Primer trimming leads to a diminished sequence depth in the trimmed regions causing a diminished sequence quality.
Trimming the amino acid alignment of ambiguously aligned regions resulted in a truncated alignment of 592 characters, with most trimmed regions in Domain I and the interdomain region between Domain I and Domain III.
After separating individuals by barcode and trimming regions with an error probability higher than 5%, the multiplex PCR approach yielded high coverage data with a mean (± SD) of 466,547 ± 240,100 paired end reads per individual and mean read length of 84.7 ± 10 bp per read.
A trimmed region with a bevel angel of 11° is applied to avoid the heat load deposition at millimeter magnitude, which is caused by the fast electrons hitting due to the LHA heat driving.
Next, the hybridized samples were digested with RNase A/T1 for 45 minutes at 37°C to remove single-stranded RNA, and to trim unhybridized regions of the probes.
This last step generates quality scores ranging from 1 to 10 for each residue in each position of the alignment, and these were used to trim uncertain regions from the alignment.
For visualization of the trimmed target regions and probes, we converted the trimmed alignments into Affymetrix' "link.psl" format and viewed them in the Integrated Genome Browser, an open source, freely-available interactive desktop genome browser tool (http://igb.bioviz.org).
After aligning sequences for each cluster and trimming to regions present in at least half of the Cf sequences in the alignment, 2,667 alignments remained.
The alignments of the four orthologous gene sets were trimmed for regions with gaps and missing data using trimAl [ 70].
