Minichromosome genomes reconstruction and comparison
Due to the conserved noncoding region shared by all minichromosomes, the
Spades based assembly was not able to separate the different
minichromosomes reliably. We therefore took an alternative approach.
Using a single gene from each minichromosome as a reference (preliminary
assignment of the genes to individual minichromosomes was based on the
GenBank data available for P. spinulosa ; acc. nos.
KF647762-KF647771) we mapped the reads and extended the sequences in the
program aTRAM 2.0 (Allen at al., 2018). To obtain annotations of the
resulting sequences, we combined two methods. First method utilized the
web based server Mitos (Bernt et al., 2013). Since this method
missannotated or entirely missed some of the genes, we used an
additional blast based approach. We combined the assembled
minichromosome sequences into a custom database and used the 37 genes ofP. spinulosa as blast queries (we run discontinous megablast and
tblastx, both with E-value set to 10). We then aligned the P.
serrata minichromosome sequences together with P. asiatica andP. spinulosa by Mauve (Darling, Mau, Blattner, & Perna, 2004).
These alignments were used as a background for combining and correcting
the annotations. To prepare a concatenated matrix, we trimmed all
minichromosomes to equal lengths. Phylogenetic tree and genetic
distances were retrieved from concatenated alignments by the same
approach as for the L. polyplacis genomes.