Databases
NanoClass was designed to classify 16S or 18S rRNA amplicon sequences based on the SILVA database. Most tools implemented in NanoClass will be able to classify sequences using alternative databases. The use of custom databases has not been fully tested and comes with no warrenties, but some advise on how to prepare the UNITE database for ITS amplicon sequences, as well as custom databases is provided below.
Important
To reduce runtime, NanoClass will only run those steps in the pipeline for which no output files are detected. This means that if a database was downloaded and build during an earlier run of NanoClass, it will automatically use this database, in stead of downloading and building a new one. This saves a lot of time and computational resources, but also requires care by the user to make sure the intended database is used.
It is recommended to always use a fresh NanoClass application if a different database is used. Simply clone or download NanoClass again following the instructions in the quick start section of this documentation.
16S or 18S rRNA SILVA database
To use either of these databases, simply enter “16S” or “18S” under the common ssu object in the config.yaml, like so:
common:
ssu: "16S"
Provided no database was already used during an earlier run, NanoClass will now automatically download and use the “16S” database for classification.
In addition, make sure that the minlen and maxlen parameters specified in the config.yaml are appropriate for the marker gene of interest. Reads that are shorter than minlen or longer then maxlen will be discarded during length filtering by nanofilt. Users should carefully choose this value to prevent spurious sequences (if too low) or valid data loss (if too high).
Appropriate values depend on the length range of the marker gene of interest (for most 16S rRNA-based projects a minlen of 1400 and maxlen of 1600 is reasonable).
nanofilt:
minlen: 1400
maxlen: 1600
ITS UNITE database
Although not automatically implemented in NanoClass, it is possible to manually download the ITS UNITE database to be used in NanoClass. Note though that this database has only been tested with a subset of the classification tools implemented in NanoClass, i.e. [“blastn”,”megablast”,”minimap”,”spingo”]
First download the UNITE database from https://files.plutof.ut.ee/public/orig/98/AE/98AE96C6593FC9C52D1C46B96C2D9064291F4DBA625EF189FEC1CCAFCF4A1691.gz Unzip it and look for the reference sequences (sh_refs_qiime_ver8_99_04.02.2020.fasta) and the reference taxonomy (sh_taxonomy_qiime_ver8_99_04.02.2020.txt).
Move these two files to the to a db/common/ directory and rename them to ref-seqs.fna and ref-taxonomy.txt, respectively.
mkdir -p db/common/
mv sh_refs_qiime_ver8_99_04.02.2020.fasta db/common/ref-seqs.fna
mv sh_taxonomy_qiime_ver8_99_04.02.2020.txt db/common/ref-taxonomy.txt
Lastly, you need to create a file called db/common/ref-seqs.aln, which can be empty.
touch db/common/ref-seqs.aln
This is a dummy file for the multiple sequence alignment needed by Mother. As this file is empty, Mother can not be used in combination with the UNITE database. Use with methods [“centrifuge”,”kraken”] is highly discouraged as these require specially formatted databases and compatibility with the UNITE database has not been tested.
To select UNITE-compatible tools to be run in NanoClass, simply change the methods object in the config.yaml, e.g.:
methods: ["blastn","megablast","minimap","spingo"]
This UNITE reference database will now automatically be used once you start the NanoClass workflow, like so:
snakemake --use-conda --cores <ncores>
Custom databases
Most classification tools implemented in NanoClass can use alternative databases supplied by the user (but see exceptions below), provided they are formatted correctly.
These databases will not be automatically downloaded as part of the NanoClass pipeline, as is the case for the SILVA 16S or 18S databases.
Instead, users should provide the databases themselves and store them in the db/common/ subdirectory of the NanoClass directory.
These will then be automatically detected once NanoClass is started and NanoClass will create / reformat all tools-specific databases based on this user-provided database.
Two files are required:
The reference sequences
The reference nucleotide sequences should be in an unzipped, multi-fasta file called ref-seqs.fna. The fasta header should have the sequence identifier only.
Here is an example of the first two entries in the ref-seqs.fna of the default 16S SILVA reference database:
>AB302407.1.2962 TCCGGTTGATCCTGCCGGACCCGACCGCTATCGGGGTAGGGCTAAGCCATGCGAGTCGCGCGCCCGGGGGCGCCCGGGAGCGGCGCACGGCTCAGTAACACGTGCCCAACCTACCCTCGGGAGGGGGACACCCCCGGGAAACTGGGGCCAATCCCCCATAGGGGAAGGGCGCTGGAAGGCCCCTTCCCCAAAAGGGGTTGCGGCCGATCCGCCGCAGCCCGCCCGAGGATGGGGGCACGGCCCATCAGGTAGTTGGCGGGTTAAAGGCCCGCCAAGCCGAAGACGGGTAGGGGCGGTGAGAGCCGCGAGCCCCGAGATCGGCACTGAGACAAGGGCCGAGGCCCTACGGGGTGCAGCAGGCGCGAATACTCCGCAAATGGGGAGAGGCCGGCCCCTCAGTTTTCTCATGAAGGACGCCCATCGGTTTGATTTAAGCGAAGTGGTATATTCGAAATTTGACAAAAAGCGTGGTATACGGATTCCGGAGGAGCCCTCGGAGCTCCTAGCAGAGGAGACGGCGATTCATCTAGGAGATGGGTATCTATTCTACGACGAAAAAGATCGGAGCTACAGGTTCGGCATAGGCCTCAATCCGAAGACGGAAATGAGCTACGCATACGCGGTGGCGGAGCTCATAGGACAGCTCTACGGCTACATGCCGCCGGTGAGAGGAGCGCGTATTGAAATAATGTCGCTAGCCATAGGGACGTTCAAGCATAAAGTCCTCGCCTTACCTATTGGCACCAGAACCGGAGGCGAGGATCTTCCGAGGATCGAGTGGGTTTTAACAGATGAGAGGTTTATTACGGCGTTTGTACGCGGGCTTGTCGATACGGAGGGCTCAGTAAAAAAGATCTCAAGGACGATAGGTGTCGTCGTAAAACAGAGGAATAGGAAAATAATCGAGTTCTATGCCCAATGCCTCGCGGCCTTGGGATTCCGGTCAAGGATATATGTATGGAACGAGAGAAACAAACCCATATATGTCTCGGCGTTGTTGGGCAAAGAAGTCGTGGAACAATTCATACAACTTATCAAGCCGAGAAACCCCTCAAAATATCCTCCTTTTTAGCTCCCACCCCTCGCCGGCCCTCCCCAGCGGAATGCGGGCAACCGCGACGGGGCTACCCCGAGTGCCGGGCGAAGAGCCCGGCTTTTGCCCGGTCTAAAACGCCGGGCGAATAAGCGGGGGGCAAGTCTGGTGTCAGCCGCCGCGGTAATACCAGCCCCGCGAGTGGTCGGGGTGTTTACTGGGCTTAAAGCGCCCGTAGCCGGCCCGGCAAGTCGCTCCTGAAATCCCCGGGCCCAACCCGGGGGCGGGGGGCGATACTGCCGGGCTAGGGGGCGGGAGAGGCCGCCGGTACTCCGGGGGTAGGGGCGAAATCCGTTAATCCCCGGAGGACCACCAGTGGCGAAAGCGGGCGGCCAGAACGCGCCCGACGGTGAGGGGCGAAAGCCGGGGGAGCAAACCGGGCCAAGGCCGGCAAAAATTTAAAAAAGCCCCAAGGTCTCGATATGGGGTGCGATCGTTATGAGGTGCTAAGAGTTGTGCCTTTCGTTCTATCGGACGGGGGAGTATACAATAACAGGGAGATCTATTTCACATCAACAGACGCCGCCTTGGTTAGCCACTTCAGAACTGTTGCAACGGTTGCGTTTGGCTACGGAGGCTACGTTGTGAAGCGGAGCAACGGGGCATATGTGGTGAAGATAAAGGGCTATGACTACTTCAAATGTCTAGCCGAGGTTATGCCAGACATATTATATAAGAACGATAAAGGCCGCGCCATTAGGTTGCCGGACGAGCTATACAGCGACAAGGATCTAGCCAAGTGGTTCATAAAGGTATATGTCTCATGCGACGGCGGTGTATCGGTCATGTTGGGTAGAAGAGGCAATATCACGTTTTACGTCAGAAGAGTATCCATAACATCCAAAAACCCATATCTACGGCGCCAGATCGGGGATTTGCTCAAAGCCTTAAGCTTTGCGCCGCGAGATGATGGCGATAAACACATCTACCTATCACGAAGAGAGGACATAGTTAGATACGCAGAAGAGATAAGATTTCTAGAGCAGGTAAAGGTAACGAAAAATTCCAAAAGATTCCGCGGAATGGAGAAAAACCAGCTCCTAGATTTAGTCGTGCGATCCTACGGAAATCCCCACCTGTTAGACCCCTTTTTTCCACCATCATCTTTCTCCCACAACGCCGGCCTAGCCCGGGCTCAAAGGGGATTAGATACCCCTGTAGTCCCGGCCGTAAACGATGCGGGCTAGCTGTCGGTCGGGCTTAGGGCCCGGCCGGTGGCGAAGGGAAACCGTTAAGCCCGCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTTAAAGGAATTGGCGGGGGGGCACCACAAGGGGTGAAGCTTGCGGCTTAATTGGAGTCAACGCCGGAAACCTCACCCGGGGCGACAGCAGGATGAAGGCCAGGCTAACGACCTTGCCGGACGAGCTGAGAGGAGGTGCATGGCCGTCGTCAGCTCGTGCCGTGAGGTGTCCGGTTAAGTCCGGCAACGAGCGAGACCCCCGCCCCTAGTTGCTACCCCGTCCTACGGGACGGGGGGCACACTAGGGGGACTGCCGGCGTAAGCCGGAGGAAGGAGGGGGCCACGGCAGGTCAGTATGCCCCGAAACCCCGGGGCTGCACGCGAGCTGCAATGGCGGGGACAGCGGGAACCGACCCCGAAAGGGGGAGGCAATCCCGTAAACCCCGCCCCAGTAGGGATCGAGGGCTGCAACTCGCCCTCGTGAACGTGGAATCCCTAGTAACCGCGTGTCACCAACGCGCGGTGAATACGTCCCTGCCCCTTGCACACACCGCCCGTCGCACCACCCGAGGGAGCTCCCTGCGAGGCCCCTTGCCGAAAGGTGGGGGGACGAGCAGGGGGCTCCCAAGGGGGGTGAAGTCGTAACAAGGTAACCGT >KU725476.45629.48552 AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCATGCTTAACACATGCAAGTCGGACGTTGTCTTCAAATTAGAATAGAAGCAAAGTGGCGAACGGGTGCGTAACACGTAGGAATCTGCCAAACAGTTTGGGCCAAATCCTAAATAAAGAAAAGCTAAAAAGCGCTGTTTGATGAGCCCACGTAGTATTAGCTAGTTGGTGAGGCAATGGCTTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGAGTGAAGAAGGGCAACGCAGCTTGTAAAGCTCTTTCATCGAATGTGCGATTATGACAAGCCTCGAATAATAAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAAGCGTTATCCGGAATGATTGGGCGTAAAGGGCGCGTAGGCGGTAAATTTGGTCGAAAGTGAAAGTCGTCAGCTCAACTGGCGGAATGCTTTCAAAACCAATTTACTTGAGTAAGATAGAGGATAGTGGAATTTCGTGTGTAGAGATAAAATTCACAGATATACGAAGGAACACCAAAAGCGAAGGCGGCTACCTGGGTCTTTACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATAGATCCGGCCGGGCTCTAAATAAAAGACGGCCAAAGGTGTCCTCTCCGATTGCGAGGTCAGAGTGTGTATTCAGCTATTTCGGGGAAACTTTGATGCAGATGTACCATTCAATATCAGACAATCCCGAGGGAAGTGCTTTGATGATATTCTATCAAGAGCACCCCGTAGAGACTATGCAAATAGGAAAATAGAAGATATTAAAAAAAATAGGCAGAGGCCAGCTAAGCTAACAGAGCCTCAAAGACGAAAAAGCAAGGCTTTTTTTTTAGGCAAAGCTCGAGCTCCTAACCTGCACTTAAAAAGACTCAAGCAAATATCTTTGTCGAATTCAATGAAATCAATTATCTCAAGATTACGGGAGATGGCTCACTGCAAATAGCTAAAAGCTACAAAAATGCAAGCTCCGTTCATCATAGCCAAATAGAGGAAGATTTCTTTCGATGGAAAGTAGGATCACTACATGAAATCTCGGCACCAAATTCCGTGCAAAAGCAAGCCGCCAATGGCGCCAAAACAAGAAGCTTTTGGTCTAAAGTTGAGCCCTCAAGGCACTTACAAGTATTTATGCCATCACGCATAAAGACAATTGCTTACTCATCTGACGGCGATAGTTAAACTATTTGATATCGCTAAGGATAGTGCTTTGGTAGTGTGATGATGGTTTGATTACTTTTAATGGCCGTGGGACTGTGCTATGCATTGATGGCTTTGACAAAAAATATGTTTAGCTTTTAGCTCAATACTTAGACACTTTTTGGAAGGTTTACGCTCATGTAGAACCTATCCAAATAAATTGCAAGTGCGGTAATCATAGCCAAGAGTGTTACTATTGCCTTCGTTTTGGTATTGAGGAATTTGAGAAAGTACAATAATCTTACCACACCTTGGCGAGCAGGAAAGCCTTATTAGTTTACAAAGATTCCGAAATTCAACAACGCTGGATTTCTGAGATGAAGGAAGCTCTGCCTCTTTTTTTTGAGTGCATCGACTAAGAGTTGTTAGCCAATAAAAAGAGAGCAACTGTAAAAGGTTTATTTCAGAAAATGATATAGTCCATTTTTGGAGATAGTTATCATGAAGTGATGGGATTTTTTATTTTCCATTATGACCTTGAAAAGCAGTTAAGCCACTGAAAAAAGGGCTCAATGCTGGCAAAAAAGCATCTTCAATATGACCCCGGTAGTCCATGCCGTAAACGATGAGTGTTCGTCCTTGGTTTGCGCTGTATGCAAAACGGCTGATCAGGGGCCCAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACGGTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCAGGGCTTGACATATAAATAAGTTTGCTTGTCCTTAACAGGATGGTACGAAAATTTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGCCGTAAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTGTTTAGTTGCTAAGACATGTGCTTTGGATTAAAAGGTCAATTATTAACTACTTAAGACTGACGAAGACCACGCCATTGAAAAAGGATACCATAACGCCGTGTGGCTACGATCATACACAGTTGTTCAAGGGTACTTCGACGAGCACAGTTCTCATAGCGCAGCACACATAACAATGTGGCACTCGGTCTTTGTAAAAGTGATTGACAATCTATACCATGTACTGCACTCATAAACAGACTGCCGGTGATAAGCCGGAGGAAGGTGAGGATGACGTCAAGTCAGCATGCCCCTTACGCCCTGGGCGACACACGTGCTACAATGGCGATTACAATAGGAAGCAAGGCTTTAAGGCAGAGCGAATCCAGAAAGATGGCCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCCGGTCAGCCATACGGCGGTGAATCCGTTCCCGGGCCTTGTACACACCGCCCGTCACACTATGGGAGCTGGCTTCGCCCAAAACATCAGACCAAAGATCACCCATTACTTTAATGTTCCACGCTGTTATTGAGTGTGGTCTACTGGAGTAATTGGTGGTCTAATGTAGGATACTAAGGTGGGGCCTTTAACTGGGGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTG
The reference taxonomy
The reference taxonomy should be in an unzipped tab-delimited file called ref-taxonomy.txt. The first column contains the sequence identifier corresponding to the identifiers used in the ref-seqs.fna. The second column contains a “;”-separated 7-level taxonomy string with domain;phylum;class;order;family;genus;species.
Here is an example of the first two entries in ref-taxonomy.txt of the default 16S reference database.
KF494428.1.1396 D_0__Bacteria;D_1__Epsilonbacteraeota;D_2__Campylobacteria;D_3__Campylobacterales;D_4__Thiovulaceae;D_5__Sulfuricurvum;D_6__Sulfuricurvum sp. EW1
AF506248.1.1375 D_0__Bacteria;D_1__Cyanobacteria;D_2__Oxyphotobacteria;D_3__Nostocales;D_4__Nostocaceae;D_5__Nostoc PCC-73102;D_6__Nostoc sp. 'Nephroma expallidum cyanobiont 23'
Exceptions
- Mother
For mother an additional fasta file is required with the multiple sequence alignments of all reference sequences in the database. The fasta header should be the same as the fasta header of the sequence file.
If you do not have an alignment for your custom database, you can simply skip mother in the NanoClass run by removing it from the methods list in the config.yaml
- Kraken2
Kraken2 cannot be used within NanoClass in combination with custom databases at this stage.
- Centrifuge
Centrifuge cannot be used within NanoClass in combination with custom databases at this stage.