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

Abstract

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.

The genomic landscape of fibrolamellar hepatocellular carcinoma: whole genome sequencing of ten patients

“…This gene has been implicated in several other epithelial cancers of the gastrointestinal system, and remains an important possible target for further investigation [30–32]. There were mutations detected by VarScan2 that we were unable to verify by Sanger sequencing, due to tandem repeats or sequence homology, a difficulty encountered when sequencing several of the MUC genes [33]. Activation of MUC4 by its external EGF domain can drive ERBB2 signaling, a transcript that was amplified in RNA-seq data from the same cohort of patients (data unpublished) [31].…”

Control of lung defence by mucins and macrophages: ancient defence mechanisms with modern functions

“…They have cysteine-rich N- and C- terminal von Willebrand factor (vWF) type D-like and C- terminal cysteine knot disulfide bonding domains that are critical for inter molecular mucin assembly (50–52). Additional highly conserved cysteine-rich CysD domains are interspersed in varying numbers in polymeric mucin carbohydrate-rich repeats (53–55). Through intra molecular disulfide linkages, CysD domains are proposed to form hydrophobic loop structures that facilitate mucin alignment and regulate mucus mesh spacing (56).…”

Technological advances in precision medicine and drug development

“…With the SMRT technology’s ability to generate long reads, a complete sequence of the region was generated; following this de novo sequencing of the region, SMRT sequencing further identified genetic variation in the troublesome exon in four individuals [143]. Also of considerable interest is the use of the MinION system in 2015 for surveillance during the West Africa Ebola virus outbreak [144].…”