Next-Generation Sequencing Strategies Enable Routine Detection of Balanced Chromosome Rearrangements for Clinical Diagnostics and Genetic Research

Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
The American Journal of Human Genetics (Impact Factor: 10.93). 04/2011; 88(4):469-81. DOI: 10.1016/j.ajhg.2011.03.013
Source: PubMed


The contribution of balanced chromosomal rearrangements to complex disorders remains unclear because they are not detected routinely by genome-wide microarrays and clinical localization is imprecise. Failure to consider these events bypasses a potentially powerful complement to single nucleotide polymorphism and copy-number association approaches to complex disorders, where much of the heritability remains unexplained. To capitalize on this genetic resource, we have applied optimized sequencing and analysis strategies to test whether these potentially high-impact variants can be mapped at reasonable cost and throughput. By using a whole-genome multiplexing strategy, rearrangement breakpoints could be delineated at a fraction of the cost of standard sequencing. For rearrangements already mapped regionally by karyotyping and fluorescence in situ hybridization, a targeted approach enabled capture and sequencing of multiple breakpoints simultaneously. Importantly, this strategy permitted capture and unique alignment of up to 97% of repeat-masked sequences in the targeted regions. Genome-wide analyses estimate that only 3.7% of bases should be routinely omitted from genomic DNA capture experiments. Illustrating the power of these approaches, the rearrangement breakpoints were rapidly defined to base pair resolution and revealed unexpected sequence complexity, such as co-occurrence of inversion and translocation as an underlying feature of karyotypically balanced alterations. These findings have implications ranging from genome annotation to de novo assemblies and could enable sequencing screens for structural variations at a cost comparable to that of microarrays in standard clinical practice.

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Available from: Toshiro K. Ohsumi
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    • "Furthermore, DNA-Seq does not require a priori knowledge of the genome being sequenced and thus, can be used for de novo genome assembly of multiple species (Hurd and Nelson 2009). Lastly, NGS also allows for the analysis of repetitive DNA sequences (Gisselsson et al. 2001), balanced chromosomal rearrangements, and an initial map of insertion and deletion (INDEL) variation in the genome (Talkowski et al. 2011). Until bioinformatics pipelines and NGS analysis software are developed and simplified for use in IVF clinics, however, microarray-based technologies are likely to continue to dominate the PGS field (Van der Aa et al. 2013). "
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    ABSTRACT: Formation of a totipotent blastocyst capable of implantation is one of the first major milestones in early mammalian embryogenesis, but less than half of in vitro fertilized embryos from most mammals will progress to this stage of development. Whole chromosomal abnormalities, or aneuploidy, are key determinants of whether human embryos will arrest or reach the blastocyst stage. Depending on the type of chromosomal abnormality, however, certain embryos still form blastocysts and may be morphologically indistinguishable from chromosomally normal embryos. Despite the implementation of pre-implantation genetic screening and other advanced in vitro fertilization (IVF) techniques, the identification of aneuploid embryos remains complicated by high rates of mosaicism, atypical cell division, cellular fragmentation, sub-chromosomal instability, and micro-/multi-nucleation. Moreover, several of these processes occur in vivo following natural human conception, suggesting that they are not simply a consequence of culture conditions. Recent technological achievements in genetic, epigenetic, chromosomal, and non-invasive imaging have provided additional embryo assessment approaches, particularly at the single-cell level, and clinical trials investigating their efficacy are continuing to emerge. In this review, we summarize the potential mechanisms by which aneuploidy may arise, the various detection methods, and the technical advances (such as time-lapse imaging, "-omic" profiling, and next-generation sequencing) that have assisted in obtaining this data. We also discuss the possibility of aneuploidy resolution in embryos via various corrective mechanisms, including multi-polar divisions, fragment resorption, endoreduplication, and blastomere exclusion, and conclude by examining the potential implications of these findings for IVF success and human fecundity.
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    • "We fine-mapped 14 breakpoint regions from 12 translocations to LINEs and attempted to capture these loci by SureSelect. Discordant reads spanned the junction from LINE to unique sequence in only five of these breakpoints (EGL002, EGL064, EGL306, EGL317, and EGL319), which is consistent with the previously recognized limitation in LINE breakpoint sequencing (Talkowski et al. 2011). Surprisingly, our SureSelect approach was successful in mapping informative reads to three segmental duplications . "
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    ABSTRACT: Unbalanced translocations are a relatively common type of copy number variation and a major contributor to neurodevelopmental disorders. We analyzed the breakpoints of 57 unique unbalanced translocations to investigate the mechanisms of how they form. Fifty-one are simple unbalanced translocations between two different chromosome ends, and six rearrangements have more than three breakpoints involving two to five chromosomes. Sequencing 37 breakpoint junctions revealed that simple translocations have between 0 and 4 base pairs (bp) of microhomology ( n = 26), short inserted sequences ( n = 8), or paralogous repeats ( n = 3) at the junctions, indicating that translocations do not arise primarily from nonallelic homologous recombination but instead form most often via nonhomologous end joining or microhomology-mediated break-induced replication. Three simple translocations fuse genes that are predicted to produce in-frame transcripts of SIRPG-WWOX , SMOC2-PROX1 , and PIEZO2-MTA1 , which may lead to gain of function. Three complex translocations have inversions, insertions, and multiple breakpoint junctions between only two chromosomes. Whole-genome sequencing and fluorescence in situ hybridization analysis of two de novo translocations revealed at least 18 and 33 breakpoints involving five different chromosomes. Breakpoint sequencing of one maternally inherited translocation involving four chromosomes uncovered multiple breakpoints with inversions and insertions. All of these breakpoint junctions had 0–4 bp of microhomology consistent with chromothripsis, and both de novo events occurred on paternal alleles. Together with other studies, these data suggest that germline chromothripsis arises in the paternal genome and may be transmitted maternally. Breakpoint sequencing of our large collection of chromosome rearrangements provides a comprehensive analysis of the molecular mechanisms behind translocation formation.
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    • "These data were calculated by using the cost shown in Talkowski et al. (2011). Since the cost of 76-cycle paired-end multiplex sequencing on Hiseq 2000 platform is $3,280 per lane, the cost of PE50 and PE25 are simply calculated as $ "

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