We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase–polymer–nucleotide complexes bound to clonal copies of DNA targets. These polymer–nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates. Avidity sequencing achieves high accuracy, with 96.2% and 85.4% of base calls having an average of one error per 1,000 and 10,000 base pairs, respectively. We show that the average error rate of avidity sequencing remained stable following a long homopolymer.

We present a novel sequencing chemistry implemented as part of the AVITI system. Relying on the proximal DNA binding sites created through DNA amplification on a solid support, avidity sequencing uses multivalent nucleotide ligands on dye-labeled cores to simultaneously form polymerase-polymer nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides by 100x and yield a negligible dissociation rate. We demonstrate the use of avidites within a novel sequencing technology that surpasses Q40 accuracy and enables a diversity of applications that include single cell RNA-seq and whole human genome sequencing.

Breast ductal carcinoma in situ (DCIS) is a localized form of breast cancer that can progress to invasive breast cancer. Diallyl trisulfide (DATS) is a bioactive compound from Allium vegetables reported to induce anticancer effects in several cancer models. The objective of this study was to characterize DATS-induced apoptosis in breast DCIS and minimally invasive breast cancer cells. Breast DCIS cells SUM 102PT (ductal carcinoma in situ with areas of micro-invasion) and SUM 225CWN (chest wall recurrence of ductal carcinoma in situ) were used in this study. DATS induced a dose-dependent reduction in the colony formation ability of breast DCIS cells. DATS inhibited DCIS cell growth by inducing apoptosis as shown by a dose-dependent increase in cytoplasmic histone-associated DNA fragmentation. Induction of apoptosis was more pronounced in SUM 102PT cells than in SUM 225CWN cells at similar concentrations of DATS. DATS-induced apoptosis was characterized by a dose-dependent increase in cleaved poly-ADP ribose polymerase (PARP). DATS treatment resulted in an increase in the cytochrome c levels and cleavage of caspases 3, 7, and 9. This study shows that DATS inhibits cell proliferation and induces apoptosis in breast DCIS derived and minimally invasive breast cancer cells, and supports further investigation of DATS as a potential chemopreventive agent for DCIS.

Objectives Breast ductal carcinoma in situ (DCIS) is an early stage, localized form of breast cancer that can progress to an invasive breast cancer phenotype.  Diallyl trisulfide is a bioactive organosulfur compound derived from Allium vegetables that has been shown to have anticancer effects in various cancer models.  We have previously shown that diallyl trisulfide inhibits alpha secretases and Notch signaling pathway components in breast cancer cells. The objective of this study was to investigate the mechanisms of action of diallyl trisulfide in breast DCIS cells, with a primary focus on characterizing the induction of apoptosis in breast DCIS cells. Methods Breast DCIS cells SUM102PT [ER(−) and PR(+)] and SUM225CWN [ER(−) and PR(−)] were used in this study.  Clonogenic assay was used to determine the colony formation ability of DCIS cells exposed to diallyl trisulfide.  Apoptosis was quantified using a cell death detection ELISAPLUS kit from Roche according to the manufacturer's instructions.  Western immunoblotting was used to determine the effect of diallyl trisulfide on apoptosis molecular markers. Results Diallyl trisulfide, a bioactive compound derived from Allium vegetables, induced a dose-dependent reduction in colony formation ability of breast DCIS cells. Diallyl trisulfide inhibited DCIS cell growth by inducing apoptosis as shown by a dose-dependent increase in DNA fragmentation as determined by ELISA assay. Induction of apoptosis was more pronounced in SUM102PT cells than in SUM225CWN cells at similar concentrations of diallyl trisulfide. Induction of apoptosis was characterized by a dose-dependent increase in cleaved PARP, cleaved caspase 3, and cleaved caspase 7. Expression levels of anti-apoptotic proteins Bcl2 and p-Bcl2 were decreased in DCIS cells exposed to diallyl trisulfide in a dose-dependent manner. Conclusions Diallyl trisulfide inhibits growth and induces apoptosis in breast DCIS cells. This study supports further investigation of diallyl trisulfide as a potential chemopreventive agent for breast DCIS. Funding Sources This work was supported by the USDA National Institute of Food and Agriculture, Hatch project NEV00765.  Core facility used for research was supported by NIH grant P20 GM103650.