Amplicon sorter is a tool for reference-free sorting of ONT sequenced amplicons based on their similarity in sequence and length and for building solid consensus sequences. The limit for separating closely related species within a sample is currently around 95 - 96%.

For more detailed explanation, please read Amplicon_sorter_manual.pdf.

2 versions:

• Linux, Unix and Mac without M1 processor: "amplicon_sorter.py" (multiprocessing version).
• Windows, Mac with M1 processor: "amplicon_sorter_single.py" (single core processing version).

Requirements:

• Python 3
• edlib: Lightweight, super fast C/C++ library for sequence alignment using edit (Levenshtein) distance (https://pypi.org/project/edlib/#description) (pip: python3 -m pip install edlib or conda: conda install bioconda::python-edlib) (for Win64: pip: python -m pip install edlib or conda: conda install conda-forge::edlib)
• biopyton (pip: pip install biopython or conda: conda install biopython or in linux sudo apt-get install python3-biopython)
• matplotlib (pip: pip install matplotlib or conda: conda install matplotlib or in Linux: sudo apt-get install python3-matplotlib)

Vierstraete, A. R., & Braeckman, B. P. (2022). Amplicon_sorter: A tool for reference-free amplicon sorting based on sequence similarity and for building consensus sequences. Ecology and Evolution, 12, e8603. https://doi.org/10.1002/ece3.8603

GNU GPL 3.0

amplicon sequencing, MinION, Oxford Nanopore Technologies, consensus, reference free, biodiversity, DNA barcoding, metabarcoding, metagenetics, PCR, sorting

-i, --input: Input file in fastq, fasta or compressed .gz format (auto detect the format). Also a folder can be given as input and will be scanned for .fasta, .fastq or .gz files to process. Make sure the input file(s) is (are) named as *.fasta, *.fastq, *.fasta.gz or *.fastq.gz because it replaces the extension in parts of the script.

-o, --outputfolder: Save the results in the specified outputfolder. Default = same folder as the inputfile in a subfolder with the name of the input file.

-c, --compressed: Save the output files in compressed *.gz format. (a few files (consensus file, result file and .csv file will not be compressed)

-min, --minlength: Minimum readlenght to process. Default=300

-max, --maxlength: Maximum readlenght to process. Default=No limit

-maxr, --maxreads: Maximum number of reads to process. Default=10000

-ar, --allreads: Use all reads from the inputfile between length limits. This argument is still limited with --maxreads to have a hard limit for large files.

-np, --nprocesses: Number of processors to use. Default=1

-sfq, --save_fastq: Save the results also in fastq files (fastq files will not contain the consensus sequence)

-ra, --random: Takes random reads from the inputfile. The script does NOT compare al sequences with each other, it compares batches of 1.000 with each other. You can use this option and sample reads several times and compare them with other reads in other batches. So it is possible to have an inputfile with 10.000 reads and sample random 20.000 reads from that inputfile. The script will run 20 batches of 1.000 reads. This way, the chance to find more reads with high similarity is increasing when there are a lot of different amplicons in the sample. No need to do that with samples with 1 or 2 amplicons.

-aln, --alignment: option to save the alignment that is used to create the consensus (max 200 reads, fasta format). Can be interesting to check how the consensus is created.

-amb, --ambiguous: option to save the consensus with ambiguous nucleotides, e. g. to find SNP positions (this is still a bit experimental, sometimes errors at the very beginning and end of the consensus).

-a, --all: Compare all selected reads with each other. Only advised for a small number of reads (< 10000) because it is time-consuming. (In contrast with the default settings where it compares batches of 1.000 with each other)

-ldc, --length_diff_consensus: Length difference (in %) allowed between consensuses to COMBINE groups based on the consensus sequence (value between 0 and 200). Default=8.0. This can be interesting if you have amplicons of different length, the shorter ones are nested sequence of the longer ones and you want to combine those in one group.

-sg, --similar_genes: Similarity to sort genes in groups (value between 50 and 100). Default=80.0

-ssg, --similar_species_groups: Similarity to CREATE species groups (value between 50 and 100). Default=Estimate

-ss, --similar_species: Similarity to ADD sequences to a species group (value between 50 and 100). Default=85.0

-sc, --similar_consensus: Similarity to COMBINE groups based on the consensus sequence (value between 50 and 100). Default=96.0

-ho, --histogram_only: Only makes a read length histogram. Can be interesting to see what the minlength and maxlength setting should be.

-mac, --macOS: Option to try if amplicon_sorter crashes on Mac with a M1 processor (I did not get confirmation from users if this works or not).

-v, --version: Print the version and exit.

1. The script reads the inputfile and can optionally create a read length histogram of all the reads in the file.
2. It starts processing a selection of the reads (based on minlength, maxlenght, maxreads). It saves the result in .group files that contain reads of the same gene (e.g. group_1 with 18S reads, group_2 with COI reads, group_3 with ITS reads).
3. It processes the group files to sort out the genes to species or genus level and saves this to different files. (e.g. file_1_1.fasta is 18S from species1, file1_2.fasta is 18S from species2, ...)
4. Each outputfile contains at the end the consensus file of the sequences in the file.
5. Files are produced per group with all consensus sequences for that group. A file is produced with all consensus sequences of all groups.
6. Files are produced with 'unique' sequences (script does not find a group where it belongs to according to the settings)

Filter your inputfile for reads >= Q12 with NanoFilt (https://github.com/wdecoster/nanofilt) or other quality filtering software. Use that Q12 inputfile for Amplicon_sorter. (Lower quality reads can be used but will result in longer processing time and a lower percentage of reads that will assigned to a species.

Copy the Amplicon_sorter.py script in the same folder as your inputfile.

Process several files in inputfolder:

python3 amplicon_sorter.py -i infolder -min 650 -max 1200 -ar -maxr 100000 -np 8:

Process all files in 'infolder' with length between 650 and 1200 bp, use all reads available, with a maximum of 100000 reads if more are available, process on 8 cores. The result will be saved in the 'infolder' in subfolders with the same name as the inputfiles.

Produce a read length histogram of your inputfile:

python3 amplicon_sorter.py -i infile.fastq –o outputfolder -min 650 -max 750 -ho:

produce the readlength histogram of infile.fastq in folder outputfolder. This gives you the information on the number of reads between 650 and 750 bp.

Sample with one species amplicon of 750 bp:

python3 amplicon_sorter.py -i infile.fastq -o outputfolder -np 8 -min 700 -max 800 -maxr 1000

process infile.fastq with default settings, save in folder outputfolder, run on 8 cores, minimum length of reads = 700, max length of reads = 800, use 1000 reads. This will sample the first 1000 reads between 700 and 800 bp of the inputfile. If you add the -ra (random) option to the command line, it will sample 1000 random reads between 700 and 800 bp.

Sample with 2 species: an amplicon of 700 bp and one of 1200 bp:

python3 amplicon_sorter.py -i infile.fastq -o outputfolder -np 8 -min 650 -max 1250 -maxr 2000

Metagenetic sample with several amplicons between 600 and 3000 bp, unknown number of species, 30000 reads in the inputfile:

python3 amplicon_sorter.py -i infile.fastq -o outputfolder -np 8 -min 550 -max 3050 -maxr 30000

Metagenetic sample with several amplicons between 600 and 3000 bp, unknown number of species, 30000 reads in the inputfile, one low abundant species (< 2% reads):

python3 amplicon_sorter.py -i infile.fastq -o outputfolder -np 8 -min 550 -max 3050 -ra -maxr 600000

By random sampling 20x the maximum number of reads, it is possible to find low abundant species.

Guppy v5.xx has a High Accuracy (HAC) and Super Accuracy (SupHAC) option to do the basecalling and sequencing is possible on a 9.4.1 and R10 type of flow cell.

If you are working with species that are more than 95 – 96% similar, it is important to change or finetune some settings of Amplicon_sorter:

• --similar_species_groups: this is used to create species groups. The script is looking for the highest similarities between species and uses those to create species groups. When a better basecaller or flow cell is used, the higher this value can be. By default, the setting to create groups is 93% which is only a small portion of the reads with the HAC data, but a big part of the reads with the SupHAC data. If you increase that value for HAC data, you will find less species in the sample. If you decrease the value, it will result in more groups of the same species. For SupHAC and/or R10 data, it is better to increase the value to 94% (or more?). From Amplicon_sorter version 2021-11-16 and later, when no value is entered (default), the script estimates this value based on the dataset.
• --similar_consensus: this parameter is used to merge species groups if the consensus is more than 96% (default for HAC) similar. When you increase this value, you will get more groups from the same species that are not merged. When decreasing this value, it is possible that closely related species are merged in one group. For the SupHAC and/or R10 data, this value can be increased to 98%.

2025/10/09:

• The option -v, --version has been added.

2025/05/28:

• The program can now read *.gz compressed files as input files (Automatically detected).
• The option -c, --compressed has been added to store the output files in compressed .gz format.

2025/05/24:

• Temporary files were written in the input folder. Changed this to the output folder when users use the -o, --outputfolder option. This fixes an issue when users do not have write permission to the input folder.

2025/03/15:

• Fixed bug that when the option -aln --alignment was used, when a group contained subgroups, it was only saving the alignment of the last subgroup. (noticed by Beilin)

2025/03/10:

• Fixed hang when it had 'no reads to compare'. (noticed by Beilin)

2024/10/16:

• Added 'amplicon_sorter_single.py' version. This is a single core version that works in Windows and Mac with an M1 processor.

2024/10/13:

• I still noticed some rare occurring the program hangs without error messages. This version with a small change did not hang on the same testfiles.

2024/10/07:

• a 'result.csv' file is saved with the number of reads per barcode.
• a global 'consensusfile.fasta' is saved with all consensus sequences from the barcodes that were processed in one go.
• The consensus file per group is no longer produced, only the consensus file for all groups per barcode is saved.
• If the sample does not contain many reads, the "finetune" cycle sometimes ends in no result. In this case, the finetune cycle is skipped.
• On a few rare occasions, the programs hangs. In the 2024_02_20 version, the program exited. This was problematic when you processed a folder with several barcodes, it did not process the remaining barcodes. Now amplicon_sorter will continue with the remaining files.

2024/02/20:

• automatic download of latest version did not work anymore. Fixed.
• fixed a rarely occurring hang of the program if only a few reads were used as input that were too different to compare. Now the program exits.

2023/06/19:

• added option -aln, --alignment to save the alignment used to create the consensus.
• added option -amb, --ambiguous to save the consensus with ambiguous nucleotides, e. g. to find SNP positions (this is still a bit experimental).
• a few minor bug fixes (load all sequences in capital letters (was issue with Minibar de-multiplexed files where primer sequences were in small letters), ssg value could be incorrect when processing several files from different flow cell types).
• improvement of the consensus in homo-polymer region.
• removed the -so, --species_only command line option and better cleanup of temporary files.
• better multiprocessing at the first step for smaller files.
• give a more logic % of the reads assigned in the result.txt file.
• save the reads that were not assigned in any groups to a separate file (xxx_no_group_unique.fasta)

2023/03/24:

• changed the -i, --input possibilities. A file or folder can be input. When it is a file, only that file will be processed. When it is a folder, it will scan the folder for .fasta or .fastq files and process them all. Keep in mind that all files in a folder will be processed with the same options.
• changed the -o', '--outputfolder option. By default it will save the data in same folder as the inputfolder in a subfolder that has the same name as the inputfile. (Results from BC103.fasta will automatically be saved in the folder BC103) This is done for all files in a input folder that are processed. When another outputfolder is given as option, the results will be saved in a subfolder in that folder with the name of the inputfile.
• fixed a small error in the -h, --help display (Thanks russellsmithies for noticing).

2023/03/12:

• option to use all reads -ar, --allreads. This option is still limited by the -maxr, --maxreads to have a hard limit.
• option -ldc, --length_diff_consensus: Length difference (in %) allowed between consensuses to COMBINE groups based on the consensus sequence. This can be interesting if you have amplicons of different length, the shorter ones are nested sequences of the longer ones and you want to combine those in one group.
• catch "hang of amplicon_sorter" if only one read is present in the inputfile. Now if less than 5 reads are present in the inputfile, the program exits.

2022/03/28:

• a little speed improvement (1,2 x) by using another consensus calling method (edlib). Levenshtein plugin no longer needed.
• added a "finetune cycle" to remove reads that are too different from the consensus. This results sometimes in a better consensus, can sometimes separate species with a high similarity, removes groups with a low number of reads.

2021/12/24: (version of the publication of March 2022 (https://doi.org/10.1002/ece3.8603)

• limit number of comparisons (for large data files).
• checks to cleanup temporary files.
• limit number of comparisons to merge files.
• memory improvement to process large data files. Still needs more improvement.

2021/12/19:

• minor changes to increase speed a little bit.
• filtering of the reads to species groups changed from list to dict format: speed increase and more memory efficient.
• parallelizing comparing consensus and making consensus to get speed increase for larger datasets
• merging groups changed from list to dict format: speed increase and more memory efficient.

2021/12/01:

• major speed improvement by using the edlib library to do the comparison between sequences (see requirements for installation) (first tests between 5 - 10 x faster).
• minor speed increase in merging groups (4 x).
• bugfix when non existing path was given as output folder. Now a multilevel path can be given as input and output.
• creation of read length histogram -ho, --histogram_only is now optional, no longer default.

2021/11/16:

• fixed rarely occurring bug
• program checks if a newer version of Amplicon_sorter is available.
• fixed bug when entering a "path/infile.fastq" as input. The program crashed halfway with an error. Now it is possible to use a path as input.
• changed the default for --similar_species_groups: the programs estimates the value from the dataset instead of the default value of 0.93.

2021/09/21:

• fixed bug that is important for sorting closely related species. (was wrongfully removed in previous version)

2021/09/11:

• speed and memory improvement when sorting the compared reads for best matches
• added option --similar_consensus
• added option --all
• save all parameters in results.txt to know afterwards with which parameters a run is performed.

2021/08/08:

• speed improvement by changing allowed difference in length from 1.1 to 1.05 (less comparisons to be done) and 1.08 for comparison of consensus sequences
• change default of --similar_species_groups from 0.92 to 0.93

2021/07/13:

• speed improvement by changing allowed difference in length from 1.3 to 1.1 (less comparisons to be done)
• little changes that improve speed a bit

2021/05/28:

• improvement on combining groups
• small speed improvement when creating consensus to compare groups

2021/05/19:

• small speed improvement when comparing remaining sequences with consensus in groups

2021/05/13:

• chunck number of comparisons in groups of 500K to save memory and process parallel
• minor bug fixes and small speed improvements
• improvements in number of groups created

2021/05/06:

• If no max number of reads is given, use all reads
• write version of script in results.txt
• write number of reads in length selection to readlength histogram
• combine groups if consensus is at least 99% similar
• save time by making consensus of max 500 random reads in group
• major speed improvement when comparing a lot of sequences. When the number of sequences is x 100, calculation time is x 10.000. Subsampling in batches of 1000 sequences decreases calculation time x 10 (it does NOT compare all sequences with each other, it compares batches of 1000 with each other)

2020/5/20:

• Save a file "results.txt" in outputfolder with how many reads are in which file.
• Catching error when there are less reads available than asked in --maxreads.

2020/5/6:

• Little cosmetic change in read length histogram.
• Corrected a minor bug with (-ho --histogram_only) option.

2020/4/27:

• Little change in step 3 that improves the processing speed.
• Made names of temporary files unique in case you want to process different files in one folder.

2020/4/17:

• Added vertical lines to the read length histogram with the minimum and maximum read length that were given as read length selections.
• Option to create a read length histogram only (--histogram_only).
• Option to change the similarity when CREATING species groups (--similar_species_groups).
• Option (--species_only) to play with the --similar_species and --similar_species_groups parameters without having to start all over.

2020/4/3:

• Option to save results in a subfolder (-o --outputfolder).
• More little improvement to sort out the groups in step 2. (The result is still not what I had in mind: sometimes reads of ITS and 18S are in the same group. The reason for this is that ITS can be very different from species to species. If I set similarity to 55%, it can put some ITS in the 18S group. If I set it higher, than COI is not in one group. This has no influence on the end results in step 3-4).

2020/3/12:

• Option to take a random selection of the reads from the inputfile (--random).
• Little improvement to sort out the groups in step 2.

2020/3/5:

• Fasta or fastq files possible as input (autodetect).
• Fastq files as output option (when input is fastq) (--save_fastq).

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