Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk+/− cells identified with next generation sequencing

- An efficient and flexible next generation sequencing method is developed for mutation analysis.
- The method is used to analyze the Pig-a gene from B[a]P-treated CD90-deficient L5178YTk+/−-3.7.2C single-cell-derived clones.
- The spectrum of analyzed Pig-a mutations in CD90-deficient clones is consistent with B[a]P exposure.
- The results validate the use of L5178YTk+/− mouse lymphoma cells for performing the in vitro Pig-a assay.

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk+/− cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P). Pig-a mutations were identified in all clones developed from CD90-deficient cells; no Pig-a mutations were found in clones of CD90-proficient cells. The spectrum of B[a]P-induced Pig-a mutations was dominated by basepair substitutions, small insertions and deletions at G:C, or at sequences rich in G:C content. We observed high concordance between Pig-a mutations determined by Sanger sequencing and by NGS, but NGS was able to identify mutations in samples that were difficult to analyze by Sanger sequencing (e.g., mixtures of two mutant clones). Overall, the NGS method is a cost and labor efficient high throughput approach for analysis of a large number of mutant clones.