Sequencing the unsequenceable: Expanded CGG-repeat alleles of the fragile X gene

Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine
Genome Research (Impact Factor: 14.63). 10/2012; 23(1). DOI: 10.1101/gr.141705.112
Source: PubMed


The human fragile X mental retardation 1 (FMR1) gene contains a (CGG)(n) trinucleotide repeat in its 5' untranslated region (5'UTR). Expansions of this repeat result in a number of clinical disorders with distinct molecular pathologies, including fragile X syndrome (FXS; full mutation range, > 200 CGG repeats) and fragile X-associated tremor/ataxia syndrome (FXTAS; premutation range, 55-200 repeats). Study of these diseases has been limited by an inability to sequence expanded CGG repeats, particularly in the full mutation range, with existing DNA sequencing technologies. Single molecule real time (SMRT) sequencing provides an approach to sequencing that is fundamentally different from other "next-generation" sequencing platforms, and is well suited for long, repetitive DNA sequences. We report the first sequence data for expanded CGG-repeat FMR1 alleles in the full mutation range that reveal the confounding effects of CGG-repeat tracts on both cloning and PCR. A unique feature of SMRT sequencing is its ability to yield real-time information on the rates of nucleoside addition by the tethered DNA polymerase; for the CGG-repeat alleles, we find a strand-specific effect of CGG-repeat DNA on the inter-pulse distance. This kinetic signature reveals a novel aspect of the repeat element; namely, that the particular G bias within the CGG/CCG-repeat element influences polymerase activity in a manner that extends beyond simple nearest-neighbor effects. These observations provide a baseline for future kinetic studies of repeat elements, as well as for studies of epigenetic and other chemical modifications thereof.

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Available from: Erick W Loomis, May 14, 2014
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    • "For analysis of large gene inserts mediated by HDR, we have overcome this obstacle using embedded primers that distinguish between targeted and WT allele sequences while producing similar PCR amplicon sizes (Voit et al., 2014). Simultaneous measurement of amplicons with different lengths has also been achieved by adding a size standard ladder to the SMRT sequencing reaction, and a similar strategy could be used for quantification of large gene additions or NHEJ-mediated integrations of the donor template (Loomis et al., 2013). "
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