1.1.0

NextITS v.1.1.0 [2026-01-22]

• Fixed handling of unknown barcode combinations (in dual asymmetric mode); thanks to Alice Retter for reporting
• Refactored and optimized the tag-jump removal step
• Fixed a bug with duplicated sequences in the tag-jump removal step; thanks to Valentin Étienne for reporting
• Implemented a chunking option for splitting the dataset into smaller parts prior to clustering in Step-2 (pre-clustering, clustering, and denoising moved to a separate sub-workflow), using MMseqs2
• Added possibility to disable reference-based and/or de novo chimera removal steps and tag-jump removal
• New parameters added:
  • lima_remove_unknown (default, false; if true, unknown barcode combinations are removed from demultiplexed data)
  • chunking_n (number of chunks to split the dataset into prior to clustering)
  • chunking_id (minimum sequence identity used for splitting the dataset into chunks)
  • chimera_methods (specifies which chimera removal methods to use - "ref" for reference-based, "denovo" for de novo, or "ref,denovo" for both; could be also "none" or null to disable chimera removal)
  • tj (specifies whether to run tag-jump removal - "true" or "false")
• Added DADA2 denoising (--preclustering dada2; also works with --clustering none)
• Implemented automated documentation for analysis procedures (generates README_Step1_Methods.txt and README_Step2_Methods.txt in the pipeline_info directory)
• Refactored parameter validation (using nf-schema plugin)
• Refactored the runtime parameter summary and help message
• Added test profiles (test, test1, test2)
• Improved run summary for Step-1
• Default parameters changed:
  • ITSx now checks only a single strand (option ITSx_complement set to F). This should be safe for most cases, as amplicons were re-oriented using primers during the pipeline run. However, we recommend checking the results carefully (e.g., columns ITSx_Extracted_Reads and ITSx_Yield_Percent in the run summary)
  • Prior to tag-jump removal, sequences are now dereplicated at 100% identity (option tj_id set to 1). It is possible to pre-cluster sequences at a lower similarity threshold (e.g., --tj_id 0.99) but this will take much longer. This change should also be safe for most cases, as amplicons undergo homopolymer-correction
• Fixed a minor bug in extraction of sample IDs at the ref-based chimera rescue step; thanks to Valentin Étienne for reporting

1.0.0

NextITS v.1.0.0 [2025-03-24]

• Added support of asymmetric barcoding scheme for demultiplexing of PacBio data
• Added support of BAM files (CCS) as input
• Added support for SWARM d=1 pre-clustering
• Changed the selection of representative sequences (sequence with the highest quality score is taken as the representative; using phredsort)
• Refactored sequence quality estimation
• Improved processing speed (using DuckDB and Parquet format)
• Improved tag valiadion for demultiplexing
• Improved compression speed for output files (runs in parallel using pigz)
• Update of the database for reference-based chimera detection (using the EUKARYOME database)
• New parameters added:
  • step (specifies which pipeline step to run - "Step1" or "Step2")
  • storagemode (Adjusts how files are directed to the results folder)
  • gzip_compression (Controls GZIP compression level in output files)
  • categorical lima_barcodetype replaces boolean lima_dualbarcode
  • lima_minendscore (For asymmetric and dual barcoding scheme)
  • lima_minrefspan (Controls barcode coverage)
  • lima_minscoringregions (Controls the number of reqired barcodes for demultiplexing using dual barcodes)
• Added auxilarry output files:
  • [Step-1] All rRNA parts extracted by ITSx (pooled within sequencing run - useful for extracting these regions for representative sequences)
  • [Step-1] File with quality scores for full-length sequences (after QC and trimming)
  • [Step-2] File with joined sequence memebership (dereplication, pre-clustering, and clustering)
• Primer trimming prior ITSx is now default (sequence quality is also estimated on trimmed sequence)
• Fixed VSEARCH clustering on denoised reads
• Resolved an issue where no de novo chimeras were detected
• Reconfigured parameter specification
• Introduced a parameter schema and enhanced parameter validation
• Container updates to included the latest versions of dependencies
• New dependencies - specialized tools written in Go to speed up the processing:
  • phredsort (https://github.com/vmikk/phredsort) (Sorts sequences by quality score)
  • seqhasher (https://github.com/vmikk/seqhasher) (Hashes sequences)
  • ucs (https://github.com/vmikk/ucs) (Parses UC files and converts them to parquet format)

v.0.5.0 [2023-08-08]

• New seqstats sub-workflow (only dereplication, primer validation, and basic run stats)
• Add SWARM clustering (Mahé et al., 2022 DOI:10.1093/bioinformatics/btab493)
• Add post-clustering curation with LULU (Frøslev et al., 2017 DOI:doi.org/10.1038)
• Add barcode validation step
• Add SSU and LSU region-based output sequences
• Add support for UNOISE-only output (without clustering)
• Add merge_replicates parameter (Step-2) for merging or keeping separate sample replicates
• Update Step-1 run summary (add homopolymer stats)
• Deprecate taxonomy annotation workflow at Step-1
• Fixed different extensions in demultiplexed input
• Experimental: UNITE-style dereplication (allows query sequences to vary in length at 100% similarity)
• Experimental: support of alternative alignment penalty scores (ITS-specific feature)

v.0.4.0 [2023-05-08]

• Add Step-2 workflow for pooling, dereplicating, and clustering sequences from Step-1
  • Read clustering with VSEARCH (Rognes et al., 2016 DOI:10.7717/peerj.2584)
  • Error-correction with UNOISE2 (Edgar, 2016 DOI:10.1101/081257)
• Add run summary for Step-1 (read counts at different pipeline stages)
• Separate config for HPC clusters
• Add Docker container

v.0.3.0 [2023-03-02]

• Add support for pre-demultiplexed data as input
• Add option for semi-full-length ITS (especially useful when forward primer is located at the very end of SSU and the HMM site can not be recognized by ITSx)
• Add removal of long homopolymer artifacts at QC stage
• Correct handling of a case with no valid sequences at primer checking step (thank to Taavi Riit for reporting the bug)
• Bug fixed in assemble_its (thanks to Kadri Põldmaa for discovering the error)
• Addition of ITSx detailed results (with information on the HMM profile used for ITS extraction)
• Fixed sample names for the rescued chimeric sequences
• Minor fixes related with the Singularity container, output directory, help message, and single-end QC

v.0.2.0 [2022-09-30]

• Add Ilumina-based workflow (see --seqplatform flag)
• Publish Singularity image to Singularity library
• Minor bugfixes in primer_check (multiprimer artefacts), pool_seqs (sequence headers), and prep_asvtab (aggregation of non-unique joined Illumina sequences) processes
• New logo design (thanks to Olesya Dulya)