Genome Reference and Sequence Variation in the Large Repetitive Central Exon of Human MUC5AC

作者：Xueliang Guo, Shuo Zheng, Hong Dang, Rhonda G. Pace, Jaclyn R. Stonebraker, Corbin D. Jones, Frank Boellmann, George Yuan, Prashamsha Haridass, Olivier Fédrigo, David L. Corcoran, Max A. Seibold, Swati Ranade, Michael R. Knowles, Wanda K. O’Neal, Judith A. Voynow · 发表于：American Journal of Respiratory Cell and Molecular Biology · 年份：2013 · DOI：10.1165/rcmb.2013-0235oc · 被引用次数：37 · 研究领域：Glycosylation and Glycoproteins Research、Genomic variations and chromosomal abnormalities、Cancer Genomics and Diagnostics

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 t...