Genome reference and sequence variation in the large repetitive central exon of human MUC5AC.

Despite modern sequencing efforts, the difficulty in assembly of highly repetitive sequences has prevented resolution of human genome gaps, including some in the coding regions of genes with important biological functions. One such gene, MUC5AC, encodes a large, secreted mucin, which is one of the two major secreted mucins in human airways. The MUC5AC region contains a gap in the human genome reference (hg19) across the large, highly repetitive, and complex central exon. This exon is predicted to contain imperfect tandem repeat sequences and multiple conserved cysteine-rich (CysD) domains. To resolve the MUC5AC genomic gap, we used high-fidelity long PCR followed by single molecule real-time (SMRT) sequencing. This technology yielded long sequence reads and robust coverage that allowed for de novo sequence assembly spanning the entire repetitive region. Furthermore, we used SMRT sequencing of PCR amplicons covering the central exon to identify genetic variation in four individuals. The results demonstrated the presence of segmental duplications of CysD domains, insertions/deletions (indels) of tandem repeats, and single nucleotide variants. Additional studies demonstrated that one of the identified tandem repeat insertions is tagged by nonexonic single nucleotide polymorphisms. Taken together, these data illustrate the successful utility of SMRT sequencing long reads for de novo assembly of large repetitive sequences to fill the gaps in the human genome. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin.

Genome-wide analysis of DNA methylation and gene expression changes in the mouse lung following subchronic arsenate exposure.

Alterations in DNA methylation have been proposed as a mechanism for the complex toxicological effects of arsenic. In this study, whole-genome DNA methylation and gene expression changes were evaluated in lungs from female mice exposed for 90 days to 50 ppm arsenate (As) in drinking water. DNA methylation changes were measured using reduced representation bisulfite deep sequencing. Differential methylation was observed in approximately 700 and 1900 start and transcribed regions, respectively. The start regions showed bias toward decreased methylation. No bias was observed in the transcribed region. A comparison of absolute methylation levels in the control animals with treatment-related changes in methylation showed that baseline methylation levels play a role in determining which genes are methylated. Genes with low absolute methylation levels in the start region showed a trend toward increased As-related methylation and decreased expression. Genes with high levels of methylation in the transcribed region showed a trend toward decreased As-related methylation, but no change in expression. No overall correlation between treatment-related changes in methylation and expression was identified. Among genes showing differential methylation in the start region and differential expression, only 57% showed an inverse correlation. The results suggest that differential methylation following As treatment may only play a permissive role in regulating expression. Despite the low correlation, the subset of 17 genes that showed an inverse relationship between As-related methylation and expression included a substantial number that has been demonstrated to play a functional role in cancer-related processes and other effects consistent with arsenic exposure.