For cloning of level 0 modules, the designated sequences are PCR-amplified, adding the respective fusion site and a BpiI recognition site as part of the primers used for amplification, and cloned via a BpiI Golden Gate cloning reaction.
Over 16,000 rbcL sequences were found to be orthologous to the designated sequences sampled throughout flowering plants.
We BLAST all of the potential sequences from the database against these designated sequences and other potential sequences that are determined to match with a certain threshold (i.e. according to both coverage and identity).
This isolate was designated sequence type ST404 [ 34].
Here, we determine orthologous sequences using designated sequences representing the breadth of variation observed in the gene region of interest across the clade of interest.
Among these, 16 were newly designated sequences.
For SSD pairs, the designated outgroup sequence was a single-copy ortholog in an outgroup genome closely-related to S. cerevisiae.
After reprocessing with WebTraceMiner, only 30.03% of the designated 3' EST sequences were found to have an authenticated 5' terminus in the non-sense strand (i.e., 5TNS, authenticated polyT tails consistent with the expected structures), while fewer than 5.34% of the designated 5' EST sequences had an in-silico verified 5' terminus in the sense strand (i.e., 5TSS)(see Table 1).
Also, as shown in the Table 1, only about 3.16% of the designated 5' EST sequences had authenticated 3' termini in the sense strand (i.e., 3TSS, authenticated polyA tail, consistent with the expected structures).
Consistent with an evolved binding site of the Fem/ Am-Tra2 proteins, we identified several amino acid replacements in the RBD that affect the designated binding nucleotide sequence of the RNA.
The designated protein-coding sequences, together with several CDSs collected from NCBI (http://www.ncbi.nlm.nih.gov/nuccore), served as a reference set for the BlastN method, which was used to select and eliminate mRNA degradation products from reads of each sample.
