Clean up kit

The PCR amplifications were performed in 25 μl reaction mixture containing 50 ng of the DNA template, 1U DreamTaq Green DNA Polymerase (Thermo Fisher Scientific), 2.5 μl of 10× buffer, 0.6 μl of 10 mM dNTPs, and 0.6 μl of each primer (10 μM). The reaction conditions were as follows: 94 °C for 5 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 180 s repeated 35 times; and 72 °C for 5 min in the case of fragments 1, 2, 3, and 4. For fragments 5 and 6, we used the following program: 94 °C for 5 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 120 s repeated 35 times; and 72 °C for 5 min. For each fragment, the appropriate amount of the PCR reaction mixtures was cleaned with the use of Clean-up Kit (A&A Biotechnology) to obtain the final volume of 100 μl with the concentration of at least 50 ng/μl. Two DNA strands of the cleaned PCR products were sequenced using Primer Walking method (Wyzer Biosciences Inc., Cambridge, MA). Overlaps between the amplified fragments from 1 to 6 were sufficient to assemble the whole circular genomic sequence with the use of appropriate reference mitogenomes of Cacatuidae taxa or N. notabilis. The annotation of genes was performed in MITOS (Bernt et al. 2013 (link)). Intergenic and long CRs were searched for remnant CRs or pseudogenes using the optimal global: local algorithm (glsearch) form FASTA package version 36.3.8g (Pearson et al. 1997 (link)).