period of time predominantly in vivo during the life

period of time, predominantly in vivo during the life
of the cancer patient, although a small proportion
may have been acquired in vitro if establishment of
the MRCA cell occurred in culture. Sequences
from the single-cell-derived parent clones include
the same set of mutations, with an addition of
mutations acquired between the establishment
of the MRCA cell of the stock cell line and isolation
of the single parent PKC412 (period 2). Duration of this
period is unknown as it depends on timing of the
establishment of the MRCA cell and hence may
include an in vivo time frame. Mutations generated
during this period were revealed by subtracting
sequences of stock cell lines from those of
parent clones. Sequences from single-cell-derived
daughter clones include the mutations from their
parent clones and, in addition, mutations acquired
in vitro during the defined cultivation time frames
spanning the two single-cell isolation events (up to
161 days, period 3). Subtraction of the sequences of parent clones from those of daughter clones therefore reveals mutations acquired during the examined
in vitro periods. Clones in Figures 3–5 follow the outlined experimental design, but the numbers of obtained clones and generations may vary.
gastric and esophageal models; SBS18, potentially due to reac-tive-oxygen-species-induced DNA damage, in neuroblastoma cell lines; SBS9, which might result from aberrant processing of AID-induced cytidine deamination by polymerase h, in lym-phoma cell lines; and SBS28, SBS34, SBS39, and SBS40, which were found mainly in the cancer types in which they had been previously reported. SBS1 (associated with deamination of 5-methyl cytosine) and SBS5 (of unknown origin) are ubiquitous among cancer types (Alexandrov et al., 2015) and were present in most cancer cell lines and PDXs. However, some SBS1 and SBS5 mutations are likely attributable to residual germline vari-ants, which remain because of the non-availability of normal DNAs from the same individuals for most cancer cell lines (STAR Methods) and which are also constituted of these two sig-natures (Rahbari et al., 2016).
A small subset of signatures was absent from the examined datasets (SBS7c, SBS12, SBS16, SBS24) or found less often than expected (e.g., SBS3) (Alexandrov et al., 2018). These may be due to the small numbers of somatic mutations in exome sequences, the small numbers of mutations some sig-natures contribute to individual cancers, the obscuring pres-ence of residual germline variants, the relatively featureless profiles of some signatures that may be more difficult to detect, and/or the genuine absence of the signatures (Alexan-drov et al., 2013b). Some signatures were detected in a small proportion of models from cancer classes in which they have not been previously reported (Alexandrov et al., 2018). Such instances likely reflect past misclassification, past cross-contamination, or minor misattribution of mutational signatures. 
Investigating Continuing Mutagenesis in Cancer Cell Lines
To investigate the patterns of activity of mutational processes underlying a wide range of signatures, we selected 28 cell lines derived from cancers of the breast, colorectum, uterus, lung, stomach, cervix, ovary, head and neck, skin (melanoma and squamous), white blood cells (B cell lymphoma and leukemia), and neuroblastoma (Figure 3A). One or more of these had high contributions from mutational signatures of tobacco smoke (SBS4); ultraviolet light (SBS7a-d); aberrant APOBEC cytidine deaminase activity (SBS2 and SBS13); defective DNA MMR with MSI (SBS6, SBS15, SBS21, SBS26); concurrent loss of MMR and proofreading functions in POLD (SBS20) and POLE (SBS14); aberrant POLE activity (SBS10a-b); deficiency of HR-DSB repair (SBS3); and signatures of uncertain origin including SBS17a-b (frequently found in esophageal and gastric cancers), SBS18 (found commonly in neuroblastoma), and SBS28 (com-mon in colorectal and endometrial cancers with mutations in POLE). SBS1 and SBS5 were detected in most cell lines.