Protein-coding and ribosomal RNA genes were firstly identified using BLAST searches at GenBank, and then by alignment with previously published mt genomes from species of Crassostrea, Mytilus and other closely related mollusks [ 33].
All protein-coding, rRNA, and tRNA genes, and intron boundaries within genes in the novel mitochondrial genomes were confirmed by multiple sequence alignment with published red algal data using the T-Coffee package (http://www.tcoffee.org; Di Tommaso et al. 2011).
The 12S rRNA (rrnL) gene was assumed to start from the end of trnV, and its end was roughly identified by alignment with the three published dipluran sequences.
These sequences were aligned with published sequences of FCoV using ClustalW Multiple alignment (Bioedit version 7.0.9).
Two rounds of PCR amplification were carried out with nested pairs of degenerate primers targeting conserved gene regions determined by alignment of published sid-1 homologs from distantly related species (see Additional data file 1) (primer sequences available on request).
Adapter-containing and low-quality reads were trimmed, followed by alignment with TopHat.
Using a recently published Chinook salmon complete sdY mRNA sequence (GB: KC756279), we were able to hypothesize a tiled alignment with the published OTY3 contig (GB: DQ393568.1).
To confirm the PCR results, we sequenced the amplicon in both directions in 2 independent setups and compared the obtained 299-bp sequence with published sequences from GenBank by performing a multiple sequence alignment.
The repeat sequences distinguishing the paralogous copies of the defensin duplication were assigned to macaque repeat families by alignment using ClustalW2 (Larkin et al. 2007) and manual comparison of the sequence with published consensus sequences (Liu et al. 2009).
It is important to develop methodology for assessing alignment of published guidelines with current best evidence.
