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Changelog

2.3.0 10th September 2024

  • Add option --compress_sample_folder to command hybpiper assemble. Tarball and compress the sample folder after assembly has completed i.e. <sample_name>.tar.gz.

    • This is useful when running HybPiper on HPC clusters with file number limits.
    • If both an uncompressed and compressed folder exist for a sample, a warning is shown and HybPiper exits.
    • All HybPiper subcommands (stats, recovery_heatmap, retrieve_sequences, paralog_retriever, filter_by_length) work with either compressed or uncompressed sample files/folders, or a combination of both.
    • If a <sample_name>.tar.gz file already exists for a sample, it will be extracted and used for the current run of hybpiper assemble, and the <sample_name>.tar.gz file will be deleted.

  • When using BWA for read mapping, the command samtools flagstat is now run during the hybpiper assemble step, rather than during hybpiper stats, and the results are written to a <sample_name>_bam_flagstat.tsv \ <sample_name>_unpaired_bam_flagstat.tsv file(s).

    • If the <sample_name>_bam_flagstat.tsv \ <sample_name>_unpaired_bam_flagstat.tsv file(s) are not present in a sample directory (i.e. the sample was assembled with HybPiper version <2.3.0), samtools flagstat will be run during hybpiper stats. If the sample is a *.tar.gz file, the *.bam file(s) will first be extracted to disk to a temporary directory called temp_bam_files, within your current working directory. This temporary directory will be deleted after samtools flagstat has been run.

  • Add option --not_protein_coding to hybpiper assemble. When this option is provided, sequences matching your target file references will be extracted from SPAdes contigs using BLASTn, rather than Exonerate. This should improve recovery when using a target file with non-protein-coding sequences. Note that this feature is new and might have bugs - please report any issues.

    • Only nucleotide *.FNA sequences will be produced (i.e. no amino-acid sequences).
    • Intronerate will not be run; intron and supercontig sequences will not be produced.
    • If BLASTx or DIAMOND is selected for read mapping (i.e. protein vs translated-nucleotide searches), a warning will be displayed and read mapping will switch to BWA.

  • Add the following options to control BLASTn searches of SPAdes contigs when option --not_protein_coding is used:

    • --extract_contigs_blast_task. Task to use for blastn searches (blastn, blastn-short, megablast, dc-megablast). Default is blastn.
    • --extract_contigs_blast_evalue. Expectation value (E) threshold for saving hits. Default is 10.
    • --extract_contigs_blast_word_size. Word size for wordfinder algorithm (length of best perfect match).
    • --extract_contigs_blast_gapopen. Cost to open a gap.
    • --extract_contigs_blast_gapextend. Cost to extend a gap.
    • --extract_contigs_blast_penalty. Penalty for a nucleotide mismatch.
    • --extract_contigs_blast_reward. Reward for a nucleotide match.
    • --extract_contigs_blast_perc_identity. Percent identity.
    • --extract_contigs_blast_max_target_seqs. Maximum number of aligned sequences to keep (value of 5 or more is recommended). Default is 500.

  • The final step of the hybpiper assemble pipeline has been renamed from exonerate_contigs to extract_contigs (as either Exonerate or BLASTn can now be used).

  • Reorganised grouping of help options when running hybpiper assemble --help to improve clarity.

  • Changed option --timeout_assemble for hybpiper assemble to --timeout_assemble_reads to match the step name.

  • Changed option --timeout_exonerate_contigs for hybpiper assemble to --timeout_extract_contigs to match the step name.

  • Changed option --exonerate_hit_sliding_window_size for hybpiper assemble to --trim_hit_sliding_window_size. This option now applies to either Exonerate hits (and is measured in amino-acids) or BLASTn (measured in nucleotides). Defaults are 5 amino-acids (Exonerate; changed from previous default of 3) or 15 nucleotides (BLASTn).

  • Changed option --exonerate_hit_sliding_window_thresh for hybpiper assemble to --trim_hit_sliding_window_thresh. This option now applies to either Exonerate hits (and is measured via amino-acid similarity) or BLASTn (measured via nucleotide similarity). Defaults are 75 for amino-acids (Exonerate; changed from previous default of 55) or 65 for nucleotides (BLASTn).

  • Fixed a bug in fix_targetfile.py - MAFFT is now called via subprocess rather than Bio.Align.Applications.MafftCommandline when checking for best match translations (see issue#156).

  • Added a more informative error message if running hybpiper retrieve_sequences or hybpiper paralog_retriever from HybPiper version >=2.2.0 on sample folders from HybPiper version >2.2.0. This error occurs because the sample folders do not contain a <prefix>_chimera_check_performed.txt file (see issue#155).

  • When extracting coding sequences from SPAdes contigs using Exonerate, changed the initial Exonerate run to not use the option --refine full (see Exonerate docs), unless the option --exonerate_refine_full is provided to hybpiper assemble. Although the Exonerate option --refine full 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.

2.2.0 17th July, 2024

  • Add option --end_with to command hybpiper assemble. Allows the user to end the assembly pipeline at a chosen step (map_reads, distribute_reads, assemble_reads, exonerate_contigs).
  • Add option --exonerate_skip_hits_with_frameshifts to command hybpiper assemble. If provided, skip Exonerate hits where the SPAdes contig contains frameshifts when considering hits for assembly of an *.FNA sequence. Default behaviour in HybPiper v2.2.0 is to include these hits; previous versions allowed them automatically.
  • Add option --exonerate_skip_hits_with_internal_stop_codons to command hybpiper assemble. If provided, skip Exonerate hits where the SPAdes contig contains internal in-frame stop codon(s) when considering hits for assembly of an *.FNA sequence. A single terminal stop codon is allowed. Default behaviour in HybPiper v2.2.0 is to include these hits; previous versions allowed them automatically.
  • Add option --exonerate_skip_hits_with_terminal_stop_codons to command hybpiper assemble. If provided, 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 behaviour in HybPiper v2.2.0 is to include these hits; previous versions allowed them automatically.
  • Add option --chimeric_stitched_contig_check to command hybpiper assemble. If provided, HybPiper will attempt to determine whether a stitched contig is a potential chimera of contigs from multiple paralogs. Default behaviour in HybPiper v2.2.0 is to skip this check; previous versions performed the check automatically. Skipping this check speeds up the final 'exonerate_contigs' step of the pipeline, significantly.
  • Add option --no_pad_stitched_contig_gaps_with_n to command hybpiper assemble. If provided, 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. Default behaviour in HybPiper v2.2.0 is to pad gaps with Ns; previous versions did this automatically.
  • Add option --skip_targetfile_checks to command hybpiper assemble. 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).
  • Add option --no_spades_eta to command hybpiper assemble. 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, silencing both ETA output and the error message.
  • Fixed a bug in exonerate_hits.py that could (rarely) result in a duplicated region in the output *.FNA sequence.
  • Fixed a bug in exonerate_hits.py that occurred when more than two Exonerate hits had identical query ranges and similarity scores; this could result in a sequence not being returned for the given gene.
  • Added tests folder containing initial unit tests. Some tests require python package pyfakefs to run.
  • Refactor of the hybpiper package. New module hybpiper_main.py with entry point (moved from assemble.py), and some assemble.py functions moved to utils.py. Target file checking functionality has been consolidated.
  • HybPiper now logs to stdout rather than stderr.
  • Commands hybpiper check_targetfile and hybpiper assemble now write a report file when checking the target file (check_targetfile_report-<target file name>.txt), rather than logging details to the main sample log. Command hybpiper check_targefile writes the report to the current working directory, whereas command hybpiper assemble writes it to the sample directory.
  • If the option --cpu is not specified for hybpiper assemble, HybPiper will now use all available CPUs minus one, rather than all available CPUs.
  • Command hybpiper assemble now checks for output from previous runs for the pipeline steps selected via --start_from and --end_with (default is to select all steps). If previous output is found, HybPiper will exit with an error unless the option --force_overwrite is provided.
  • Corrected the reading frame of sequence Artocarpus-gene660 in the test dataset target file.
  • Command hybpiper assemble now writes the file <prefix>_chimera_check_performed.txt to the sample directory. This is a text file containing 'True' or 'False' depending on whether the option --skip_chimeric_genes was provided to command hybpiper assemble. Used by hybpiper retrieve_sequences and hybpiper paralog_retriever.

2.1.8 25th June, 2024

  • Add new subcommand hybpiper filter_by_length, used to filter the sequence output of hybpiper retrieve_sequences by absolute length and/or length relative to mean length in target file representatives. This is done on a per-sample/per-gene basis, rather than the sample-level filtering available in hybpiper retrieve_sequences. See wiki for more information.
  • Update the regex used to check target file fasta header formatting, to capture scenarios where a name contains multiple dashes and also ends with a dash.
  • In the fix_targetfile.py module, remove the import of Bio.Align.Applications.MafftCommandline and call MAFFT via subprocess (see issue#147).
  • In the gene_recovery_heatmap.py module, cast the dataframe from the seq_lengths_file to object dtype to avoid a deprecation warning .
  • Add option --no_heatmap to command hybpiper paralog_retriever (see issue#150).
  • Fix an Exonerate-related debug message in exonerate_hits.py.

2.1.7 09th May, 2024

  • The flag --run_intronerate was removed from the hybpiper assemble command in the run_hybpiper_test_dataset.sh file.
  • Removed the legacy check and attempted download of the test dataset in the run_hybpiper_test_dataset.sh file.
  • Added a check to hybpiper stats and hybpiper retrieve_sequences to ensure sample names in the namelist.txt file do not contain forward slashes issue#143.
  • When checking for putative chimeric gene sequences in hybpiper retrieve_sequences and hybpiper paralog_retriever, generate a warning rather than an error if the file <sample_name>_genes_derived_from_putative_chimeric_stitched_contig.csv can't be found for a given sample. This file will not be written if no gene sequences were produced for this sample (i.e. no reads mapped, no SPAdes contigs, no sequences extracted from SPAdes contigs via Exonerate).
  • Check that target file FASTA headers do not contain quotation marks (" or '); issue#125.
  • Updated the installation instructions in the README and Wiki to use the Bioconda package, and added installation instruction for Macs with Apple Silicon (M1/M2/M3 chips).
  • Fixed a bug in exonerate_hits.py that meant that hits were not always trimmed to start with the first amino-acid with full alignment identity. This bug could potentially have had an effect on output sequences only if the values for --exonerate_hit_sliding_window_size and/or --exonerate_hit_sliding_window_thresh were changed from default values.
  • Use importlib.metadata rather than pkg_resources for module version checks, due to deprecation of the latter.

2.1.6 19th July, 2023

  • Intronerate is now run by default. The flag --run_intronerate for subcommand hybpiper assemble has been changed to --no_intronerate.
  • If Intronerate fails, failed genes and errors will be printed and logged; the exonerate_contigs step of the pipeline will continue.
  • Updated error handling and logging for the exonerate_contigs step of the pipeline.
  • Change default DPI of heatmaps to 100 (previously 150) for hybpiper recovery_heatmap and hybpiper paralog_retriever
  • Enforce rendering of all loci (x-axis) and sample (y-axis) labels in heatmaps; previously, matplotlib/seaborn would dynamically drop labels if they were too closely spaced.
  • Added flags --no_xlabels and --no_ylabels for hybpiper recovery_heatmap and hybpiper paralog_retriever; turns off rendering of the corresponding labels in the saved figures.
  • If the auto-calculated size of heatmaps for hybpiper recovery_heatmap and hybpiper paralog_retriever is greater than the maximum number of pixels (65536) in either/or length and height, resize the figure to 400 inches and 100 DPI. Note that large datasets can fail to render fully in the saved figure even if the pixel dimensions are less than the maximum (see e.g. https://stackoverflow.com/questions/64393779/how-to-render-a-heatmap-for-a-large-array), but reducing the size/DPI further allows the full figure to be rendered.
  • Added module version.py for a single location of HybPiper version number.
  • Print and log HybPiper version when calling all subcommands.
  • Added column 'TotalBasesRecovered' to the hybpiper stats report, listing the total number of nucleotides recovered for each sample (not counting N characters). Added 'TotalBasesRecovered' as a filtering option in hybpiper retrieve_sequences.

2.1.5 21st June, 2023

  • Bugfix: fixed an issue in exonerate_hits.py that could result in initial Exonerate hits being trimmed too aggressively at their 3' ends.
  • Bugfix: fixed an issue in exonerate_hits.py that could introduce minor insertions in to the supercontig (concatenated exon and partial intron) sequence used when running Intronerate.

2.1.4 5th June, 2023

  • Bugfix: fixed an issue when using --run_intronerate that could cause an error and result in no *.FNA sequence being produced for some genes.

2.1.3 23rd March, 2023

  • Log platform and ulimit details for debugging purposes.
  • Added parameter --exonerate_hit_sliding_window_thresh to hybpiper assemble. This value (default is 55) is used as the similarity threshold within the sliding window when trimming the 5' and 3' termini of Exonerate hits (i.e., the filter added in version 2.1.2). Previously, this filter used the value from the --thresh parameter, which is also used to perform filtering of entire Exonerate hits based on global alignment similarity.
  • Bugfix: in exonerate_hits.py, ensure that similarity-filtered Exonerate hits are ordered by query start THEN query end; in cases where hits have identical query start coordinates, sorting only by query start can return the hits in a different order from one run to another, sometimes causing issues when trimming overlaps and concatenating to create a stitched-contig.
  • Add parameter --hybpiper_output / -o to hybpiper assemble. If a directory is supplied using this parameter, hybpiper assemble will create it and use it for all output.

2.1.2 31st January, 2023

  • Removed the call to time when running SPAdes assemblies via parallel (see #109).
  • Added a filter/trimming step to exonerate_hits.py. This process applies a sliding window to Exonerate hit alignments that have already passed a global similarity threshold (parameter --thresh for command hybpiper assemble). The 5' and 3' termini of SPAdes contig hits are trimmed if the similarity within the sliding window is below the --thresh value. This removes putative spurious 5' and 3' hit sequence produced by Exonerate alignments, and results in more accurate output *.FNA and *.FAA sequences. The sliding window size can be adjusted using the hybpiper assemble parameter --exonerate_hit_sliding_window_size; default value is 3 (i.e., 3 amino acids / 9 nucleotides).
  • HybPiper now provides a warning if any output sequence contains internal stop codons, and writes the corresponding gene names to the file <prefix>_genes_with_non_terminal_stop_codons.txt.
  • Fixed a bug that meant reads with older format headers (i.e., suffixes /1 or /2) weren't processed properly when using BLASTx/DIAMOND (see #108).

2.1.1 12th December, 2022

  • When mapping reads with DIAMOND via hybpiper assemble --diamond, remove the gunzip step and on-the-fly fastq to fasta conversion (as DIAMOND supports both *.fastq and *.gz input). Further, pass the value of the hybpiper assemble parameter --cpu directly to the --threads parameter of the diamond blastx command; do not run diamond via GNU parallel. See issue #104.

2.1.0 1st December, 2022

  • The subcommand hybpiper check_targetfile now writes a *.ctl file; see wiki for details.
  • Added the new subcommand hybpiper fix_targetfile; see wiki for details.
  • New dependency: MAFFT. Used for optional alignments when running hybpiper fix_targetfile; see wiki for details.
  • The flag ---distribute_high_mem has been changed to --distribute_low_mem for subcommand hybpiper assemble. Default is off, i.e., reads are now distributed using the faster, more memory intensive approach.
  • The time command has been removed from all BWA and BLAST command strings (see #89).
  • Update minimum Python version from 3.6 to 3.7, to match minimum requirements for Biopython 1.80 release.

2.0.3 16th November, 2022

  • The calculation for automatically determining the default heatmap dimensions has been changed, to prevent label trimming when using large numbers of samples and genes (pull request from LPDagallier).
  • The default DPI of the heatmap *.png file has been reduced from 300 to 150.

2.0.2 14th November, 2022

  • Added the --run_profiler flag to the check_dependencies subcommand (bugfix).
  • Moved to correct semantic versioning; removed the 'release candidate build x' string from version description, and bumped to version 2.0.2.

2.0.1 release candidate build 13 12th September, 2022

  • Added a --run_profiler option to all subcommands; when used, the given subcommand is run with cProfile and the data is saved to a *.csv file.
  • If a DNA target file is provided but the flag --bwa is omitted, a translated target file is now written directly to the sample directory (rather than the same directory as the input DNA file) with the generic name translated_target_file.fasta. This prevents file overwrite/access issues when processing multiple samples concurrently (e.g. using Nextflow).
  • Added a check for sequence fasta files that exist but are empty to stats, paralog_retriever and retrieve_sequences; a warning is printed if such a file is found.

2.0.1 release candidate build 12 6th July, 2022

  • When running hybpiper stats, empty lines in the namelist.txt file are ignored.

2.0.1 release candidate build 11 29th June, 2022

  • Adding hybpiper_dir search for single sample recovery when running hybpiper retrieve_sequences. Adding sample name to recovered fasta file.
  • Adding --memory 1024 to spades.py command (macOS Monterey compatibility pre SPAdes version 3.15.4)

2.0.1 release candidate build 8 24th June, 2022

  • Capture errors relating to malformed input *.fastq files during read distribution, and log error to file.
  • When running hybpiper stats, if no input read count file can be found, log issue and sample to screen along with instructions, and exit.

2.0.1 release candidate build 8 16th June, 2022

  • If mapping is performed with BWA and the BWA mapping step fails, remove any *.bam file that has been produced.

2.0 release candidate March, 2022

This update involves a substantial refactor of the HybPiper pipeline, with changes to the internal code, additional functionality, and additional output. Changes include:

New and updated dependencies

Code refactor:

  • HybPiper now has an installation process (conda or pip via setup.py), rather than scripts being called directly.
  • After installation, all HybPiper commands are accessed via the main command hybpiper, followed by a subcommand e.g. assemble.
  • Thereads_first.py module has been renamed to assemble.py, and now imports other pipeline modules rather than calling them as external Python scripts.
  • The exonerate_hits.py module has been substantially re-written to use the BioPython SearchIO Exonerate text parser, as this allows much more data recovered from Exonerate search results.
  • The intronerate.py module has been removed; this functionality has been moved to exonerate_hits.py.
  • The paralog_investigator.py module has been removed; this functionality has been moved to exonerate_hits.py.
  • The gene_recovery_heatmap_ggplot.R and gene_recovery_heatmap.R R scripts have been replaced by the Python module gene_recovery_heatmap.py.
  • The get_seq_lengths.py script has been removed; this functionality is now performed by the module hybpiper_stats.py (command hybpiper stats). The file seq_lengths.tsv is written by default; this filename can be changed via the parameter --seq_lengths_filename.

Additional features/functionality:

  • HybPiper can now check target files for sequences with problematic low-complexity regions via command hybpiper check_targetfile.
  • The hybpiper assemble command checks that the provided target file is formatted correctly and can be translated as expected (in the case of a nucleotide target file). Any issues are printed to screen and details are logged to file.
  • The program DIAMOND can be used in place of BLASTX when mapping reads to target sequences.
  • When using BLAST or DIAMOND, the hybpiper stats command now calculates the enrichment efficiency; previously this was only calculated when using BWA.
  • The hybpiper assemble commands can now accept read files in compressed gzip format (suffix *.gz).
  • Logging when running hybpiper assemble has been unified and extended to provide additional debugging information. A single log file is written per-sample in the sample directory e.g. EG30_hybpiper_assemble_2021-12-02-10_45_56.log.
  • Checks for all dependencies are now run by default when hybpiper assemble is run.
  • Gene assemblies can no be performed using the SPAdes MDA (single cell) mode.
  • All Exonerate searches are now performed with the option --refine full; in the case of failure, a fallback run without this parameter is performed.
  • In some situations HybPiper creates a gene sequence by 'stitching' together Exonerate hits from different SPAdes contigs. In some scenarios this can result in hit from different paralogs being joined together. HybPiper now performs a test (work in progress, currently sensitivity is low) to search for such 'chimeric' sequences, and provides warnings in the file {sample_name}_genes_derived_from_putative_chimera_stitched_contig.csv within each sample folder. Note that this chimera test is only performed in cases where a stitched contig has been created from multiple contigs, and paired-end reads are provided.
  • By default, the SPAdes assembly folder is now deleted for each gene after contigs have been recovered. The user no longer needs to run cleanup.py after each run, and this script has been removed. Deleting the SPAdes directory dramatically reduces the total number of files produced by a completed run of HybPiper, which can be very useful when running it on and HPC with file number limits. To retain the SPAdes directory (i.e. for debugging purposes), the flag --keep_intermediate_files can be used.
  • When running Intronerate (via flag -run_intronerate with command hybpiper assemble, a block of 10 'N' characters is inserted into supercontigs at locations where different SPAdes contigs have been concatenated. This behaviour can be turned off via the flag --no_padding_supercontigs.
  • In cases where HybPiper recovers sequence for multiple non-contiguous segments of a gene, the gaps between the segments will be padded with a number of 'N' characters. The number of Ns corresponds to the number of amino acids in 'best' protein reference for that gene that do not have corresponding SPAdes contig hits, multiplied by 3 to convert to nucleotides.
  • The command hybpiper stats now writes pipeline run statistics directly to file, rather than to standard out.
  • The command hybpiper recover_sequences now supports sequence recovery from a single sample.
  • The command hybpiper recover_sequences now supports filtering of samples based on statistics generated via the hybpiper stats command (e.g. number of genes recovered, number of genes with paralogs, etc).
  • The command hybpiper paralog_retriever now writes a table of gene-vs-sample with a matrix of paralog counts, and produces a heatmap image file from this table. A text report file is also produced, listing gene and sample names that contain paralogs in above a given threshold percentage.

New options/flags:

For subcommand hybpiper assemble:

  • --diamond. When provided, DIAMOND (with default sensitivity fast) will be used to map reads against the bait/target file, rather than the default BLASTX.
  • --diamond_sensitivity. DIAMOND will be run with the provided sensitivity (options are mid-sensitive, sensitive, more-sensitive, very-sensitive, ultra-sensitive).
  • --run_intronerate. When used, the function intronerate() in module exonerate_hits.py will be run to recover introns and supercontigs where possible.
  • --merged. When used, R1 and R2 reads will be merged using BBmerge.sh, where possible. Both the merged and the remaining unmerged reads will be used for SPAdes assembly.
  • --no_stitched_contigs. When used, gene sequences will comprise the longest Exonerate hit from a single SPAdes contig; no contig/hit stitching will be attempted.
  • --paralog_min_length_percentage. Corresponds to the minimum percentage length for a SPADes contig Exonerate hit (vs the reference query sequence length) for it to be flagged and recovered as a 'long' paralog. Previously this parameter was hardcoded to 0.75. This parameter effected both types of paralog warnings (by length, and by depth).
  • --bbmap_subfilter. Ban BBmap alignments with more than this many substitutions when searching for chimeric stitched contigs. Default is 7.
  • --bbmap_threads. The number of threads to use for BBmap when searching for chimeric stitched contigs. Default is 2.
  • --bbmap_memory. The amount of memory (RAM) in MB to use for BBmap when searching for chimeric stitched contigs. Default is 1000.
  • --chimeric_stitched_contig_edit_distance. Minimum number of differences between one read of a read pair vs a stitched contig reference for a read pair to be flagged as discordant. Default is 5.
  • --chimeric_stitched_contig_discordant_reads_cutoff. Minimum number of discordant reads pairs required to flag a stitched contig as a potential chimera of contigs from multiple paralogs. Default is 5.
  • --keep_intermediate_files. 1) If used, the SPAdes assembly folder for each gene will not be deleted. Note that previous versions of HybPiper retained the SPAdes assembly folders by default; they could previously be removed after running reads_first.py using the cleanup.py script. 2) If used, retain the exonerate_hits.py module log within each gene folder. Default behaviour is to delete each log after it has been copied to the main log file in the sample directory.
  • --distribute_hi_mem. When used, distributing and writing reads to individual gene directories will be 40-50 percent faster, but can use more memory/RAM with large input files.
  • --single_cell_assembly. Run SPAdes assemblies using MDA (single-cell) mode.
  • --verbose_logging. When used, the pipline logging will be much more verbose (particularly in the Exonerate stage), which can increase log file size dramatically.

For subcommand hybpiper retrieve_sequences:

  • --single_sample_name. Allows the user to specify a single sample name; sequences from this sample only will be recovered in single *.fasta file.
  • --skip_chimeric_genes. If used, skip retrieval of any genes/sample for which the stitiched contig was flagged as chimeric (potentially derived from multiple paralogs).
  • --fasta_dir. User can specify a directory for output sequences.
  • --stats_file. User can supply the stats fil produced by the command hybpiper stats for filtering samples (see below).
  • --filter_by. User can filter samples based on statistics in the stats file.

For subcommand hybpiper stats:

  • A positional parameter for the sequence type has been added, with the options {gene, supercontig}. Note that all length statistics are now provided as number of nucleotides, regardless of whether an amino-acid or nucleotide bait file was used.
  • --stats_filename. The statistics are now written directly to file. This parameter can be used to specify the filename; default is hybpiper_stats.tsv.

For subcommand hybpiper recovery_heatmap:

  • seq_lengths_file. A positional parameter providing the filename for the seq_length.tsv (default) output of hybpiper stats
  • --heatmap_filename. Supply a filename for the heatmap image file. Default is heatmap.
  • --figure_length. Supply a length dimension (in inches) for the heatmap image file. Default is auto-calculated.
  • --figure_height. Supply a height dimension (in inches) for the heatmap image file. Default is auto-calculated.
  • --sample_text_size. Supply a text size (in points) for the heatmap image file sample names. Default is auto-calculated.
  • --gene_text_size. Supply a text size (in points) for the heatmap image file gene names. Default is auto-calculated.
  • --heatmap_filetype. Provide a filetype for the heatmap image file {png,pdf,eps,tiff,svg}. Default is png.
  • --heatmap_dpi. Supply a Dots Per Inch value (in DPI) for the heatmap image file. Default is 300.

For subcommand hybpiper paralog_retriever:

  • --fasta_dir_all. Supply a directory name for the paralog *.fasta files when recovering all sequences included putative chimeric stitched contig sequences (the latter are recovered when paralogs are not present). Default is paralogs_all.
  • --fasta_dir_no_chimeras. Supply a directory name for the paralog *.fasta files when recovering all sequences except for putative chimeric stitched contig sequences. Default is paralogs_no_chimeras.
  • --paralogs_above_threshold_report_filename. Specify the filename for a text report file listing genes and samples with paralogs in greater than a given percentage threshold of genes/samples. Default is genes_with_paralogs.txt.
  • --paralogs_list_threshold_percentage. Percent of total number of samples and genes that must have paralog warnings to be reported in the genes_with_paralogs.txt (default) file.
  • --heatmap_filename. Supply a filename for the heatmap image file. Default is paralog_heatmap.
  • --figure_length. Supply a length dimension (in inches) for the heatmap image file. Default is auto-calculated.
  • --figure_height. Supply a height dimension (in inches) for the heatmap image file. Default is auto-calculated.
  • --sample_text_size. Supply a text size (in points) for the heatmap image file sample names. Default is auto-calculated.
  • --gene_text_size. Supply a text size (in points) for the heatmap image file gene names. Default is auto-calculated.
  • --heatmap_filetype. Provide a filetype for the heatmap image file {png,pdf,eps,tiff,svg}. Default is png.
  • --heatmap_dpi. Supply a Dots Per Inch value (in DPI) for the heatmap image file. Default is 300.

The following options/flags have been changed or removed:

  • The parameter --baitfile or -b has been replaced with --targetfile_dna/targetfile_aa or -t_dna/t-aa, to more accurately describe the contents of the file provided.
  • The parameter --length_pct has been removed as a parameter to hybpiper assemble and is no longer used in internal code.
  • The parameter --thresh (percent identity threshold for retaining Exonerate hits) for hybpiper assemble now defaults to 55 (previously 65).
  • The flag --check-depend has been replaced by hybpiper check_dependencies. Dependency checking is now also performed every time the hybpiper assemble command is run.
  • The flags --no-blast, --no-distribute, --no-exonerate, and --no-assemble have been replaced with the parameter --start_from.
  • The parameter --timeout has been replaced with --timeout_assemble and --timeout_exonerate_contigs, providing more fine-grained control over different stages of the assemble pipeline.

The following output files have been changed or removed:

  • ToDo

The following output files/folders have been added:

  • The command hybpiper paralog_retreiver now write paralogs to two folders - one with all sequences, and the other without putative chimeric sequences.
  • The command hybpiper paralog_retreiver now produces a heatmap showing paralogs detected.
  • The command hybpiper paralog_retreiver now produces a report file in *tsv format listing genes with paralogs in above user-provided thresholds.
  • The file containing paralog warnings (produced when multiple long contigs are present for a gene) has been renamed from genes_with_paralog_warnings.txt to <sample_name>_genes_with_long_paralog_warnings.txt.
  • In addition to the standard paralog warning produced when multiple long contigs are present for a gene, the hybpiper assemble command now provides a paralog warning when multiple short contigs are present which together cover the reference sequence for a given gene at a depth >1, across a given percentage length (default 75%) of the reference. These warning are written to each sample directory to the file <sample_name>_genes_with_paralog_warnings_by_contig_depth.csv
  • chimera_test_diagnostic_reads.sam.
  • XXX Other intronerate files, if we end up keeping them?
  • The hybpiper retrieve_sequences command can now produce an output folder to contain fasta files.

1.3.2 February, 2020

  • Fix for Issue 41 a problem in intronerate.py when attempting to resolve overlapping gene annotations.
  • Add support in retrieve_sequences.py for a list of HybPiper outputs rather than everything in a directory
  • Add support for supercontigs in get_seq_lengths.py
  • Remove integer requirement for --cov_cutoff to accommodate auto and off settings in Spades.

1.3.1 IMPORTANT BUG FIX August, 2018

  • Reverts a change in 1.3 that changed the behavior when there is only one hit in exonerate. Strandedness was not handled properly, leading some sequences to be returned in reverse complement. Sequences recovered using 1.3 should be re-run with reads_first.py --no-blast --no-distribute --no-assemble to repeat just the exonerate step.

1.3 The Herbarium Update January, 2018

Features

  • Added --exclude flag to be the inverse of --target: all sequences with the specified string will not be used as targets for exon extraction (they will still be used for read-mapping). Useful if you want to add supercontig sequence to the target file, but not use it for exon extraction.

  • Added --addN to intronerate.py. This feature will add 10 N characters in between joined contig when recovering the supercontig. This is useful for identifying where the intron recovery fails, and for annotation processing (i.e. for GenBank).

  • Added a new version of the heatmap script, gene_recovery_heatmap_ggplot.R. This script is much simpler and produces nice color PNG images, but struggles a bit on PDF output. The original heatmap script is stil included. Thanks to Paul Wolf for the ggplot code!

Bug Fixes

  • Fixed misassembly of supercontigs when there are multiple alignments to different parts of the same exon.
  • Fixed poor filtering of GFF results to produce intron/exon annotation.
  • Fixed non-propogation of exonerate parameters

1.2.1 September, 2017

Bug Fixes

  • Fixed assembly issue when gene does not have unpaired reads.
  • Fixed distribution of targets when unpaired reads present.
  • Fixed use of unpaired reads to detect best target.

1.2 May, 2017

Features

  • Added --unpaired flag. When using paired-end sequencing reads, a third read file may be specified with this flag. Reads will be mapped to targets separately, but will be used along with paired reads in contig assembly.

  • Added --target flag. Adds the ability to choose which of the reference sequences is used for each gene. If --target is a file (tab-delimited file with one gene and one target name per line), HybPiper will use that. Otherwise --target can be the name of one reference. HybPiper will only use targets with the specified name in the Alignment/Exon Extraction phase. All other targets for that locus will only be used in the Mapping/Read Sorting phase.

  • Added --timeout flag, which uses GNU Parallel to kill processes (i.e. Spades or Exonerate) if they take X percent longer than average. Use if there are a lot of stuck jobs (--timeout 1000)

  • Python 3 compatibility

Bug Fixes

  • Can accommodate Solexa FASTQ paired headers
  • Fixed spades_runner.py not recognizing --cpu on redos
  • Prints more meaningful messages for some common errors
  • Can accommodate prefix not being in current directory
  • Deletes sorted reads on restart to prevent double counting reads.
  • spades_runner.py will now respect --kvals
  • Added initial call to log for reads_first.py

1.1 May, 2016: Release associated with manuscript in Applications in Plant Sciences.

  • Added paralog_investigator.py, which detects and extracts long exons from putative paralogs in all genes in one sample.

  • Added paralog_retriever.py, which retrieves sequences generated by paralog_investigator.py for many samples (or the coding sequence generated by exonerate_hits.py if no paralogs are detected).

  • Added a test_dataset of 13 genes for 9 samples, and a shell script to run the test data through the main script and several post-processing scripts.

  • Fixed bug involving calling HybPiper with a relative path such as: ../reads_first.py

  • reads_first.py --check_depend now checks for SPAdes, BWA, and Samtools

  • Full revision of README, which is now shorter. Full tutorials on installing and running HybPiper are now on the Wiki.

1.0 Feb, 2016: Initial fully-featured release associated with submission of manuscript to Applications in Plant Sciences.

-- Sequence assembly now uses SPAdes rather than Velvet + CAP3