. Default is automatically calculated based on the
number of samples.
--sample_text_size INTEGER
Size (in points) for the sample text labels in the
output heatmap file. Default is automatically
calculated based on the number of samples.
--gene_text_size INTEGER
Size (in points) for the gene text labels in the
output heatmap file. Default is automatically
calculated based on the number of genes.
--heatmap_filetype {png,pdf,eps,tiff,svg}
File type to save the output heatmap image as. Default
is: png.
--heatmap_dpi INTEGER
Dots per inch (DPI) for the output heatmap image.
Default is: 100.
--no_xlabels If supplied, do not render labels for x-axis (loci) in
the saved heatmap figure. Default is: False.
--no_ylabels If supplied, do not render labels for y-axis (samples)
in the saved heatmap figure. Default is: False.
--run_profiler If supplied, run the subcommand using cProfile. Saves
a *.csv file of results. Default is: False.
NOTE: if paralogs are detected for a given gene/sample, the sequence in the paralog folder with the suffix *.main will not necessarily be identical to the corresponding *.FNA sequence. This is because each paralog sequence is recovered from a single SPAdes contig only, whereas the *.FNA sequence could be derived from a stitched contig (comprising sequence from more than one SPAdes contig).
usage: hybpiper check_dependencies [-h] [--run_profiler]
optional arguments:
-h, --help show this help message and exit
--run_profiler If supplied, run the subcommand using cProfile. Saves a
*.csv file of results
usage: hybpiper check_targetfile [-h]
(--targetfile_dna TARGETFILE_DNA | --targetfile_aa TARGETFILE_AA)
[--no_terminal_stop_codons]
[--sliding_window_size SLIDING_WINDOW_SIZE]
[--complexity_minimum_threshold COMPLEXITY_MINIMUM_THRESHOLD]
[--run_profiler]
optional arguments:
-h, --help show this help message and exit
--targetfile_dna TARGETFILE_DNA, -t_dna TARGETFILE_DNA
FASTA file containing DNA target sequences for each
gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--targetfile_aa TARGETFILE_AA, -t_aa TARGETFILE_AA
FASTA file containing amino-acid target sequences for
each gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--no_terminal_stop_codons
When testing for open reading frames, do not allow a
translated frame to have a single stop codon at the
C-terminus of the translated protein sequence. Default
is False.
--sliding_window_size INTEGER
Number of characters (single-letter DNA or amino-acid
codes) to include in the sliding window when checking
for sequences with low-complexity-regions.
--complexity_minimum_threshold COMPLEXITY_MINIMUM_THRESHOLD
Minimum threshold value. Beneath this value, the
sequence in the sliding window is flagged as low
complexity, and the corresponding target file sequence
is reported as having low-complexity regions.
--run_profiler If supplied, run the subcommand using cProfile. Saves
a *.csv file of results
Additional information:
--sliding_window_size
By default, the sliding window size used to scan across each sequence in the target file is 50 characters (amino-acids) for a protein file, and 100 characters (nucleotides) for a DNA file.
--complexity_minimum_threshold
By default, the threshold below which a DNA sequence within the sliding window will be flagged as low-complexity is set to 1.5. However, for a DNA target file containing only ATCG characters (i.e. assuming no N or ambiguity characters), the maximum 'complexity value' achievable is 2.0, and so alternative values up to 2.0 can be used.
By default, the threshold below which a protein sequence within the sliding window will be flagged as low-complexity is set to 3.0. However, for a protein target file containing only ARNDCQEGHILKMFPSTWYV characters (i.e. assuming no ambiguity characters), the maximum 'complexity value' achievable is 4.0, and so alternative values up to 4.0 can be used.
See here for additional details.
usage: hybpiper fix_targetfile [-h]
(--targetfile_dna TARGETFILE_DNA | --targetfile_aa TARGETFILE_AA)
[--no_terminal_stop_codons]
[--allow_gene_removal]
[--reference_protein_file REFERENCE_PROTEIN_FILE]
[--maximum_distance FLOAT]
[--filter_by_length_percentage FLOAT]
[--keep_low_complexity_sequences]
[--alignments]
[--concurrent_alignments INTEGER]
[--threads_per_concurrent_alignment INTEGER]
[--write_all_fasta_files] [--verbose_logging]
[--run_profiler]
control_file
positional arguments:
control_file The *.ctl file, as output by the command "hybpiper
check_targetfile".
optional arguments:
-h, --help show this help message and exit
--targetfile_dna TARGETFILE_DNA, -t_dna TARGETFILE_DNA
FASTA file containing DNA target sequences for each
gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--targetfile_aa TARGETFILE_AA, -t_aa TARGETFILE_AA
FASTA file containing amino-acid target sequences for
each gene. The fasta headers must follow the naming
convention: >TaxonID-geneName
--no_terminal_stop_codons
When testing for open reading frames, do not allow a
translated frame to have a single stop codon at the
C-terminus of the translated protein sequence. Default
is False. If supplied, this parameter will override
the setting in the *.ctl file.
--allow_gene_removal Allow frame-correction and filtering steps to remove
all representative sequences for a given gene. Default
is False; HybPiper will exit with an information
message instead. If supplied, this parameter will
override the setting in the *.ctl file.
--reference_protein_file REFERENCE_PROTEIN_FILE
If a given DNA sequence can be translated in more than
one forward frame without stop codons, choose the
translation that best matches the corresponding
reference protein provided in this fasta file. The
fasta headers must follow the naming convention:
>TaxonID-geneName
--maximum_distance FLOAT
When comparing candidate DNA translation frames to a
reference protein, the maximum distance allowed
between the translated frame and the reference
sequence for any candidate translation frame to be
selected. Useful to filter out sequences with
frameshifts that do NOT introduce stop codons. 0.0
means identical sequences, 1.0 means completely
different sequences. Default is 0.5
--filter_by_length_percentage FLOAT
If more than one representative sequence is present
for a given gene, filter out sequences shorter than
this percentage of the longest gene sequence length.
Default is 0.0 (all sequences retained).
--keep_low_complexity_sequences
Keep sequences that contain regions of low complexity,
as identified by the command "hybpiper
check_targetfile". Default is to remove these
sequences.
--alignments Create per-gene alignments from the final
fixed/filtered target file sequences. Note that DNA
sequences will be translated prior to alignment.
--concurrent_alignments INTEGER
Number of alignments to run concurrently. Default is
1.
--threads_per_concurrent_alignment INTEGER
Number of threads to run each concurrent alignment
with. Default is 1.
--write_all_fasta_files
If provided, *.fasta files will be written for
sequences removed from the fixed/filtered target file,
according to filtering categories (length threshold,
low-complexity regions, etc.). By default, these files
will not be written.
--verbose_logging If supplied, enable verbose logging. NOTE: this will
increase the size of the log files.
--run_profiler If supplied, run the subcommand using cProfile. Saves
a *.csv file of results
See here for additional details.
usage: hybpiper filter_by_length [-h]
(--denylist DENYLIST | --seq_lengths_file SEQ_LENGTHS_FILE)
[--length_filter LENGTH_FILTER]
[--percent_filter PERCENT_FILTER]
[--sequence_dir SEQUENCE_DIR]
[--filtered_dir FILTERED_DIR]
[--run_profiler]
{dna,aa,supercontig,intron}
positional arguments:
{dna,aa,supercontig,intron}
File sequence type for all FASTA files to filter in
current directory. For example, the amino-acid output
of HybPiper would be: aa
optional arguments:
-h, --help show this help message and exit
--denylist DENYLIST Text file containing gene-sample combinations to omit.
The format of the file should be one gene per line, a
tab, and then a comma-delimited list of samples to
disallow: gene[tab]sample,sample,sample
--seq_lengths_file SEQ_LENGTHS_FILE
Filename for the seq_lengths file (output of the
"hybpiper stats" command), with a list of genes in the
first row, mean target lengths in the second row, and
sample recovery in other rows.
--length_filter LENGTH_FILTER
Minimum length to allow a sequence in nucleotides for
DNA or amino acids for protein sequences
--percent_filter PERCENT_FILTER
Minimum fraction (between 0 and 1) of the mean target
length to allow a sequence for a gene. Lengths taken
from HybPiper stats file.
--sequence_dir SEQUENCE_DIR
Specify directory containing sequences output by the
"hybpiper retrieve_sequences" command. Default is to
search in the current working directory
--filtered_dir FILTERED_DIR
Specify directory for output filtered FASTA files.
Default is to write to the current working directory
--run_profiler If supplied, run the subcommand using cProfile. Saves
a *.csv file of results