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Protocol publication

[…] investigation allows us to isolate the effects of DNA methylation on gene evolution and provide direct insight into how epigenetic information affects molecular evolution., Single-copy orthology was assigned (i) across seven ant species (C. floridanus, Harpegnathos saltator, Linepithema humile, Pogonomyrmex barbatus, Solenopsis invicta, Acromyrmex echinator, and Atta cephalotes), and (ii) between C. floridanus and D. melanogaster by orthoDB (; )., Multiple sequence alignment was performed with PRANK (), as implemented by GUIDANCE (). PhyML () was used to impute trees from multiple sequence alignments, modifying branch lengths and rate variables, but keeping topology the same as input trees. Gblocks () was then used to filter alignment columns, using default settings, prior to further analyses., Coding sequence substitution rates for D. melanogaster, as summed over species from the D. melanogaster species subgroup, were calculated previously (). Substitution rates for ants were averaged across all aligned codons for a given protein, with free dN/dS ratios for each branch, using PAML with the F3×4 codon model (). We filtered out genes for which dN or dS values were greater than 14 across the seven ant tree, as well as genes that had an aligned length of less than 50 codons. In order to mask CpG dinucleotides for an additional analysis, a separate dataset was produced wherein alignment columns with a CpG in any of the aligned species were masked before running PAML., We used ChIP-seq data that were generated previously for C. floridanus (). We remapped these data to the C. floridanus genome (Cflo_3.3) using bowtie (), after filtering for adapter contamination and read quality using Trimmomatic (). We allowed one mismatch in the “seed” region, and only accepted the most valid alignment for each mapping read., MACS2 () was then used to estimate the read enrichment relative to an input control (as well as bulk histone H3 profiles for histone modifications to histone H3) for each ChIP library after removal of duplicate reads. We only allowed one of each duplicated read when running MACS in an effort to minimize bias introduced through PCR amplification. ChIP enrichment scores were assigned to a coding sequence (CDS) as fold enrichment value over normalized read counts overlapping the given CDS for merged libraries from major workers, minor workers, and males ()., ChIP-seq data from D. melanogaster embryos were obtained for each histone modification from modEncode (; modENCODE ID numbers: 3955, 4120, […]

Pipeline specifications

Software tools Gblocks, PAML, Bowtie, Trimmomatic, MACS