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Exact(5)

Sequence alignment for each locus was conducted with MAFFT 6.611 [62] and manually checked with the Genetic Data Environment GDEE) software [63].

Discrepant results were manually checked with Artemis [ 52].

These scaffolds were manually checked with stringent paired read mapping (100% identity) to either confirm the assembly or identify points where chimeras might occur.

Sequences were aligned with OPV sequences from GenBank by using ClustalW (www.ncbi.nlm.nih.gov/pmc/articles/PMC308517), and alignments were manually checked with MEGA version 4.0 software (Arizona State University, Phoenix, AZ, USA).

The automatic annotation was then manually checked, with additional data from the literature, including the glycosomal [ 36, 37] and flagellar [ 38] proteomes, as well as published metabolic pathway information [ 39, 40].

Similar(55)

We manually checked those with a somatic score above 100 (representing a probability of 1-10(100/-10) for a difference between samples).

As an additional quality control, we manually checked variants with Phred-scaled variant quality scores <20 using the Integrated Genomics Viewer Broad Institutee) and discarded those with poor read support.

To identify the IRs that are transcribed, the coverage plot was manually checked and compared with bioinformatic analysis of the putative ORFs and with the putative ribosome binding sites (RBSs).

All hits were fully translated and manually checked for homology with the target query.

All predicted cloverleaf secondary structures were manually checked and compared with other known ascidian tRNAs.

The resulting topologies were manually checked for congruence with systematics of analyzed taxa.

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