Worthy to note is the size reached by the SRA archive in a short time span when compared to the entire nucleotide collection currently available (Fig. 1).
The PCR products were sequenced by GATC Biotech corporation (Konstanz, Germany) and identified by comparison with the GenBank® database (settings: nucleotide collection (nr/nt), exclude uncultured/environmental sample sequences, default megablast algorithm parameters).
Retrieved sequences were submitted to the taxonomic identification servers BLAST (16S ribosomal RNA database for 16S rRNA or Nucleotide collection nr/nt for gyrB sequences) and EzBioCloud (http://www.ezbiocloud.net/identify) (Yoon et al. 2017).
The gene sequence of pfkA was obtained from the nucleotide collection of the National Centre for Biotechnology Information (NCBI) and the primers for the gene were designed using the NCBI Primer-Blast.
Obtained fragment sequences were compared with the NCBI nucleotide collection (nr/nt) database using MegaBlast.
If a novel transcript was predicted but failed to yield homologous fragments in either BLAST search, it was realigned against the NCBI nucleotide collection (nr/nt) database to exclude an origin from local region-specific sequences.
We used nucleotide collection database, and identified up to 5 highly conserved sequences (RNAz requires at least 2 and can take into account at most 6 sequences) with conserved length greater than 20 nt, and E-value<10.
Taxonomic assignment of the retrieved sequences to the Sebacinales was done by using BLAST [36], [37] against the nucleotide collection of the National Center for Biotechnology Information (NCBI, GenBank; www.ncbi.nlm.nih.gov).nih.gov
To verify the identity of the amplified regions, the National Center for Biotechnology Information (NCBI) nucleotide collection (nr/nt) database was queried with the sequence of the most common haplotype using a BlastN search with default settings (NCBI, http://www.ncbi.nlm.nih.gov).nih.gov
Simple repeats accounted for 0.3% of the BES nucleotide collection.
A BLASTn search was performed against the nucleotide collection of NCBI aiming to remove contaminating contigs.
