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Besides comparative analysis, a round of database queries was also performed to integrate additional information.
Related family members were identified by a second round of database searching against GenBank to ensure all available genomic data was utilized.
To use all available sequence data, CTG proteins with a top database hit to a bacterial protein in UniProt were extracted for a second round of database searching against GenBank (E value of 10-20).
Following a first round of database searching and on-target recrystallization of the sample spots, MS/MS spectra were collected on selected precursors (see Additional file 3 for detailed information).
Peptide mass fingerprint (PMF) data were collected on an UltraflexIII MALDI ToF/ToF mass spectrometer (Bruker Daltonics, Bremen, Germany) and used for a first round of database searching using MASCOT 2.3 (http://www.matrixscience.com) against NCBI nr core collection and the potato genome sequencing consortium (PGSC) Solanum tuberosum group Phureja DM protein database.
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The PSSM is then submitted to a round of sequence database search, same as in PSI-BLAST.
Sequences having hits in the former database did not go to the next round of searching against a later database.
The functional annotation of the selected sequences was then again confirmed by another round of BLAST search in the NCBI database.
The consensus sequences from all validated contigs were used as query sequences in a second round of TBLASTN analysis of the Pinophyta database subset to identify sequences that were not initially identified in the original TBLASTN searches, both to extend the contigs and to ensure that no CLE or CLEL sequences were overlooked due to truncated sequences lacking a CLE or CLEL domain.
This second round of clustering resulted in a Sanger-based database of 12,329 unigenes from Alexandrium.
A prospectively maintained database of the first round of screening in our geographical area was analysed.
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