Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

This study tested the hypothesis that bromate (KBrO3)-induced renal cell death is mediated by epigenetic mechanisms. Global DNA methylation, as assessed by 5-methylcytosine staining, was not changed in normal rat kidney cells treated with acute cytotoxic doses of KBrO3 (100 and 200 ppm), as compared with controls. However, KBrO3 treatment did increase p38, p53 and histone 2AX (H2AX) phosphorylation, and p21 expression. Treatment of cells with inhibitors of DNA methyltransferase (5-azacytidine or 5-Aza) and histone deacetylase (trichostatin A or TSA) in addition to KBrO3 increased cytotoxicity, as compared with cells exposed to KBrO3 alone. 5-Aza and TSA co-treatment did not alter p38 or p53 phosphorylation, but slightly decreased H2AX phosphorylation and significantly decreased p21 expression. We also assessed epigenetic changes in cells treated under sub-chronic conditions with environmentally relevant concentrations of KBrO3. Under these conditions (0-10ppm KBrO3 for up to 18 days), we detected no increases in cell death or DNA damage. In contrast, slight alterations were detected in the phosphorylation of H2AX, p38, and p53. Sub-chronic low-dose KBrO3 treatment also induced a biphasic response in p21 expression, with lower concentrations increasing expression, but higher concentrations decreasing expression. Methylation-specific PCR demonstrated that sub-chronic KBrO3 treatment altered the methylation of cytosine bases in the p21 gene, as compared with controls, correlating to alterations in p21 protein expression. Collectively, these data show the novel finding that KBrO3-induced renal cell death is altered by inhibitors of epigenetic modifying enzymes and that KBrO3 itself induces epigenetic changes in the p21 gene.

Women of advanced maternal age account for an increasing proportion of live births in many developed countries across the globe. Offspring of older mothers are at an increased risk for a variety of subsequent health outcomes, including outcomes that do not manifest until childhood or adulthood. The molecular underpinnings of the association between maternal aging and offspring morbidity remain elusive. However, one possible mechanism is that maternal aging produces specific alterations in the offspring's epigenome in utero, and these epigenetic alterations persist into adulthood. We conducted an epigenome-wide association study (EWAS) of the effect of a mother's age on blood DNA methylation in 2,740 adult daughters using the Illumina Infinium HumanMethylation450 array. A false discovery rate (FDR) q-value threshold of 0.05 was used to identify differentially methylated CpG sites (dmCpGs). We identified 87 dmCpGs associated with increased maternal age. The majority (84%) of the dmCpGs had lower methylation in daughters of older mothers, with an average methylation difference of 0.6% per 5-year increase in mother's age. Thirteen genomic regions contained multiple dmCpGs. Most notably, nine dmCpGs were found in the promoter region of the gene LIM homeobox 8 (LHX8), which plays a pivotal role in female fertility. Other dmCpGs were found in genes associated with metabolically active brown fat, carcinogenesis, and neurodevelopmental disorders. We conclude that maternal age is associated with persistent epigenetic changes in daughters at genes that have intriguing links to health.