Kapa hifi hotstart uracil dna polymerase

For MethylC-seq library generation, genomic DNA was sonicated to an average size of 200 bp using the Covaris sonicator. Sonicated DNA was then purified and end repaired, followed by ligation to methylated Illumina TruSeq sequencing adaptors. Library amplification was performed with KAPA HiFi HotStart Uracil+ DNA polymerase (Kapa Biosystems), using six cycles of amplification. Single-read MethylC-seq libraries (for details, see Supplementary Table 1) were sequenced on the Illumina HiSeq 1500 platform. The sequenced reads in FASTQ format were mapped to in silico bisulfite-converted reference genomes (danRer7, JGI.71 or mm10 for zebrafish, Xenopus and mouse, respectively) using the Bowtie alignment algorithm with the following parameters: -e 120 -l 20 -n 0 (ref. 70 (link)), as previously reported^{71 (link)}. Published paired-read MethylC-seq data^{6 (link)–8 (link)} were mapped with the following parameters: -e 120 -l 20 -n 1 -k 10 -o 4 -I 0 -X 1000. To estimate the bisulfite non-conversion frequency the frequency of all cytosine base calls at reference cytosine positions in the lambda phage genome (unmethylated spike-in control) was normalized by the total number of base calls at reference cytosine positions in the lambda phage genome (Supplementary Table 1).

We followed the MethylC-seq protocol for library preparation^{66 (link)}. In brief, for each species, 500 ng to 1 μg of brain genomic DNA was mixed with 0.1% to 0.5% (w/w) of unmethylated lambda phage genomic DNA. The mixed DNA was sheared into 200 bp fragments using a Covaris Sonicator S220. Then methylated Illumina adaptors (Nextflex Bisulfite-seq adaptors, BIOO scientific) were ligated to sheared DNA, and bisulfite conversion was performed using EZ DNA Methylation-Gold kit (Zymo Research) following the manufacturer’s instructions. After bisulfite treatment, DNA was purified and amplified using universal Illumina primers and KAPA HiFi HotStart Uracil+ DNA polymerase (Kapa Biosystems). The honeybee library was obtained using the same protocol with minor modifications, MethylCode Bisulfite Conversion Kit (Thermo Fisher) was used for bisulfite conversion and the PfuTurbo Cx Hotstart DNA Polymerase (Agilent) was used for library amplification. All libraries but the honeybee and amphioxus samples were sequenced in a Illumina HiSeq 1500 instrument in single-end mode, with reads spanning 100 bp. The honeybee samples were sequenced with an Illumina Genome Analyzer IIx in single-end mode, with reads spanning 84 bp, and amphioxus were sequenced in a NovaSeq 6000 in a paired-end 28-87 bp format.