Examining the database

This example follows on from Different primers, and assumes you have used thapbi_pict import with the provided FASTA file (based on Supplementary Table 3 in Redekar et al. 2019), and created a THAPBI PICT database named Redekar_et_al_2019_sup_table_3.sqlite.

As the extension might suggest, this is an Sqlite v3 database, and can be examined directly at the command line if you are very curious. However, we will briefly review the provided commands within THAPBI PICT for checking a database.

Database export

The thapbi_pict dump command is intended for database export and/or answering simple queries without needing to use SQL to query the database. It defaults to giving plain text tab separated tables, but FASTA is also supported:

$ thapbi_pict dump -h
...

By default it outputs all the sequences, but you can do simple taxonomic filtering at genus or species level, for example:

$ thapbi_pict dump -d Redekar_et_al_2019_sup_table_3.sqlite \
  -g Phytophthora -s fallax -o P_fallax.tsv
Wrote 5 txt format entries to 'P_fallax.tsv'
$ cut -c 1-84 P_fallax.tsv
<SEE TABLE BELOW>

This gives a short table, with the sequence truncated for display:

#Marker	Identifier	Genus	Species	TaxID	MD5	Sequence
ITS1-long	DQ297398.1	Phytophthora	fallax	360399	693cf88b7f57bcc7a3532a6b7ff0268a	CCA
ITS1-long	HQ261557.1	Phytophthora	fallax	360399	693cf88b7f57bcc7a3532a6b7ff0268a	CCA
ITS1-long	HQ261558.1	Phytophthora	fallax	360399	693cf88b7f57bcc7a3532a6b7ff0268a	CCA
ITS1-long	HQ261559.1	Phytophthora	fallax	360399	693cf88b7f57bcc7a3532a6b7ff0268a	CCA
ITS1-long	DQ297392.1	Phytophthora	fallax	360399	da7ff4ae11bdb6cc2b8c2aea3937481f	CCA

The final columns give the amplicon marker sequence and its MD5 checksum.

Adding -m or --minimal to the command gives instead:

$ thapbi_pict dump -d Redekar_et_al_2019_sup_table_3.sqlite \
  -g Phytophthora -s fallax -o P_fallax.tsv -m
Wrote 2 txt format entries to 'P_fallax.tsv'
$ cut -c 1-56 P_fallax.tsv
<SEE TABLE BELOW>

Now the table only has one data row per unique marker sequence, again showing this with the sequence truncated:

#MD5	Species	Sequence
693cf88b7f57bcc7a3532a6b7ff0268a	Phytophthora fallax	CCA
da7ff4ae11bdb6cc2b8c2aea3937481f	Phytophthora fallax	CCA

Alternatively, we can ask for FASTA output:

$ thapbi_pict dump -d Redekar_et_al_2019_sup_table_3.sqlite \
  -g Phytophthora -s fallax -f fasta -o P_fallax.fasta
Wrote 2 fasta format entries to 'P_fallax.fasta'

This produces a short FASTA file as follows (with line wrapping added for display):

$ cat P_fallax.fasta
>DQ297398.1 Phytophthora fallax taxid=360399;HQ261557.1 Phytophthora fallax
taxid=360399;HQ261558.1 Phytophthora fallax taxid=360399;HQ261559.1 Phytophthora
fallax taxid=360399
CCACACCTAAAAAAATTCCACGTGAACTGTATTGTCAACCAAATTCGGGGATTCCTTGCTAGCGTGCCTTCGGGCGTGCC
GGTAGGTTGAGACCCATCAAACGAAAACATCGGCTGAAAGGTCGGAGCCAGTAGTTACCTTTGTAAACCCTTTACTAAAT
ACTGAAAAACTGTGGGGACGAAAGTCCTTGCTTTTACTAGATAGCAACTTTCAGCAGTGGATGTCTAGGCTC
>DQ297392.1 Phytophthora fallax taxid=360399
CCACACCTTAAAAAATTCCACGTGAACTGTATTGTCAACCAAATTCGGGGATTCCTTGCTAGCGTGCCTTCGGGCGTGCC
GGTAGGTTGAGACCCATCAAACGAAAACATCGGCTGAAAGGTCGGAGCCAGTAGTTACCTTTGTAAACCCTTTACTAAAT
ACTGAAAAACTGTGGGGACGAAAGTCCTTGCTTTTACTAGATAGCAACTTTCAGCAGTGGATGTCTAGGCTC

To be clear, each FASTA record is written as two potentially very long lines. The first title line consists of the FASTA new record > marker and then four semi-colon separated accessions with species. The sequence shared by those four entries is given on the second line (without line breaks as markers tend not to be overly long, and it facilitates command line analysis/debugging).

Using the optional -m or --minimal switch changes the FASTA output to:

$ thapbi_pict dump -d Redekar_et_al_2019_sup_table_3.sqlite \
  -g Phytophthora -s fallax -f fasta -o P_fallax_minimal.fasta -m
Wrote 2 fasta format entries to 'P_fallax_minimal.fasta'
$ cat P_fallax_minimal.fasta
>693cf88b7f57bcc7a3532a6b7ff0268a Phytophthora fallax
CCACACCTAAAAAAATTCCACGTGAACTGTATTGTCAACCAAATTCGGGGATTCCTTGCTAGCGTGCCTTCGGGCGTGCC
GGTAGGTTGAGACCCATCAAACGAAAACATCGGCTGAAAGGTCGGAGCCAGTAGTTACCTTTGTAAACCCTTTACTAAAT
ACTGAAAAACTGTGGGGACGAAAGTCCTTGCTTTTACTAGATAGCAACTTTCAGCAGTGGATGTCTAGGCTC
>da7ff4ae11bdb6cc2b8c2aea3937481f Phytophthora fallax
CCACACCTTAAAAAATTCCACGTGAACTGTATTGTCAACCAAATTCGGGGATTCCTTGCTAGCGTGCCTTCGGGCGTGCC
GGTAGGTTGAGACCCATCAAACGAAAACATCGGCTGAAAGGTCGGAGCCAGTAGTTACCTTTGTAAACCCTTTACTAAAT
ACTGAAAAACTGTGGGGACGAAAGTCCTTGCTTTTACTAGATAGCAACTTTCAGCAGTGGATGTCTAGGCTC

This discards the original accessions and instead uses >, MD5 checksum, space, semi-colon separated list of taxonomic assignments, new line, sequence, new line. Again, there is deliberately no sequence line wrapping in the file itself.

Edit graph

In the worked example with the default database, we introduced the edit-graph command for use with CytoScape to examine the sequence space of the samples. It can also be run on a database alone provided you include the -k or --marker switch:

$ thapbi_pict edit-graph -k ITS1-long \
  -d Redekar_et_al_2019_sup_table_3.sqlite \
  -o Redekar_et_al_2019_sup_table_3.xgmml
Loaded 838 unique ITS1-long sequences from DB.
Computed Levenshtein edit distances.
Will draw 533 nodes with at least one edge (305 are isolated sequences).

Of the 838 unique sequences in the database, just over three hundred are isolated sequences (over 3bp edits away from anything else). The remaining five hundred plus give us an interesting edit distance graph.

Opening this in CytoScape the first thing that struck me was the largest two components are both for Pythium regulare - suggesting if these are truly all from one species that it has at least two distinct ITS1 markers in the genome?

Another use of this view would be to consider the genus conflicts reported by the thapbi_pict conflicts command - most of the handful of Lagenidium and Brevilegnia nodes are isolated.