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Open Access Research article

Efficient high-throughput sequencing of a laser microdissected chromosome arm

Eva Seifertova1*, Lyle B Zimmerman2, Michael J Gilchrist2, Jaroslav Macha1, Svatava Kubickova3, Halina Cernohorska3, Vojtech Zarsky1, Nick DL Owens2, Abdul K Sesay2, Tereza Tlapakova1 and Vladimir Krylov1

Author Affiliations

1 Charles University in Prague, Faculty of Science, Vinicna 7, Prague 2, 128 44, Czech Republic

2 MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

3 Veterinary Research Institute, Hudcova 70, Brno, 621 00, Czech Republic

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BMC Genomics 2013, 14:357  doi:10.1186/1471-2164-14-357

Published: 28 May 2013

Abstract

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.

Keywords:
Xenopus; Tropicalis; Chromosomes; Next generation sequencing; WGA; Genetic map