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, Oct 05, 2015
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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.
    Genome Research 06/2015; 25(7). DOI:10.1101/gr.191247.115 · 14.63 Impact Factor
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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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    • "Each guide RNA target site (n = 5) and predicted off-target sites (n = 126) was selected for capture sequencing (Table S1) using the Agilent SureSelectXT Target Enrichment System. Capture Sequencing was performed as described earlier (Talkowski et al., 2011). For details on experimental procedures, see Supplemental Information. "
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    ABSTRACT: Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.
    Cell Stem Cell 11/2014; 15(5):p643–652. DOI:10.1016/j.stem.2014.10.004 · 22.27 Impact Factor
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