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Custom reference databases

LotuS3 can use custom reference databases for taxonomic annotation. A custom database requires both a FASTA-formatted reference sequence file and a tab-delimited taxonomy file.

Required flags

Use:

  • -refDB for the FASTA reference database;
  • -tax4refDB for the taxonomy file.

The format is the same as for databases installed by the LotuS3 autoinstaller. Useful examples include:

DB/SLV_138_SSU.fasta
DB/SLV_138_LSU.tax

Taxonomy format

The taxonomy file uses seven fixed levels:

  1. kingdom
  2. phylum
  3. class
  4. order
  5. family
  6. genus
  7. species

Levels are denoted by tags such as k__, p__, c__, and are separated by semicolons. If taxonomy information is missing, use ?.

Example taxonomy line:

FJ588878	k__Eukaryota; p__Phragmoplastophyta; c__?; o__?; f__?; g__?; s__Osyris wightiana

Example: using SILVA138 as a custom database

./lotus3 -tax4refDB DB/SLV_138_SSU.tax \
  -refDB DB/SLV_138_SSU.fasta \
  -i Example/ \
  -m Example/miSeqMap.sm.txt \
  -o myTestRun_customDB \
  -forwardPrimer GTGYCAGCMGCCGCGGTAA \
  -reversePrimer GGACTACNVGGGTWTCTAAT \
  -CL uparse \
  -taxAligner vsearch

This example uses SILVA138 as a custom database, VSEARCH as the taxonomic search algorithm and UPARSE for OTU clustering.

Multiple reference databases

Multiple complementary reference databases can be searched by providing comma-separated FASTA and taxonomy files:

./lotus3 -tax4refDB DB/SLV_138_SSU.tax,DB/HITdb/HITdb_taxonomy.txt \
  -refDB DB/SLV_138_SSU.fasta,DB/HITdb/HITdb_sequences.fna \
  -i Example/ \
  -m Example/miSeqMap.sm.txt \
  -o myTestRun_multiDB \
  -forwardPrimer GTGYCAGCMGCCGCGGTAA \
  -reversePrimer GGACTACNVGGGTWTCTAAT \
  -CL uparse \
  -taxAligner vsearch

For built-in databases, the shorter syntax can be used:

./lotus3 -refDB SLV,HITdb \
  -i Example/ \
  -m Example/miSeqMap.sm.txt \
  -o myTestRun_multiDB \
  -forwardPrimer GTGYCAGCMGCCGCGGTAA \
  -reversePrimer GGACTACNVGGGTWTCTAAT \
  -CL uparse \
  -taxAligner vsearch

The order of databases can affect results. For example, -refDB GG2,SLV and -refDB SLV,GG2 can differ because the first database is treated as the primary annotation source.