whole genome re-sequencing of Phaeodactylum tricornutum
At the end of the 4-year experimental evolution experiments, three replicates (~109 cells for each replicate) samples from ambient, high CO2, warming, or high CO2 + warming conditions at the same time point (i.e., same cell cycle stage) in the middle of light phase were pooled by culture condition and then were harvested by filtration and centrifugation (8000 g, 10 min). The samples were then flash-frozen in liquid nitrogen and stored at -80 oC until further analysis. Total genomic DNA was extracted and at least 3 g genomic DNA was used to construct paired-end libraries with an insert size of 500 bp using Paired-End DNA Sample Prep kit (Illumina Inc., San Diego, CA, USA). These libraries were sequenced using Novaseq 6000 (Illumina Inc., San Diego, CA, USA) NGS platform at Genedenovo company (Guangzhou, China). Quality trimming is an essential step to generate high confidence of variant calling. Raw reads would be processed to get high quality clean reads according to four stringent filtering standards: 1) removing reads with10 % unidentified nucleotides (N); 2) removing reads with>50 % bases having phred quality scores of 20; 3) removing reads aligned to the barcode adapter.