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NAR Top Articles - Genomics

Genomics

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May 2015


Optimization of scarless human stem cell genome editing
Yang, LH; Guell, M; Byrne, S; Yang, JL; De Los Angeles, A; Mali, P; Aach, J; Kim-Kiselak, C; Briggs, AW; Rios, X; Huang, PY; Daley, G; Church, G
Nucleic Acids Res. 2013, 41, 9049-9061
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Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8x higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%)...

Single-cell paired-end genome sequencing reveals structural variation per cell cycle
Voet, T; Kumar, P; Van Loo, P; Cooke, SL; Marshall, J; Lin, ML; Esteki, MZ; Van der Aa, N; Mateiu, L; McBride, DJ; Bignell, GR; McLaren, S; Teague, J; Butler, A; Raine, K; Stebbings, LA; Quail, MA; D'Hooghe, T; Moreau, Y; Futreal, PA; Stratton, MR; Vermee
Nucleic Acids Res. 2013, 41, 6119-6138
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The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants...

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer
L'Abbate, A; Macchia, G; D'Addabbo, P; Lonoce, A; Tolomeo, D; Trombetta, D; Kok, K; Bartenhagen, C; Whelan, CW; Palumbo, O; Severgnini, M; Cifola, I; Dugas, M; Carella, M; De Bellis, G; Rocchi, M; Carbone, L; Storlazzi, CT
Nucleic Acids Res. 2014, 42, 9131-9145
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The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples...

A comprehensive survey of non-canonical splice sites in the human transcriptome
Parada, GE; Munita, R; Cerda, CA; Gysling, K
Nucleic Acids Res. 2014, 42, 10564-10578
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We uncovered the diversity of non-canonical splice sites at the human transcriptome using deep transcriptome profiling. We mapped a total of 3.7 billion human RNA-seq reads and developed a set of stringent filters to avoid false non-canonical splice site detections. We identified 184 splice sites with non-canonical dinucleotides and U2/U12-like consensus sequences. We selected 10 of the herein identified U2/U12-like non-canonical splice site events and successfully validated 9 of them via reverse transcriptase-polymerase chain reaction and Sanger sequencing. Analyses of the 184 U2/U12-like non- canonical splice sites indicate that 51% of them are not annotated in GENCODE. In addition, 28% of them are conserved in mouse and 76% are involved in alternative splicing events, some of them with tissue-specific alternative splicing patterns. Interestingly, our analysis identified some U2/U12-like non-canonical splice sites that are converted into canonical splice sites by RNA A-to-I editing. Moreover, the U2/U12-like non-canonical splice sites have a differential distribution of splicing regulatory sequences, which may contribute to their recognition and regulation...

metaseq: a Python package for integrative genome-wide analysis reveals relationships between chromatin insulators and associated nuclear mRNA
Dale, RK; Matzat, LH; Lei, EP
Nucleic Acids Res. 2014, 42, 9158-9170
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Here we introduce metaseq, a software library written in Python, which enables loading multiple genomic data formats into standard Python data structures and allows flexible, customized manipulation and visualization of data from high-throughput sequencing studies. We demonstrate its practical use by analyzing multiple datasets related to chromatin insulators, which are DNA-protein complexes proposed to organize the genome into distinct transcriptional domains. Recent studies in Drosophila and mammals have implicated RNA in the regulation of chromatin insulator activities. Moreover, the Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role of RNA in this process remains unclear. Better understanding of chromatin insulator regulation requires integration of multiple datasets, including those from chromatin-binding, RNA-binding, and gene expression experiments. We use metaseq to integrate RIP-and ChIP-seq data for Shep and the core gypsy insulator protein Su(Hw) in two different cell types, along with publicly available ChIP-chip and RNA-seq data...

Mapping of six somatic linker histone H1 variants in human breast cancer cells uncovers specific features of H1.2
Millan-Arino, L; Islam, AMMK; Izquierdo-Bouldstridge, A; Mayor, R; Terme, JM; Luque, N; Sancho, M; Lopez-Bigas, N; Jordan, A
Nucleic Acids Res. 2014, 42, 4474-4493
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Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagged recombinant H1 variants expressed in breast cancer cells, we have investigated the distribution of six H1 variants in promoters and genome-wide. H1 is depleted at promoters depending on its transcriptional status and differs between variants. Notably, H1.2 is less abundant than other variants at the transcription start sites of inactive genes, and promoters enriched in H1.2 are different from those enriched in other variants and tend to be repressed. Additionally, H1.2 is enriched at chromosomal domains characterized by low guanine-cytosine (GC) content and is associated with lamina-associated domains. Meanwhile, other variants are associated with higher GC content, CpG islands and gene-rich domains...

Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay
Larman, HB; Scott, ER; Wogan, M; Oliveira, G; Torkamani, A; Schultz, PG
Nucleic Acids Res. 2014, 42, 9146-9157
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A sensitive and highly multiplex method to directly measure RNA sequence abundance without requiring reverse transcription would be of value for a number of biomedical applications, including high throughput small molecule screening, pathogen transcript detection and quantification of short/degraded RNAs. RNA Annealing, Selection and Ligation (RASL) assays, which are based on RNA template-dependent oligonucleotide probe ligation, have been developed to meet this need, but technical limitations have impeded their adoption. Whereas DNA ligase-based RASL assays suffer from extremely low and sequence-dependent ligation efficiencies that compromise assay robustness, Rnl2 can join a fully DNA donor probe to a 3'-diribonucleotide-terminated acceptor probe with high efficiency on an RNA template strand. Rnl2-based RASL exhibits sub-femtomolar transcript detection sensitivity, and permits the rational tuning of probe signals for optimal analysis by massively parallel DNA sequencing (RASL-seq)...

Stability, delivery and functions of human sperm RNAs at fertilization
Sendler, E; Johnson, GD; Mao, SH; Goodrich, RJ; Diamond, MP; Hauser, R; Krawetz, SA
Nucleic Acids Res. 2013, 41, 4104-4117
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Increasing attention has focused on the significance of RNA in sperm, in light of its contribution to the birth and long-term health of a child, role in sperm function and diagnostic potential. As the composition of sperm RNA is in flux, assigning specific roles to individual RNAs presents a significant challenge. For the first time RNA-seq was used to characterize the population of coding and non-coding transcripts in human sperm. Examining RNA representation as a function of multiple methods of library preparation revealed unique features indicative of very specific and stage-dependent maturation and regulation of sperm RNA, illuminating their various transitional roles. Correlation of sperm transcript abundance with epigenetic marks suggested roles for these elements in the pre- and post-fertilization genome....

Transcriptome-wide investigation of genomic imprinting in chicken
Fresard, L; Leroux, S; Servin, B; Gourichon, D; Dehais, P; San Cristobal, M; Marsaud, N; Vignoles, F; Bed'hom, B; Coville, JL; Hormozdiari, F; Beaumont, C; Zerjal, T; Vignal, A; Morisson, M; Lagarrigue, S; Pitel, F
Nucleic Acids Res. 2014, 42, 3768-3782
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Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing similar to 200 Gb of sequences...

Genome-wide reorganization of histone H2AX toward particular fragile sites on cell activation
Seo, J; Kim, K; Chang, DY; Kang, HB; Shin, EC; Kwon, J; Choi, JK
Nucleic Acids Res. 2014, 42, 1016-1025
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gamma H2AX formation by phosphorylation of the histone variant H2AX is the key process in the repair of DNA lesions including those arising at fragile sites under replication stress. Here we demonstrate that H2AX is dynamically reorganized to preoccupy gamma H2AX hotspots on increased replication stress by activated cell proliferation and that H2AX is enriched in aphidicolin-induced replisome stalling sites in cycling cells. Interestingly, H2AX enrichment was particularly found in genomic regions that replicate in early S phase. High transcription activity, a hallmark of early replicating fragile sites, was a determinant of H2AX localization. Subtelomeric H2AX enrichment was also attributable to early replication and high gene density. In contrast, late replicating and infrequently transcribed regions, including common fragile sites and heterochromatin, lacked H2AX enrichment. In particular, heterochromatin was inaccessible to H2AX incorporation, maybe partly explaining the cause of mutation accumulation in cancer heterochromatin. Meanwhile, H2AX in actively dividing cells was intimately colocalized with INO80...

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