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Efficient high-throughput sequencing of a laser microdissected chromosome arm
E. Seifertova, LB. Zimmerman, MJ. Gilchrist, J. Macha, S. Kubickova, H. Cernohorska, V. Zarsky, ND. Owens, AK. Sesay, T. Tlapakova, V. Krylov,
Language English Country England, Great Britain
Document type Journal Article, Research Support, Non-U.S. Gov't
NLK
BioMedCentral
from 2000-12-01
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Directory of Open Access Journals
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Free Medical Journals
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- MeSH
- Chromosomes genetics MeSH
- Genomics MeSH
- Nucleic Acid Hybridization MeSH
- Lasers * MeSH
- Chromosome Mapping MeSH
- Microdissection * MeSH
- Sequence Analysis, DNA methods MeSH
- Nucleic Acid Amplification Techniques MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Xenopus genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Genomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly. RESULTS: We laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly. CONCLUSION: We present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds.
References provided by Crossref.org
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