Mercurial > repos > dereeper > pangenome_explorer
diff COG/bac-genomics-scripts/prot_finder/README.md @ 3:e42d30da7a74 draft
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| author | dereeper |
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| date | Thu, 30 May 2024 11:52:25 +0000 |
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/COG/bac-genomics-scripts/prot_finder/README.md Thu May 30 11:52:25 2024 +0000 @@ -0,0 +1,664 @@ +prot_finder +=========== + +`prot_finder.pl` is a script to search for query protein homologs in annotated bacterial genomes with **BLASTP**. A companion bash shell script pipeline is available, `prot_finder_pipe.sh`. + +Included is also the script `prot_binary_matrix.pl` to create a binary presence/absence matrix (e.g. for [**iTOL**](http://itol.embl.de/)) from the `prot_finder.pl` output. Additionally, two downstream scripts are provided to wrangle these binary presence/absence matrix: `transpose_matrix.pl` to transpose a delimited TEXT matrix and `binary_group_stats.pl` to get overall presence/absence statistics for groups of columns in a delimited binary TEXT matrix (in the style of [`po2group_stats.pl`](/po2group_stats)). + +* [Synopsis](#synopsis) + * [prot_finder synopsis](#prot_finder-synopsis) + * [prot_binary_matrix synopsis](#prot_binary_matrix-synopsis) + * [transpose_matrix synopsis](#transpose_matrix-synopsis) + * [binary_group_stats synopsis](#binary_group_stats-synopsis) +* [Description](#description) +* [Usage](#usage) + * [prot_finder usage](#prot_finder-usage) + * [Manual consecutively](#manual-consecutively) + * [cds_extractor for subject proteins](#cds_extractor-for-subject-proteins) + * [Legacy BLASTP](#legacy-blastp) + * [BLASTP plus](#blastp-plus) + * [prot_finder](#prot_finder-1) + * [prot_finder_pipe bash script pipeline](#prot_finder_pipe-bash-script-pipeline) + * [prot_binary_matrix usage](#prot_binary_matrix-usage) + * [transpose_matrix usage](#transpose_matrix-usage) + * [binary_group_stats usage](#binary_group_stats-usage) +* [Options](#options) + * [prot_finder.pl options](#prot_finderpl-options) + * [Mandatory prot_finder.pl options](#mandatory-prot_finderpl-options) + * [Optional prot_finder.pl options](#optional-prot_finderpl-options) + * [prot_finder_pipe.sh options](#prot_finder_pipesh-options) + * [Mandatory prot_finder_pipe.sh options](#mandatory-prot_finder_pipesh-options) + * [Optional prot_finder_pipe.sh options](#optional-prot_finder_pipesh-options) + * [prot_binary_matrix.pl options](#prot_binary_matrixpl-options) + * [transpose_matrix.pl options](#transpose_matrixpl-options) + * [binary_group_stats.pl options](#binary_group_statspl-options) + * [Mandatory binary_group_stats.pl options](#mandatory-binary_group_statspl-options) + * [Optional binary_group_stats.pl options](#optional-binary_group_statspl-options) +* [Output](#output) + * [cds_extractor.pl output](#cds_extractorpl-output) + * [prot_finder.pl output](#prot_finderpl-output) + * [prot_finder_pipe.sh output](#prot_finder_pipesh-output) + * [prot_binary_matrix.pl output](#prot_binary_matrixpl-output) + * [transpose_matrix.pl output](#transpose_matrixpl-output) + * [binary_group_stats.pl output](#binary_group_statspl-output) +* [Dependencies](#dependencies) + * [`prot_finder.pl`/`prot_finder_pipe.sh` dependencies](#prot_finderplprot_finder_pipesh-dependencies) + * [`binary_group_stats.pl` dependencies](#binary_group_statspl-dependencies) +* [Run environment](#run-environment) +* [Author - contact](#author---contact) +* [Citation, installation, and license](#citation-installation-and-license) +* [Acknowledgements](#acknowledgements) +* [Changelog](#changelog) + * [prot_finder changelog](#prot_finder-changelog) + * [prot_binary_matrix changelog](#prot_binary_matrix-changelog) + * [transpose_matrix changelog](#transpose_matrix-changelog) + * [binary_group_stats changelog](#binary_group_stats-changelog) + +## Synopsis + +### prot_finder synopsis + + ./prot_finder_pipe.sh -q query.faa (-s subject.faa|-f (embl|gbk)) > blast_hits.tsv + +**or** + + perl prot_finder.pl -r report.blastp -s subject.faa > blast_hits.tsv + +### prot_binary_matrix synopsis + + perl prot_binary_matrix.pl blast_hits.tsv > binary_matrix.tsv + +**or** + + perl prot_finder.pl -r report.blastp -s subject.faa | perl prot_binary_matrix.pl > binary_matrix.tsv + +### transpose_matrix synopsis + + perl transpose_matrix.pl input_matrix.tsv > input_matrix_transposed.tsv + +**or** + + perl prot_binary_matrix.pl blast_hits.tsv | perl transpose_matrix.pl > binary_matrix_transposed.tsv + +### binary_group_stats synopsis + + perl binary_group_stats.pl -i binary_matrix.tsv -g group_file.tsv -p > overall_stats.tsv + +## Description + +The script `prot_finder.pl` is intended to search for homologous proteins in annotated bacterial genomes. For this purpose, a previous [**BLASTP**](http://blast.ncbi.nlm.nih.gov/Blast.cgi), either [legacy or plus](https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download), needs to be run with query protein sequences against a **BLASTP** database of subject proteins (e.g. all proteins from several *Escherichia coli* genomes). + +The script [`cds_extractor.pl`](/cds_extractor) (with options **-p -f**) can be used to create multi-FASTA protein files of all non-pseudo CDS from RichSeq genome files to create the needed subject **BLASTP** database. Present locus tags will be used as FASTA IDs, but see [`cds_extractor.pl`](/cds_extractor) for a description of the format. Query protein sequences for the **BLASTP** need a **unique** FASTA ID. + +The **BLASTP** report file (option **-r**), the subject protein multi-FASTA file (option **-s**), and optionally the query protein (multi-)FASTA (option **-q**) file are then given to `prot_finder.pl`. Significant **BLASTP** subject hits are filtered according to the given cutoffs (options **-i**, **-cov_q**, and **-cov_s**) and the result is printed as an informative tab-separated result table to *STDOUT*. To apply global identity/coverage cutoffs to subject hits high-scoring pairs (HSPs) are tiled (see http://www.bioperl.org/wiki/HOWTO:Tiling and http://search.cpan.org/dist/BioPerl/Bio/Search/Hit/GenericHit.pm). Additionally, the subject protein sequences with significant query hits are written to result multi-FASTA files, named according to the respective query FASTA IDs (optionally including the query sequence with option **-q**). + +Optionally, [**Clustal Omega**](http://www.clustal.org/omega/) can be called (option **-a** with optional **-p**) to create multiple alignments (FASTA format) for each of the resulting multi-FASTA files. These alignments can be used to calculate phylogenies e.g. with **RAxML** (http://sco.h-its.org/exelixis/software.html) or **MEGA** (http://www.megasoftware.net/). + +Run the script [`cds_extractor.pl`](/cds_extractor) (with options **-p -f**) and the **BLASTP** manually or use the bash shell wrapper script `prot_finder_pipe.sh` (see below ['prot_finder_pipe bash script pipeline'](#prot_finder_pipe-bash-script-pipeline)) to execute the whole pipeline including `prot_finder.pl` (with optional option **-q**). For additional options of the pipeline shell script see below ['prot_finder_pipe.sh options'](#prot_finder_pipesh-options). Be aware that some options in `prot_finder_pipe.sh` corresponding to options in `prot_finder.pl` have different names (**-c** instead of **-cov_q**, **-k** instead of **-cov_s**, and **-o** instead of **-p**; also **-f** has a different meaning). If [`cds_extractor.pl`](/cds_extractor) is used in the pipeline (option **-f** of the shell script) the working folder has to contain the annotated bacterial genome subject files (in RichSeq format, e.g. EMBL or GENBANK format). Also, the Perl scripts [`cds_extractor.pl`](/cds_extractor) (only for `prot_finder_pipe.sh` option **-f**) and `prot_finder.pl`have to be either contained in the current working directory or installed in the global *PATH*. **BLASTP** (legacy and/or plus) and **Clustal Omega** binaries have to be installed in global *PATH*, or for **Clustal Omega** you can give the path to the binary with option **-o**. In the pipeline **BLASTP** is run with **disabled** query filtering, locally optimal Smith-Waterman alignments, and increasing the number of database sequences to show alignments to 500 for [**BioPerl**](http://www.bioperl.org) parsing (legacy: **-F F -s T -b 500**, plus: **-seg no -use_sw_tback -num_alignments 500**). The pipeline script ends with the *STDERR* message 'Pipeline finished!', if this is not the case have a look at the log files in the result directory for errors. + +The resulting tab-separated table with significant **BLASTP** hits (from `prot_finder.pl` or `prot_finder_pipe.sh`) can be given to the script `prot_binary_matrix.pl`, either as *STDIN* or as a file, to create a presence/absence matrix of the results. See below ['prot_binary_matrix.pl options'](#prot_binary_matrixpl-options) for the `prot_binary_matrix.pl` options. By default a tab-delimited binary presence/absence matrix for query hits per subject organism will be printed to *STDOUT*. Use option **-t** to count all query hits per subject organism, not just the binary presence/absence. This presence/absence matrix can be given to the script `transpose_matrix.pl`, either as *STDIN* or as a file, to transpose the matrix, i.e. rows will become columns and columns rows. Actually, `transpose_matrix.pl` can be used to transpose any delimited TEXT matrix (see below ['transpose_matrix.pl options'](#transpose_matrixpl-options)). + +The presence/absence matri(x|ces) can e.g. be loaded into the Interactive Tree Of Life website ([**iTOL**](http://itol.embl.de/)) to associate the data with a phylogenetic tree. [**iTOL**](http://itol.embl.de/) likes individual comma-separated input files, thus use `prot_binary_matrix.pl` options **-s -c** for this purpose. However, the organism names have to have identical names to the leaves of the phylogenetic tree, thus manual adaptation, e.g. in a spreadsheet software (like [**LibreOffice Calc**](https://www.libreoffice.org/discover/calc/)), might be needed. **Careful**, subject organisms without a significant **BLASTP** hit won't be included in the `prot_finder.pl` result table and hence can't be included by `prot_binary_matrix.pl`. If needed add them manually to the result matri(x|ces). + +At last, script `binary_group_stats.pl` can be used to categorize columns of the binary presence/absence matrix from `prot_binary_matrix.pl` according to group affiliations. `binary_group_stats.pl` is based upon [`po2group_stats.pl`](/po2group_stats), which does the same thing for genomes in an ortholog/paralog output matrix from a [Proteinortho5](http://www.bioinf.uni-leipzig.de/Software/proteinortho/) calculation. Actually, `binary_group_stats.pl` can work with any delimited TEXT **binary** matrix (option **-i**). But, all fields of the binary matrix need to be filled with either a **0** indicating absence or a **1** indicating presence, i.e. all rows need to have the same number of columns. Use option **-d** to set the delimiter of the input matrix, default is set to tab-delimited/separated matrices. + +Also, column headers in the first row and row headers in the first column are **mandatory** for the input binary matrix. Only alphanumeric (a-z, A-Z, 0-9), underscore (_), dash (-), and period (.) characters are allowed for the **column headers** and **group names** in the group file (option **-g**) to avoid downstream problems with the operating/file system. As a consequence, also no whitespaces are allowed in these! Additionally, **column headers**, **row headers**, and **group names** need to be **unique**. + +The group affiliations of the columns are intended to get overall presence/absence statistics for groups of columns and not simply single columns of the matrix. Percentage inclusion (option **-cut_i**) and exclusion (option **-cut_e**) cutoffs can be set to define how strict the presence/absence of column groups within a row are defined. Of course groups can also hold only single column headers to get single column statistics. Group affiliations are defined in a mandatory **tab-delimited** group input file (option **-g**), including the column headers from the input binary matrix, with **minimal two** and **maximal four** groups. + +See the *README.md* of [`po2group_stats.pl`](/po2group_stats) for an explanation of the logic behind the categorization and the resulting group binary matrix and venn diagram of `binary_group_stats.pl` (and of course its [options](#binary_group_statspl-options) and [output](#binary_group_statspl-output)). + +## Usage + +### prot_finder usage + +#### Manual consecutively + +##### cds_extractor for subject proteins + + for file in *.(gbk|embl); do perl cds_extractor.pl -i "$file" -p -f; done + cat *.faa > subject.faa + rm !(subject).faa + +##### Legacy **BLASTP** + + formatdb -p T -i subject.faa -n prot_finder_db + blastall -p blastp -d prot_finder_db -i query.faa -o prot_finder.blastp -e 1e-10 -F F -s T -b 500 + +**or** + +##### **BLASTP** plus + + makeblastdb -dbtype prot -in subject.faa -out prot_finder_db + blastp -db prot_finder_db -query query.faa -out prot_finder.blastp -evalue 1e-10 -seg no -use_sw_tback -num_alignments 500 + +##### prot_finder + + perl prot_finder.pl -r prot_finder.blastp -s subject.faa -cov_s 80 > blast_hits.tsv + +**or** + + perl prot_finder.pl -r prot_finder.blastp -s subject.faa -d result_dir -f -q query.faa -i 50 -cov_q 50 -b -a -p ~/bin/clustalo -t 6 > result_dir/blast_hits.tsv + +#### prot_finder_pipe bash script pipeline + + ./prot_finder_pipe.sh -q query.faa -s subject.faa > blast_hits.tsv + +**or** + + ./prot_finder_pipe.sh -q query.faa -f embl -d result_dir -p legacy -e 0 -t 12 -i 50 -c 50 -k 30 -b -a -o ~/bin/clustalo -m > result_dir/blast_hits.tsv + +### prot_binary_matrix usage + + perl prot_binary_matrix.pl -s -d result_dir -t blast_hits.tsv + +**or** + + perl prot_finder.pl -r report.blastp -s subject.faa | perl prot_binary_matrix.pl -l -c > binary_matrix.csv + +**or** + + mkdir result_dir && ./prot_finder_pipe.sh -q query.faa -s subject.faa -d result_dir -m | tee result_dir/blast_hits.tsv | perl prot_binary_matrix.pl > binary_matrix.tsv + +### transpose_matrix usage + + perl transpose_matrix.pl -d ' ' -e NA input_matrix_space-delimit.txt > input_matrix_space-delimit_transposed.txt + +**or** + + perl prot_finder.pl -r report.blastp -s subject.faa | perl prot_binary_matrix.pl -l -c | perl transpose_matrix.pl -d , > binary_matrix_transposed.csv + +**or** + + mkdir result_dir && ./prot_finder_pipe.sh -q query.faa -s subject.faa -d result_dir -m | tee result_dir/blast_hits.tsv | perl prot_binary_matrix.pl | tee result_dir/binary_matrix.tsv | perl transpose_matrix.pl > result_dir/binary_matrix_transposed.tsv + +Transpose all matrices in a folder: + + for matrix in *.tsv; do perl transpose_matrix.pl "$matrix" > "${matrix%.*}_transposed.tsv"; done + +### binary_group_stats usage + + perl binary_group_stats.pl -i binary_matrix_transposed.csv -g group_file.tsv -d , -r result_dir -cut_i 0.7 -cut_e 0.2 -b -p -co -s -u -a > overall_stats.tsv + +## Options + +### `prot_finder.pl` options + +#### Mandatory `prot_finder.pl` options + +- **-r**=_str_, **-report**=_str_ + + Path to **BLASTP** report/output + +- **-s**=_str_, **-subject**=_str_ + + Path to subject multi-FASTA protein sequence file (\*.faa) created with [`cds_extractor.pl`](/cds_extractor) (and its options **-p -f**), which was used to create the **BLASTP** database + +#### Optional `prot_finder.pl` options + +- **-h**, **-help** + + Help (perldoc POD) + +- **-d**=_str_, **-dir_result**=_str_ + + Path to result folder [default = query identity and coverage cutoffs, './results_i#_cq#'] + +- **-f**, **-force_dir** + + Force output to an existing result folder, otherwise ask user to remove content of existing folder. Careful, files from a previous analysis might not be overwritten if different to current analysis. + +- **-q**=_str_, **-query**=_str_ + + Path to query (multi-)FASTA protein sequence file (\*.faa) with **unique** FASTA IDs, which was used as query in the **BLASTP**. Will include each query protein sequence in the respective multi-FASTA 'query-ID_hits.faa' result file. + +- **-b**, **-best_hit** + + Give only the best hit (i.e. highest identity) for each subject sequence if a subject has several hits with different queries + +- **-i**=_int_, **-ident_cutoff**=_int_ + + Query identity cutoff for significant hits (not including gaps), has to be an integer number >= 0 and <= 100 [default = 70] + +- **-cov_q**=_int_, **-cov_query_cutoff**=_int_ + + Query coverage cutoff, has to be an integer number >= 0 and <= 100 [default = 70] + +- **-cov_s**=_int_, **-cov_subject_cutoff**=_int_ + + Subject/hit coverage cutoff, has to be an integer >= 0 and <= 100 [default = 0] + +- **-a**, **-align_clustalo** + + Call [**Clustal Omega**](http://www.clustal.org/omega/) for multiple alignment of each 'query-ID_hits.faa' result file + +- **-p**=_str_, **-path_clustalo**=_str_ + + Path to executable **Clustal Omega** binary if not present in global *PATH* variable; requires option **-a** + +- **-t**=_int_, **-threads_clustalo**=_int_ + + Number of threads for **Clustal Omega** to use; requires option **-a** [default = all processors on system] + +- **-v**, **-version** + + Print version number to *STDERR* + +### `prot_finder_pipe.sh` options + +#### Mandatory `prot_finder_pipe.sh` options + +- **-q**=_str_ + + Path to query protein (multi-)FASTA file (\*.faa) with **unique** FASTA IDs + +- **-f**=_str_ + + File extension for files in the **current** working directory to use for [`cds_extractor.pl`](/cds_extractor) (e.g. 'embl' or 'gbk'); excludes shell script option **-s** + +**or** + +- **-s**=_str_ + + Path to subject protein multi-FASTA file (\*.faa) already created with [`cds_extractor.pl`](/cds_extractor) (and its options **-p -f**), will not run [`cds_extractor.pl`](/cds_extractor); excludes shell script option **-f** + +#### Optional `prot_finder_pipe.sh` options + +- **-h** + + Print usage + +- **-d**=_str_ + + Path to result folder [default = query identity and coverage cutoffs,'./results_i#_cq#'] + +- **-p**=(legacy|plus) + + **BLASTP** suite to use [default = plus] + +- **-e**=_real_ + + E-value for **BLASTP** [default = 1e-10] + +- **-t**=_int_ + + Number of threads to be used for **BLASTP** and **Clustal Omega** [default = all processors on system] + +- **-i**=_int_ + + Query identity cutoff for significant hits [default = 70] + +- **-c**=_int_ + + Query coverage cutoff (corresponds to [`prot_finder.pl` option](#optional-prot_finderpl-options) **-cov_q**) [default = 70] + +- **-k**=_int_ + + Subject coverage cutoff (corresponds to [`prot_finder.pl` option](#optional-prot_finderpl-options) **-cov_s**) [default = 0] + +- **-b** + + Give only the best hit (i.e. highest identity) for each subject sequence + +- **-a** + + Multiple alignment of each multi-FASTA result file with [**Clustal Omega**](http://www.clustal.org/omega/) + +- **-o**=_str_ + + Path to executable **Clustal Omega** binary if not in global *PATH*; requires shell script option **-a** (corresponds to [`prot_finder.pl` option](#optional-prot_finderpl-options) **-p**) + +- **-m** + + Clean up all non-essential files, see below ['`prot_finder_pipe.sh` output'](#prot_finder_pipesh-output) + +### `prot_binary_matrix.pl` options + +- **-h**, **-help** + + Help (perldoc POD) + +- **-s**, **-separate** + + Separate presence/absence files for each query protein printed to the result directory [default without **-s** = *STDOUT* matrix for all query proteins combined] + +- **-d**=_str_, **-dir_result**=_str_ + + Path to result folder, requires option **-s** [default = './binary_matrix_results'] + +- **-t**, **-total** + + Count total occurrences of query proteins, not just presence/absence binary + +- **-c**, **-csv** + + Output matri(x|ces) in comma-separated format (\*.csv) instead of tab-delimited format (\*.tsv) + +- **-l**, **-locus_tag** + + Use the locus_tag **prefixes** in the subject_ID column of the `prot_finder.pl` output (instead of the subject_organism column) as organism IDs to associate query hits to organisms. The subject_ID column will include locus_tags if they're annotated for a genome (see the [`cds_extractor.pl`](/cds_extractor) format description). Useful if the [`cds_extractor.pl`](/cds_extractor) output doesn't include strain names for 'o=' in the FASTA IDs, because the prefix of a locus_tag should be unique for a genome (see http://www.ncbi.nlm.nih.gov/genbank/genomesubmit_annotation). + +- **-v**, **-version** + + Print version number to *STDERR* + +### `transpose_matrix.pl` options + +- **-h**, **-help** + + Help (perldoc POD) + +- **-d**=_str_, **-delimiter**=_str_ + + Set delimiter of input and output matrix (e.g. comma ',', single space ' ' etc.) [default = tab-delimited/separated] + +- **-e**=_str_, **-empty**=_str_ + + Fill empty cells of the input matrix with a value in the transposed matrix (e.g. 'NA', '0' etc.) + +- **-v**, **-version** + + Print version number to *STDERR* + +### `binary_group_stats.pl` options + +#### Mandatory `binary_group_stats.pl` options + +- **-i**=*str*, **-input**=*str* + + Input delimited TEXT binary matrix (e.g. *.tsv, *.csv, or *.txt), see option **-d** + +- **-g**=*str*, **-groups_file**=*str* + + Tab-delimited file with group affiliation for the columns from the input binary matrix with **minimal two** and **maximal four** groups (easiest to create in a spreadsheet software and save in tab-separated format). **All** column headers from the input binary matrix need to be included. Column headers and group names can only include alphanumeric (a-z, A-Z, 0-9), underscore (_), dash (-), and period (.) characters (no whitespaces allowed either). Example format with two column headers in group A, three in group B and D, and one in group C: + + group\_A group\_B group\_C group\_D<br> + column\_header1 column\_header9 column\_header3 column\_header8<br> + column\_header7 column\_header6  column\_header5<br> +  column\_header4  column\_header2 + +#### Optional `binary_group_stats.pl` options + +- **-h**, **-help** + + Help (perldoc POD) + +- **-d**=*str*, **-delimiter**=*str* + + Set delimiter of input binary matrix (e.g. comma ',', single space ' ' etc.) [default = tab-delimited/separated] + +- **-r**=*str*, **-result\_dir**=*str* + + Path to result folder \[default = inclusion and exclusion percentage cutoff, './results\_i#\_e#'\] + +- **-cut\_i**=*float*, **-cut\_inclusion**=*float* + + Percentage inclusion cutoff for column presence counts in a group per row, has to be > 0 and <= 1. Cutoff will be rounded according to the column header number in each group and has to be > the rounded exclusion cutoff in this group. \[default = 0.9\] + +- **-cut\_e**=*float*, **-cut\_exclusion**=*float* + + Percentage exclusion cutoff, has to be >= 0 and < 1. Rounded cutoff has to be < rounded inclusion cutoff. \[default = 0.1\] + +- **-b**, **-binary\_group\_matrix** + + Print a group binary matrix with the presence/absence column group results according to the cutoffs (excluding 'unspecific' category rows) + +- **-p**, **-plot\_venn** + + Plot venn diagram from the group binary matrix (except 'unspecific' and 'underrepresented' categories) with function `venn` from **R** package **gplots**, requires option **-b** + +- **-co**, **-core_strict** + + Include 'strict core' category in output for rows where **all** columns have a '1' + +- **-s**, **-singletons** + + Include singleton/column-specific rows for each column header in the output, activates also overall column header presence ('1') counts in final stats matrix for columns with singletons + +- **-u**, **-unspecific** + + Include 'unspecific' category in output + +- **-a**, **-all\_column\_presence\_overall** + + Report overall presence counts for all column headers (appended to the final stats matrix), also those without singletons; will include all overall column header presence counts without option **-s** + +- **-v**, **-version** + + Print version number to *STDERR* + +## Output + +### `cds_extractor.pl` output + +- \*.faa + + Multi-FASTA file(s) of subject CDS protein sequences; will be removed with [`prot_finder_pipe.sh` option](#optional-prot_finder_pipesh-options) **-m** + +For optional error files from [`cds_extractor.pl`](/cds_extractor) see its documentation. + +### `prot_finder.pl` output + +- *STDOUT* + + The resulting tab-delimited output table with the significant subject **BLASTP** hits is printed to *STDOUT*. Redirect (e.g. to a file in the result directory, options **-d -f**) or pipe into another tool as needed (e.g. `prot_binary_matrix.pl`). + +- ./results_i#_cq# + + All output files are stored in a result folder + +- ./results_i#_cq#/query-ID_hits.faa + + Multi-FASTA protein files of significant subject hits for each query protein (named after the respective query FASTA ID), optionally includes the respective query protein sequence (with option **-q**) + +- subject.faa.idx + + Index file of the subject protein file for fast sequence retrieval (can be deleted if no further **BLASTPs** are needed with these subject sequences); will be removed with [`prot_finder_pipe.sh` option](#optional-prot_finder_pipesh-options) **-m** + +- (./results_i#_cq#/queries_no_blastp-hits.txt) + + Lists all query sequence IDs without significant subject hits; with option **-b** includes also queries with significant hits but *without* a best blast hit for a subject + +- (./results_i#_cq#/clustal_omega.log) + + Optional log file of verbose **Clustal Omega** *STDOUT*/*STDERR* messages; will be removed with [`prot_finder_pipe.sh` option](#optional-prot_finder_pipesh-options) **-m** + +- (./results_i#_cq#/query-ID_aln.fasta) + + Optional **Clustal Omega** multiple alignment of each 'query-ID_hits.faa' result file in FASTA alignment format + +- (./results_i#_cq#/query-ID_tree.nwk) + + Optional, **Clustal Omega** NJ-guide tree in Newick format + +### `prot_finder_pipe.sh` output + +In addition to the [`cds_extractor.pl` output](#cds_extractorpl-output) and the [`prot_finder.pl` output](#prot_finderpl-output) the pipeline also creates the following non-essential output files, which will be removed with [`prot_finder_pipe.sh` option](#optional-prot_finder_pipesh-options) **-m**: + +- ./results_i#_cq#/cds_extractor.log + + Log file of [`cds_extractor.pl`](/cds_extractor) *STDOUT*/*STDERR* messages (with `prot_finder_pipe.sh` option **-f**) + +- ./results_i#_cq#/prot_finder.faa + + Concatenated [`cds_extractor.pl`](/cds_extractor) output files to create the subject **BLASTP** database + +- prot_finder_db.phr, prot_finder_db.pin, prot_finder_db.psq + + **BLASTP** database files from the concatenated subject sequences ('prot_finder.faa') + +- formatdb.log, error.log **or** ./results_i#_cq#/makeblastdb.log + + Legacy **BLASTP** or **BLASTP+** log files + +- ./results_i#_cq#/prot_finder.blastp + + **BLASTP** report/output + +- ./results_i#_cq#/prot_finder.log + + Log file of `prot_finder.pl` *STDOUT*/*STDERR* messages + +### `prot_binary_matrix.pl` output + +- *STDOUT* + + The resulting presence/absence matrix is printed to *STDOUT* without option **-s**. Redirect or pipe into another tool as needed. + +- (./binary_matrix_results) + + Separate query presence/absence files are stored in a result folder with option **-s** + +- (./binary_matrix_results/query-ID_binary_matrix.(tsv|csv)) + + Separate query presence/absence files with option **-s** + +### `transpose_matrix.pl` output + +- *STDOUT* + + The transposed matrix is printed to *STDOUT*. Redirect or pipe into another tool as needed. + +### `binary_group_stats.pl` output + +- *STDOUT* + + The tab-delimited final stats matrix is printed to *STDOUT*. Redirect or pipe into another tool as needed. + +- ./results_i#_e# + + All output files are stored in a results folder + +- ./results_i#_e#/[\*_specific|\*_absent|cutoff_core|underrepresented]_rows.txt + + Files including the row headers for rows in non-optional categories + +- (./results_i#_e#/[\*_singletons|strict_core|unspecific]_rows.txt) + + Optional category output files with the respective row headers + +- (./results_i#_e#/binary_matrix.tsv) + + Tab-delimited binary matrix of group presence/absence results according to cutoffs (excluding 'unspecific' rows) + +- (./results_i#_e#/venn_diagram.pdf) + + Venn diagram for non-optional categories (except 'unspecific' and 'underrepresented' categories) + +## Dependencies + +### `prot_finder.pl`/`prot_finder_pipe.sh` dependencies + +- [**BioPerl**](http://www.bioperl.org) (tested version 1.006923) +- [`cds_extractor`](/cds_extractor) (tested version 0.7.1) +- [**BLASTP**](https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download) + - legacy **BLASTP** (tested version 2.2.26) + - **BLASTP+** (tested version 2.2.28+) +- [**Clustal Omega**](http://www.clustal.org/omega/) (tested version 1.2.1) + +### `binary_group_stats.pl` dependencies + +- **Statistical computing language [R](http://www.r-project.org/)** + + `Rscript` is needed to plot the venn diagram with option **-p**, tested with version 3.2.2 + +- **gplots (https://cran.r-project.org/web/packages/gplots/index.html)** + + Package needed for **R** to plot the venn diagram, includes function `venn`. Tested with **gplots** version 2.17.0. + +## Run environment + +The scripts run under UNIX flavors. + +## Author - contact + +Andreas Leimbach (aleimba[at]gmx[dot]de; Microbial Genome Plasticity, Institute of Hygiene, University of Muenster) + +## Citation, installation, and license + +For [citation](https://github.com/aleimba/bac-genomics-scripts#citation), [installation](https://github.com/aleimba/bac-genomics-scripts#installation-recommendations), and [license](https://github.com/aleimba/bac-genomics-scripts#license) information please see the repository main [*README.md*](https://github.com/aleimba/bac-genomics-scripts/blob/master/README.md). + +## Acknowledgements + +The Perl implementation for transposing a matrix on Stack Overflow +was very useful for `transpose_matrix.pl`: https://stackoverflow.com/questions/1729824/transpose-a-file-in-bash + +## Changelog + +### prot_finder changelog + +* v0.7.1 (05.04.2016) + * bug fix: significant but non-best blast hits with option **-b** now listed in *queries_no_blastp-hits.txt* +* v0.7 (23.11.2015) + * changed script name from `blast_prot_finder.pl` to `prot_finder.pl` + * fixed bug introduced in v0.6 with a `seek`, because option **-query** didn't pull query sequences anymore + * included version switch + * included 'use autodie;' pragma + * included `pod2usage` with Pod::Usage and `pod2usage`-die for Getopt::Long call + * tab-delimited output table with filtered **BLASTP** hits now printed to *STDOUT* instead of file and can be used as *STDIN* for `prot_binary_matrix.pl` + * result files now created in result directory to unclutter working dir (new options **-dir_result** and **-force_dir**) and replaced subroutine 'file_exist' with 'empty_dir' (also no *STDOUT* message which files were created anymore) + * new output file *queries_no_blastp-hits.txt* to list all queries without **BLASTP** hits + * new output file *clustal_omega.log* to log **Clustal Omega** verbose output + * additional new options, **-path_clustalo** and **-threads_clustalo** + * major code changes/restructuring and additions: + * POD syntax changes and additions for new code + * Perl syntax changes and removing code redundancies + * including script run status messages + * changes to subroutine 'split_fasta_header' to work more robustly with `split` instead of a regex and adapted to `cds_extractor.pl` v0.7+ output format + * included several additional option and input checks + * replaced simple `blast_prot_finder_legacy.sh` with more elaborate bash script pipeline `prot_finder_pipe.sh` + * changed some output column names and moved the subject_ID column before the subject_gene column +* v0.6 (10.06.2013) + * included BioPerls 'frac\*' methods, which include hsp tiling, to correct bug in query coverage and identity calculations for **whole** hits + * corrected bug to use **whole** hit e-value and not just first hsp e-value of a significant hit + * option **-cov_subject** for subject/hit coverage cutoff (not only query coverage cutoff) + * more info in POD +* v0.5 (14.05.2013) + * optionally include query proteins in result subject multi-FASTA files + * new option **-best_hit** to include only best **BLASTP** hit (highest identity) for each subject locus_tag, if a subject protein has hits to several query proteins + * print queries with no subject **BLASTP** hits to *STDOUT* +* v0.4 (24.01.2013) + * corrected bug in hash structure to store **BLASTP** hits, query acc/IDs (keys) and array reference of subject locus_tags (values), as several queries can have the same subject locus_tag as hit + * include 'subject protein_function' in 'blast_hits.txt' output +* v0.3 (12.09.2012) + * **original** script name `blast_prot_finder.pl` + * **BLASTP** hits are stored in a hash with subject locus_tags (keys) and query accessions/IDs (values) + +### prot_binary_matrix changelog + +* v0.6 (23.11.2015) + * adapted to `prot_finder.pl` v0.7 output + * included a POD, `pod2usage` with Pod::Usage and `pod2usage`-die for Getopt::Long call + * Perl syntax changes and some simpler loop structures + * Removed option **-input** and instead accept `prot_finder.pl` output as *STDIN* or file as argument + * Removed option **-c|-collective** (actually repurposed, see below), option **-separate** to indicate separate output is enough + * **Without** option **-separate** result matrix is now printed to *STDOUT* + * **With** option **-separate** result matrices now printed to result directory (name optionally given with new option **-dir_result**) + * New option **-locus_tag** to use the locus_tag prefix in column subject_ID of the `prot_finder.pl` output as ID, which is in most cases the locus_tag, instead of the subject_organism; controls if the locus_tags follow NCBI standards + * Output now default tab-separated format (*tsv*), optionally with new option **-csv** comma-separated (*csv*) + * Removed subroutine 'result' and statement which result files have been created +* v0.5 (05.03.2014) + * option **-total** to count all occurences of query proteins within one organism (paralogs), instead of just binary presence/absence + * enforce mandatory options + * changed script name to 'prot_binary_matrix.pl' + * version switch + * included 'use autodie' + * options with Getopt::Long + * changed usage to HERE document +* v0.4 (29.04.2013) + * adapted to new *prot_finders* 'blast_hits.txt' layout, which includes an additional column for 'subject protein_function' + * changed script name to 'blast_binary_matrix.pl' +* v0.3 (15.02.2013) + * status of created result files in *STDOUT* for option **-s** +* v0.2 (21.12.2012) + * prot_finder output file 'blast_hits.txt' doesn't have to be ordered by query protein accessions/IDs anymore +* v0.1 (25.10.2012) + * **original** script name 'blastp_iTOL_binary.pl' + +### transpose_matrix changelog + +* v0.1 (12.04.2016) + +### binary_group_stats changelog + +* v0.1 (06.06.2016)