Fig. 4

Missed mutations exemplified on four isogenic subclones. a The DNA of four isogenic subclones (human HG-3 cell line) were fragmented to 130 bp for HG3CD5n_cl1 and HG3CD5p_cl7 and to 170 bp peak insert sizes for HG3CD5n_cl41 and HG3CD5n_cl48. Several mutations were missed by variant calling for samples fragmented to 130 bp, but clearly less for 170 bp. b Example mutation on gene OR5H15 (red arrow) with coverage depth of 6 × and 2 × for 130 bp and 40 × and 35 × depth for 170 bp insert samples. OR5H15 does not contain any UTRs in this single 900 bp exon. Reads were sorted in IGV to bases at the mutation site, hence all detected Ts for 130 bp samples (3 and 1, respectively) are indicated. c Specific target regions of v5+UTR and v5 in gene OR5H15 were identical to which the coverage histogram peaks map. Target regions are given in the last two lines. The amplitudes were higher for the 130 bp samples as well as the maximum read depth in the visible region compared to 170 bp samples. At the same time pronounced amplitudes were also obvious for 170 bp within the gene region of OR5H15 implying that sequencing longer DNA fragments would have gained even smaller amplitudes and a higher coverage across the gene region. The four subclones carried four further mutations (grey arrows) beside the failed mutation of 130 bp samples (red arrow) indicating sequence similarity of the subclones