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Reads that could be aligned to the human genome (H. sapiens, UCSC hg19) were removed (aligned with Bowtie2 (Langmead and Salzberg, 2012), using — reorder no-hd no-contain dovetail).

Starting at the highest hairs you want removed, align the twists of thread to that hair.

Finally, in the refinement phase, the alignment is refined by iteratively removing aligned nodes and adding new mapped ones.

For the most part these additional filters will remove aligned data lessening sensitivity but improving accuracy.

All p63, H3K27ac, and RNAPII ChIP-seq datasets were normalized to the same sequencing depth by randomly removing aligned reads.

To improve the data quality of the aligned sequences, GBLOCKs was used to remove aligned columns with more than half gaps or with low data quality [ 35, 36].

If there were no BLAST hits, another BLASTp search was performed against the NCBI nonredundant protein database to determine the sequence similarity by removing aligned regions that were less than 75% of the smaller sequence.

We applied the GUIDANCE method (Penn et al., 2010) to the original alignment of DGC GGDEF domain sequences (169) with a confidence score of 0.948650, to remove aligned positions considered unreliable.

We also constructed a trimmed version of this matrix using Block Mapping and Gathering with Entropy (BMGE) (Criscuolo and Gribaldo 2010) v1.1.1, as a means of removing aligned sites showing evidence of substitutional saturation and compositional heterogeneity.

All alignments were filtered to remove poorly aligned sequence, removing short stretches of sequence surrounded by gaps and regions with an excessive number of substitutions in a short sequence.

After using trimAl, we also visually inspected the trimmed alignments and removed poorly aligned regions.