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

Abstract

The human fragile X mental retardation 1 (FMR1 ) gene contains a (CGG) n trinucleotide repeat in its 59 untranslated region (59UTR). Expansions of this repeat result in a number of clinical disorders with distinct molecular pathologies, including fragile X syndrome (FXS; full mutation range, greater than 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 interpulse 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.

Detection of long repeat expansions from PCR-free whole-genome sequence data

“…As the throughput of WGS increases and the cost decreases, WGS may soon become the basis for frontline tests for repeat expansions and other genetic disorders. Theoretically, long reads can also identify many of the longer repeat expansions (Loomis et al 2013), but those technologies are still too expensive to be routinely used for whole-genome screening. At the same time, because the substitution and indel error rates in these long reads range from 10% to 30% (Sovićet al 2016;Bao and Lan 2017), it may be difficult to classify the repeat confidently when its size is close to the normal-premutation or premutation-expansion boundary cutoffs unless the samples are sequenced to high depth.…”

“…Because high-throughput WGS technologies are currently limited to ∼150 base pair (bp) read lengths, variantcalling methods that rely on reads aligned to the reference are subsequently limited to repeat lengths less than 150 bases (Narzisi and Schatz 2015). Many pathogenic repeat expansions have repeats spanning hundreds to thousands of base pairs (Dürr et al 1996;Gatchel and Zoghbi 2005;Kronquist et al 2008;Gijselinck et al 2016), so it has been assumed that short-read sequencing technologies may not be able to identify pathogenic repeat expansions (Loomis et al 2013;Ashley 2016).…”

Group Testing Approach for Trinucleotide Repeat Expansion Disorder Screening

“…PCR-based assays have remained the standard for measuring microsatellite expansion as the expansions in these diseases are generally much longer than the short reads produced by NGS, and single-molecule long-read sequencing approaches are too expensive and low throughput (8 ). Because of this, cost reductions have lagged for microsatellite expansion testing, and an order of magnitude decrease as described here makes a substantial step toward catching up with genetic tests accessible by short read sequencing.…”

“…The large size and low complexity of the pathogenic alleles are not amenable to typical next-generation sequencing (NGS) approaches (e.g., Illumina sequencing with a typical read length under 200 bp). Long-read NGS approaches have been described (8 ) but are substantially more expensive than the PCR fragment sizing methods. Thus, although NGS has markedly reduced the cost of many genetic tests (9 ), FXS testing has remained static in cost since the introduction of PCR-based methods.…”

The genome jigsaw

“…And, he adds, if a certain sequencing technology is the only one that will work to solve a medically important question, "then there is no price tag that can be put on this medically relevant information". Last year, researchers collaborating with Pacific Biosciences used the company's sequencer to distinguish the repetitive genomic region involved in fragile X syndrome, a developmental disorder that is caused by repeats in a particular region on the X chromosome, and that worsens in severity with higher numbers of repeats 8 . As technology developers get closer to instruments that produce longer reads, scientists will need longer DNA fragments at the beginning of their sequencing experiments.…”