Lineage-Specific Biology Revealed by a Finished Genome Assembly of the Mouse

Church, Deanna M.; Goodstadt, Leo; Hillier, LaDeana W.; Zody, Michael C.; Goldstein, Steve; She, Xinwe; Bult, Carol J.; Agarwala, Richa; Cherry, Joshua L.; DiCuccio, Michael; Hlavina, Wratko; Kapustin, Yuri; Meric, Peter; Maglott, Donna; Birtle, Zoë; Marques, Ana C.; Graves, Tina; Zhou, Shiguo; Teague, Brian; Potamousis, Konstantinos; Churas, Christopher; Place, Michael; Herschleb, Jill; Runnheim, Ron; Forrest, Daniel K.; Amos‐Landgraf, James; Schwartz, David C.; Cheng, Ze; Lindblad‐Toh, Kerstin; Eichler, Evan E.; Ponting, Chris P.

doi:10.1371/journal.pbio.1000112

Cited by 443 publications

(386 citation statements)

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“…Such activities can significantly improve the quality of genomes, including the discovery of missing genes and gene families. A recent effort to upgrade the mouse genome assembly, for example, resulted in the correction or addition of 2185 genes, 61% of which corresponded to lineage-specific segmental duplications (Church et al 2009). Within the human genome, there are >900 annotated genes mapping to large segmental duplications.…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Reconstructing complex regions of genomes using long-read sequencing technology

et al. 2014

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Obtaining high-quality sequence continuity of complex regions of recent segmental duplication remains one of the major challenges of finishing genome assemblies. In the human and mouse genomes, this was achieved by targeting large-insert clones using costly and laborious capillary-based sequencing approaches. Sanger shotgun sequencing of clone inserts, however, has now been largely abandoned, leaving most of these regions unresolved in newer genome assemblies generated primarily by next-generation sequencing hybrid approaches. Here we show that it is possible to resolve regions that are complex in a genome-wide context but simple in isolation for a fraction of the time and cost of traditional methods using long-read single molecule, real-time (SMRT) sequencing and assembly technology from Pacific Biosciences (PacBio). We sequenced and assembled BAC clones corresponding to a 1.3-Mbp complex region of chromosome 17q21.31, demonstrating 99.994% identity to Sanger assemblies of the same clones. We targeted 44 differences using Illumina sequencing and find that PacBio and Sanger assemblies share a comparable number of validated variants, albeit with different sequence context biases. Finally, we targeted a poorly assembled 766-kbp duplicated region of the chimpanzee genome and resolved the structure and organization for a fraction of the cost and time of traditional finishing approaches. Our data suggest a straightforward path for upgrading genomes to a higher quality finished state.

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confidence: 99%

Reconstructing complex regions of genomes using long-read sequencing technology

et al. 2014

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“…As one of the early advocates of a vibrant RNA componentry in extant organisms (DeChiara and Brosius 1987;Brosius 1991Brosius , 1996Petherick 2008), the writer never doubted that the number of functional non-protein-coding RNAs could easily match those for mRNAs in line with more conservative estimates being in the 3% range (Clamp et al 2007;Church et al 2009;Cabili et al 2011;Managadze et al 2013). 9 Considering genes encoding RNAs smaller than 9 ORFs amount to 1.3% in the mouse and human genomes and an additional 0.8% serve as 5 0 -and 3 0 -UTRs at their extremities (International Human Genome Sequencing Consortium 2004;Church et al 2009;Managadze et al 2013).…”

Section: The (Nucleic) Acid Test For Functionmentioning

confidence: 99%

“…9 Considering genes encoding RNAs smaller than 9 ORFs amount to 1.3% in the mouse and human genomes and an additional 0.8% serve as 5 0 -and 3 0 -UTRs at their extremities (International Human Genome Sequencing Consortium 2004;Church et al 2009;Managadze et al 2013). Accordingly, all sequences that end up in mature mRNAs cover somewhat .2% of their respective genomes.…”

Section: The (Nucleic) Acid Test For Functionmentioning

confidence: 99%

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

Brosius

2014

Cold Spring Harbor Perspectives in Biology

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“…To evaluate the performance of NIKS, we simulated 160 wholegenome sequencing experiments by first introducing ~2,000 random, homozygous, single-base mutations into the mouse and maize genome reference sequences and then sequencing at 17, 25, 35 and 50-fold genome coverage 24,25 (Supplementary Notes). The reference genomes are comparable in size-the mouse genome is 2.6 Gb and maize is 2.0 Gb-but differ drastically in their repeat content (Supplementary Fig.…”

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Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers

et al. 2013

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Genes underlying mutant phenotypes can be isolated by combining marker discovery, genetic mapping and resequencing, but a more straightforward strategy for mapping mutations would be the direct comparison of mutant and wild-type genomes. Applying such an approach, however, is hampered by the need for reference sequences and by mutational loads that confound the unambiguous identification of causal mutations. Here we introduce NIKS (needle in the k-stack), a reference-free algorithm based on comparing k-mers in whole-genome sequencing data for precise discovery of homozygous mutations. We applied NIKS to eight mutants induced in nonreference rice cultivars and to two mutants of the nonmodel species Arabis alpina. In both species, comparing pooled F 2 individuals selected for mutant phenotypes revealed small sets of mutations including the causal changes. Moreover, comparing M 3 seedlings of two allelic mutants unambiguously identified the causal gene. Thus, for any species amenable to mutagenesis, NIKS enables forward genetics without requiring segregating populations, genetic maps and reference sequences.Forward genetic screens have been of fundamental importance in elucidating biological mechanisms in model species 1 . Their success, however, has relied on the feasibility of mutant gene isolation. Identification of causal mutations typically begins with genetic mapping, followed by candidate gene sequencing and complementation studies using transformation. Advances in DNA sequencing technologies have tremendously accelerated genetic mapping by combining bulk segregant analysis, that is, pooling recombinant genomes, with whole-genome sequencing, usually referred to as mapping by sequencing 2,3 . This approach is now becoming standard for mutation mapping and identification in many model species 3-12 and has even been applied to decipher quantitative traits with complex genetic architectures 13,14 . Recently, mutagen-induced changes have been used as novel markers, allowing mapping of mutations using isogenic mapping populations 10,15 . Nevertheless, all mapping-by-sequencing methods rely on resequencing, a method for whole-genome reconstruction based on aligning sequences to a reference sequence. Therefore, this requirement restricts the application of the technique to species for which such a reference genome sequence is available.Many reference-sequence assembly projects are currently in progress, including ones for most of the major crop species and breeding animals. However, even with an existing reference sequence, extending mapping-by-sequencing methods beyond the sequenced reference accessions has proved technically challenging. Mutant alleles of genes that are not present in the reference sequence cannot be identified within resequencing data alone. In particular, fast-evolving genes, such as those involved in disease resistance, might not always be represented in the reference sequence 16,17 .Alternative solutions for mapping-by-sequencing in species without reference sequences have been proposed, such ...

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Lineage-Specific Biology Revealed by a Finished Genome Assembly of the Mouse

Abstract: A finished clone-based assembly of the mouse genome reveals extensive recent sequence duplication during recent evolution and rodent-specific expansion of certain gene families. Newly assembled duplications contain protein-coding genes that are mostly involved in reproductive function.

Cited by 443 publications

References 78 publications

Reconstructing complex regions of genomes using long-read sequencing technology

Reconstructing complex regions of genomes using long-read sequencing technology

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers

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