obitools4/Release-notes.md

OBITools release notes

Latest changes

Breaking changes

• In obimultiplex, the short version of the --tag-list option used to specify the list of tags and primers to be used for the demultiplexing has been changed from -t to -s.

• The command obifind is now renamed obitaxonomy.

• The --taxdump option used to specify the path to the taxdump containing the NCBI taxonomy has been renamed to --taxonomy.

Bug fixes

• In obipairing, correct the stats seq_a_single and seq_b_single when on right alignment mode

• Not really a bug but the memory impact of obiuniq has been reduced by reducing the batch size and not reading the qualities from the fastq files as obiuniq is producing only fasta output without qualities.

New features

• obitoaxonomy a new --dump|D option allows for dumping a sub-taxonomy.

• Taxonomy dump can now be provided as a four-columns CSV file to the --taxonomy option.

• NCBI Taxonomy dump does not need to be uncompressed and unarchived anymore. The path of the tar and gziped dump file can be directly specified using the --taxonomy option.

• Most of the time obitools identify automatically sequence file format. But it fails sometimes. Two new option --fasta and --fastq are added to allow the processing of the rare fasta and fastq files not recognized.

• In obiscript, adds new methods to the Lua sequence object:

  • md5_string(): returning the MD5 check sum as an hexadecimal string,
  • subsequence(from,to): allows to extract a subsequence on a 0 based coordinate system, upper bound expluded like in go.
  • reverse_complement: returning a sequence object corresponding to the reverse complement of the current sequence.

Change of git repositiory

• The OBITools4 git repository has been moved to the github repository. The new address is: https://github.com/metabarcoding/obitools4. Take care for using the new install script for retrieving the new version.

  curl -L https://raw.githubusercontent.com/metabarcoding/obitools4/master/install_obitools.sh \
    | bash
  

  or with options:

  curl -L https://raw.githubusercontent.com/metabarcoding/obitools4/master/install_obitools.sh \
    | bash -s -- --install-dir test_install --obitools-prefix k
  

CPU limitation

• By default, OBITools4 tries to use all the computing power available on your computer. In some circumstances this can be problematic (e.g. if you are running on a computer cluster managed by your university). You can limit the number of CPU cores used by OBITools4 or by using the --max-cpu option or by setting the OBIMAXCPU environment variable. Some strange behaviour of OBITools4 has been observed when users try to limit the maximum number of usable CPU cores to one. This seems to be caused by the Go language, and it is not obvious to get OBITools4 to run correctly on a single core in all circumstances. Therefore, if you ask to use a single core, OBITools4 will print a warning message and actually set this parameter to two cores. If you really want a single core, you can use the --force-one-core option. But be aware that this can lead to incorrect calculations.

New features

• The output of the obitools will evolve to produce results only in standard formats such as fasta and fastq. For non-sequential data, the output will be in CSV format, with the separator ,, the decimal separator ., and a header line with the column names. It is more convenient to use the output in other programs. For example, you can use the csvtomd command to reformat the csv output into a markdown table. The first command to initiate this change is obicount, which now produces a 3-line CSV output.

  obicount data.csv | csvtomd 
  

• Adds the new experimental obicleandb utility to clean up reference database files created with obipcr. An easy way to create a reference database for obitag is to use obipcr on a local copy of Genbank or EMBL. However, these sequence databases are known to contain many taxonomic errors, such as bacterial sequences annotated with the taxid of their host species. obicleandb tries to detect these errors. To do this, it first keeps only sequences annotated with the taxid to which a species, genus, and family taxid can be assigned. Then, for each sequence, it compares the distance of the sequence to the other sequences belonging to the same genus to the same number of distances between the considered sequence and a randomly selected set of sequences belonging to another family using a Mann-Whitney U test. The alternative hypothesis is that out-of-family distances are greater than intrageneric distances. Sequences are annotated with the p-value of the Mann-Whitney U test in the obicleandb_trusted slot. Later, the distribution of this p-value can be analyzed to determine a threshold. Empirically, a threshold of 0.05 is a good compromise and allows to filter out less than 1‰ of the sequences. These sequences can then be removed using obigrep.

• Adds a new obijoin utility to join information contained in a sequence file with that contained in another sequence or CSV file. The command allows you to specify the names of the keys in the main sequence file and in the secondary data file that will be used to perform the join operation.

• Adds a new tool obidemerge to demerge a merge_xxx slot by recreating the multiple identical sequences having the slot xxx recreated with its initial value and the sequence count set to the number of occurences refered in the merge_xxx slot. During the operation, the merge_xxx slot is removed.

• Adds CSV as one of the input format for every obitools command. To encode sequence the CSV file must includes a column named sequence and another column named id. An extra column named qualities can be added to specify the quality scores of the sequence following the same ascii encoding than the fastq format. All the other columns will be considered as annotations and will be interpreted as JSON objects encoding potentially for atomic values. If a calumn value can not be decoded as JSON it will be considered as a string.

• A new option --version has been added to every obitools command. It will print the version of the command.

• In obiscript a qualities method has been added to retrieve or set the quality scores from a BioSequence object.\

• In obimultuplex the ngsfilter file describing the samples can be no provided not only using the classical nfsfilter format but also using the csv format. When using csv, the first line must contain the column names. 5 columns are expected:

  • experiment the name of the experiment
  • sample the name of the sample
  • sample_tag the tag used to identify the sample
  • forward_primer the forward primer sequence
  • reverse_primer the reverse primer sequence

  The order of the columns is not important, as long as they are present and named correctly. The obiparing command will print an error message if some column is missing. It now includes a **--template ** option that can be used to create an example CSV file.

  Supplementary columns are allowed. Their names and content will be used to annotate the sequence corresponding to the sample, as the key=value; did in the nfsfilter format.

  The CSV format used allows for comment lines starting with # character. Special data lines starting with @param in the first column allow to configure the algorithm. The options --template provided an over commented example of the csv format, including all the possible options.

Enhancement

• In every OBITools command, the progress bar are automatically deactivated when the standard error output is redirected.
• Because Genbank and ENA:EMBL contain very large sequences, while OBITools4 are optimized As Genbank and ENA:EMBL contain very large sequences, while OBITools4 is optimised for short sequences, obipcr faces some problems with excessive consumption of computer resources, especially memory. Several improvements in the tuning of the default obipcr parameters and some new features, currently only available for FASTA and FASTQ file readers, have been implemented to limit the memory impact of obipcr without changing the computational efficiency too much.
• Logging system and therefore format, have been homogenized.

Bug

• In obitag, correct the wrong assignment of the obitag_bestmatch attribute.
• In obiclean, the --no-progress-bar option disables all progress bars, not just the data.
• Several fixes in reading FASTA and FASTQ files, including some code simplification and and factorization.
• Fixed a bug in all obitools that caused the same file to be processed multiple times. when specifying a directory name as input.

April 2nd, 2024. Release 4.2.0

New features

• A new OBITools named obiscript allows to process each sequence according to a Lua script. This is an experimental tool. The --template option allows for generating an example script on the stdout.

API Changes

• Two of the main class obiseq.SeqWorker and obiseq.SeqWorker have their declaration changed. Both now return two values a obiseq.BioSequenceSlice and an error. This allow a worker to return potentially several sequences as the result of the processing of a single sequence, or zero, which is equivalent to filter out the input sequence.

Enhancement

• In obitag if the reference database contains sequences annotated by taxid not referenced in the taxonomy, the corresponding sequences are discarded from the reference database and a warning indicating the sequence id and the wrong taxid is emitted.
• The bug corrected in the parsing of EMBL and Genbank files as implemented in version 4.1.2 of OBITools4, potentially induced some reduction in the performance of the parsing. This should have been now fixed.
• In the same idea, parsing of genbank and EMBL files were reading and storing in memory not only the sequence but also the annotations (features table). Up to now none of the OBITools are using this information, but with large complete genomes, it is occupying a lot of memory. To reduce this impact, the new version of the parser doesn't any more store in memory the annotations by default.
• Add a --taxonomic-path to obiannotate. The option adds a taxonomic_path tag to sequences describing the taxonomic classification of the sequence according to its taxid. The path is a string. Each level of the path is delimited by a | character. A level consists of three parts separated by a @. The first part is the taxid, the second the scientific name and the last the taxonomic rank. The first level described is always the root of the taxonomy. The latest corresponds to the taxid of the sequence. If a sequence is not annotated by a taxid, as usual the sequence is assumed having the taxid 1 (the root of the taxonomy).

Bug fixes

• Fix a bug in the parsing of the JSON header of FASTA and FASTQ files occurring when a string includes a curly brace.
• Fix a bug in the function looking for the closest match in obitag. This error led to some wrong taxonomic assignment.
• Fix a bug in the writing of the fastq files, when quality of a nucleotide was not in the range 0-93.

February 16th, 2024. Release 4.1.2

Bug fixes

• Several bugs in the parsing of EMBL and Genbank files have been fixed. The bugs occurred in the case of very large sequences, such as complete genomes. The Genbank parser is now more robust. It breaks for more errors than the previous version. This allows to detect parsing errors instead of hiding them and producing wrong results.

December 20th, 2023. Release 4.1.1

New feature

• In obimatrix a --transpose option allows to transpose the produced matrix table in CSV format.
• In obitpairing and obipcrtag two new options --exact-mode and --fast-absolute to control the heuristic used in the alignment procedure. --exact-mode allows for disconnecting the heuristic and run the exact algorithm at the cost of a speed. --fast-absolute change the scoring schema of the heuristic.
• In obiannotate adds the possibility to annotate the first match of a pattern using the same algorithm than the one used in obipcr and obimultiplex. For that four option were added :
  • --pattern : to specify the pattern. It can use IUPAC codes and position with no error tolerated has to be followed by a # character.
  • --pattern-name : To specify the names of the slot used to report the results. Default is pattern
  • --pattern-error : To specify the maximum number of error tolerated during matching process.
  • --allows-indels : By default considered errors are mismatched, this flag allows for indels. Only the first match is reported if several occurrences exist. If no match is found on direct strand then pattern is looked for on the reverse complemented strand of the sequence.

Enhancement

• For efficiency purposes, now the obiuniq command run on disk by default. Consequently, the --on-disk option has been replaced by --in-memory to ask explicitly to use memory.
• Adds an option --penalty-scale to the obipairing and obipcrtag command to fine tune the pairing score in the system of the alignment procedure by applying a scaling factor to the mismatch score and the gap score relatively to the match score.

Bug fixes

• In obicsv, the --keep count was not equivalent to --count.
• In obipairing and obipcrtag, correct a bug in the alignment procedure leading to negative scores.
• In obimultiplex, correct a bug leading to a miss-read of the ngsfilter file when tags where written in lower case.
• In obitag, correct a bug leading to the annotation by taxid 1 (root) all the sequences having a 100% match with one the reference sequence.
• Correct a bug in the EMBL reader.

November 16th, 2023. Release 4.1.0

New feature

• In the OBITools language a new gc computes the GC fraction of a sequence.
• First version of the obisummary command. It produces summary statistics of the sequence file provided as input. The statistics includes, the number of reads, of variants, the total length of the DNA sequences (equivalent to obicount), some summaries about tags used in the sequence annotations and their frequencies of usage.
• First version of the obimatrix command. It allows producing OTU tables from sequence files in CSV format.
• The obicsv command has now a --auto option, that extract automatically the attributes present in a file for inspecting the beginning of the sequence file. Only attributes that do not correspond to map are reported. To extract information from map attributes, see the obimatrix command.

Enhancement

• A new completely rewritten GO version of the fastq and fasta parser is now used instead of the original C version.
• A new file format guesser is now implemented. This is a first step towards allowing new formats to be managed by OBITools.
• New way of handling header definitions of fasta and fastq formats with JSON headers. The sequence definition is now printed in new files as an attribute of the JSON header named "definition". That's facilitates the writing of parsers for the sequence headers.
• The -D (--delta) option has been added to obipcr. It allows extracting flanking sequences of the barcode.
  • If -D is not set, the output sequence is the barcode itself without the priming sites.
  • If -D is set to 0, the output sequence is the barcode with the priming sites.
  • When -D is set to ### (where ### is an integer), the output sequence is the barcode with the priming sites,
    and ### base pairs of flanking sequences.
• A new output format in JSON is proposed using the --json-output. The sequence file is printed as a JSON vector, where each element is a map corresponding to a sequence. The map has at most four elements:
  • "id" : which is the only mandatory element (string)
  • "sequence" : if sequence data is present in the record (string)
  • "qualities" : if quality data is associated to the record (string)
  • "annotations" : annotations is associated to the record (a map of annotations).

Bugs

• in the obitools language, the composition function now returns a map indexed by lowercase string "a", "c", "g", "t" and "o" for other instead of being indexed by the ASCII codes of the corresponding letters.
• Correction of the reverse-complement operation. Every reverse complement of the DNA sequence follow now the following rules :
  • Nucleotide codes are complemented to their lower complementary base
  • . and - characters are returned without change
  • [ is complemented to ] and oppositely
  • all other characters are complemented as n
• Correction of a bug is the Subsequence method of the BioSequence class, duplicating the quality values. This made obimultiplex to produce fastq files with sequences having quality values duplicated.

Becareful

GO 1.21.0 is out, and it includes new functionalities which are used in the OBITools4 code. If you use the recommanded method for compiling OBITools on your computer, their is no problem, as the script always load the latest GO version. If you rely on you personnal GO install, please think to update.

August 29th, 2023. Release 4.0.5

Bugs

• Patch a bug in the obiseq.BioSequence constructor leading to a error on almost every obitools. The error message indicates : fatal error: sync: unlock of unlocked mutex This bug was introduced in the release 4.0.4

August 27th, 2023. Release 4.0.4

Bugs

• Patch a bug in the install-script for correctly follow download redirection.
• Patch a bug in obitagpcr to consider the renaming of the forward_mismatch and reverse_mismatch tags to forward_error and reverse_error.

Enhancement

• Comparison algorithms in obitag and obirefidx take more advantage of the data structure to limit the number of alignments actually computed. This increase a bit the speed of both the software. obirefidx is nevertheless still too slow compared to my expectation.
• Switch to a parallel version of the gzip library, allowing for high speed compress and decompress operation on files.

New feature

• In every OBITools, writing an empty sequence (sequence of length equal to zero) through an error and stops the execution of the tool, except if the --skip-empty option is set. In that case, the empty sequence is ignored and not printed to the output. When output involved paired sequence the --skip-empty option is ignored.
• In obiannotate adds the --set-identifier option to edit the sequence identifier
• In obitag adds the --save-db option allowing at the end of the run of obitag to save a modified version of the reference database containing the computed index. This allows next time using this partially indexed reference library to accelerate the taxonomic annotations.
• Adding of the function gsub to the expression language for substituting string pattern.

May 2nd, 2023. Release 4.0.3

New features

• Adding of the function contains to the expression language for testing if a map contains a key. It can be used from obibrep to select only sequences occurring in a given sample :

  obigrep -p 'contains(annotations.merged_sample,"15a_F730814")' wolf_new_tag.fasta
  

• Adding of a new command obipcrtag. It tags raw Illumina reads with the identifier of their corresponding sample. The tags added are the same as those added by obimultiplex. The produced forward and reverse files can then be split into different files using the obidistribute command.

  obitagpcr -F library_R1.fastq \
            -R library_R2.fastq \
            -t sample_ngsfilter.txt \
            --out tagged_library.fastq \
            --unidentified not_assigned.fastq
  

  the command produced four files : tagged_library_R1.fastq and tagged_library_R2.fastq containing the assigned reads and not_assigned_R1.fastq and not_assigned_R2.fastq containing the unassignable reads.

  the tagged library files can then be split using obidistribute:

  mkdir pcr_reads
  obidistribute --pattern "pcr_reads/sample_%s_R1.fastq" -c sample tagged_library_R1.fastq
  obidistribute --pattern "pcr_reads/sample_%s_R2.fastq" -c sample tagged_library_R2.fastq
  

• Adding of two options --add-lca-in and --lca-error to obiannotate. These options aim to help during construction of reference database using obipcr. On obipcr output, it is commonly run obiuniq. To merge identical sequences annotated with different taxids, it is now possible to use the following strategie :

  obiuniq -m taxid myrefdb.obipcr.fasta \
  | obiannotate -t taxdump --lca-error 0.05 --add-lca-in taxid \
  > myrefdb.obipcr.unique.fasta
  

  The obiuniq call merge identical sequences keeping track of the diversity of the taxonomic annotations in the merged_taxid slot, while obiannotate loads a NCBI taxdump and computes the lowest common ancestor of the taxids represented in merged_taxid. By specifying --lca-error 0.05, we indicate that we allow for at most 5% of the taxids disagreeing with the computed LCA. The computed LCA is stored in the slot specified as a parameter of the option --add-lca-in. Scientific name and actual error rate corresponding to the estimated LCA are also stored in the sequence annotation.

Enhancement

• Rename the forward_mismatches and reverse_mismatches from instanced by obimutiplex into forward_error and reverse_error to be coherent with the tags instanced by obipcr

Corrected bugs

• Correction of a bug in memory management and Slice recycling.
• Correction of the --fragmented option help and logging information
• Correction of a bug in obiconsensus leading into the deletion of a base close to the beginning of the consensus sequence.

March 31th, 2023. Release 4.0.2

Compiler change

OBItools4 requires now GO 1.20 to compile.

New features

• Add the possibility for looking pattern with indels. This has been added to obimultiplex through the --with-indels option.
• Every obitools command has a --pprof option making the command publishing a profiling web site available at the address : http://localhost:8080/debug/pprof/
• A new obiconsensus command has been added. It is a prototype. It aims to build a consensus sequence from a set of reads. The consensus is estimated for all the sequences contained in the input file. If several input files, or a directory name are provided the result contains a consensus per file. The id of the sequence is the name of the input file depleted of its directory name and of all its extensions.
• In obipcr an experimental option --fragmented allows for spliting very long query sequences into shorter fragments with an overlap between the two contiguous fragment insuring that no amplicons are missed despite the split. As a site effect some amplicon can be identified twice.
• In obipcr the -L option is now mandatory.

Enhancement

• Add support for IUPAC DNA code into the DNA sequence LCS computation and an end free gap mode. This impact obitag and obimultiplex in the --with-indels mode.
• Print the synopsis of the command when an error is done by the user at typing the command
• Reduced the memory copy and allocation during the sequence creation.

Corrected bugs

• Better management of non-existing files. The produced error message is not yet perfectly clear.
• Patch a bug leading with some programs to crash because of : "empty batch pushed on the channel"
• Patch a bug when directory names are used as input data name preventing the system to actually analyze the collected files.
• Make the --help or -h options working when mandatory options are declared
• In obimultiplex correct a bug leading to a wrong report of the count of reverse mismatch for sequences in reverse direction.
• In obimultiplex correct a bug when not enough space exist between the extremities of the sequence and the primer matches to fit the sample identification tag
• In obipcr correction of bug leading to miss some amplicons when several amplicons are present on the same large sequence.

March 7th, 2023. Release 4.0.1

Corrected bugs

• Makes progress bar updating at most 10 times per second.
• Makes the command exiting on error if undefined options are used.

Enhancement

• OBITools are automatically processing all the sequences files contained in a directory and its sub-directory
  recursively if its name is provided as input. To process easily Genbank files, the corresponding filename extensions have been added. Today the following extensions are recognized as sequence files : .fasta, .fastq, .seq, .gb, .dat, and .ecopcr. The corresponding gziped version are also recognized (e.g. .fasta.gz)

New features

• Takes into account the OBIMAXCPU environmental variable to limit the number of CPU cores used by OBITools in bash the below command will limit to 4 cores the usage of OBITools

  export OBICPUMAX=4
  

• Adds a new option --out|-o allowing to specify the name of an outpout file.

  obiconvert -o xyz.fasta xxx.fastq
  

  is thus equivalent to

  obiconvert  xxx.fastq > xyz.fasta
  

  That option is actually mainly useful for dealing with paired reads sequence files.

• Some OBITools (now obigrep and obiconvert) are capable of using paired read files. Options have been added for this (--paired-with FILENAME, and --paired-mode forward|reverse|and|andnot|xor). This, in combination with the --out option shown above, ensures that the two matched files remain consistent when processed.

• Adding of the function ifelse to the expression language for computing conditionnal values.

• Adding two function to the expression language related to sequence conposition : composition and gcskew. Both are taking a sequence as single argument.

February 18th, 2023. Release 4.0.0

It is the first version of the OBITools version 4. I decided to tag then following two weeks of intensive data analysis with them allowing to discover many small bugs present in the previous non-official version. Obviously other bugs are certainly persent in the code, and you are welcome to use the git ticket system to mention them. But they seems to produce now reliable results.

Corrected bugs

• On some computers the end of the output file was lost, leading to the loose of sequences and to the production of incorrect file because of the last sequence record, sometime truncated in its middle. This was only occurring when more than a single CPU was used. It was affecting every obitools.
• The obiparing software had a bug in the right aligment procedure. This led to the non alignment of very sort barcode during the paring of the forward and reverse reads.
• The obipairing tools had a non deterministic comportment when aligning a paor very low quality reads. This induced that the result of the same low quality read pair was not the same from run to run.

New features

• Adding of a --compress|-Z option to every obitools allowing to produce gz compressed output. OBITools were already able to deal with gziped input files transparently. They can now produce their results in the same format.
  • Adding of a --append|-A option to the obidistribute tool. It allows to append the result of an obidistribute execution to preexisting files. - Adding of a --directory|-d option to the obidistribute tool. It allows to declare a secondary classification key over the one defined by the '--category|-c` option. This extra key leads to produce directories in which files produced according to the primary criterion are stored.
• Adding of the functions subspc, printf, int, numeric, and bool to the expression language.