Reproducibility and linearity of barcode sequencing. (a) Comparison of the barcode sequence read numbers in two technical replicates. Aliquots of the frozen pool of library strains were processed for genomic DNA extraction and barcode PCR in two independent experiments conducted 6 months apart. The barcodes were sequenced in two separate sequencing runs. The sequence read numbers were normalized by total numbers of reads matching either uptags or dntags (listed in Additional file 3). The total matched reads were adjusted to 1 million for uptags or dntags of each sample. Only barcodes with read numbers > 0 in both samples are shown. (b) Comparison of barcode sequence read numbers in two biological replicates. Pooled library strains were grown for five generations in rich medium in two independent experiments conducted 6 months apart and the barcodes were sequenced in two separate sequencing runs. The total matched reads were adjusted to 1 million for uptags or dntags of each sample. Only barcodes with read numbers > 0 in both samples are shown. (c) Comparison of log ratios of barcode read numbers calculated using uptags and dntags. Pooled mutants grown in rich medium (YES) and minimal medium (EMM) for five generations were used for barcode sequencing analysis. We plot the log ratios of 1,881 strains, which satisfy the condition that read numbers of both uptag and dntag in YES ≥12, and read numbers of both uptag and dntag in EMM > 0. (d) The linearity and dynamic range of barcode sequencing assessed using spike-in controls. A rad32 deletion strain and a rad26 deletion strain from the Bioneer version 1.0 upgrade package (M-1030H-U) were spiked into 24 version 1.0 pooled samples that had been grown in minimal or rich medium for different generations. The ratios between the cell number of each spike-in strain and the total cell number of the version 1.0 pooled strains were 1/200, 1/1,000, 1/2,500, 1/5,000, 1/10,000, and 1/20,000. The read numbers were normalized by total matched reads of the version 1.0 strains. Only uptag reads of the rad32 strain are plotted here. See Additional file 8 for the dntag reads of the rad32 strain and the barcode reads of the rad26 deletion strain.
Han et al. Genome Biology 2010 11:R60 doi:10.1186/gb-2010-11-6-r60