XB-ART-47146BMC Genomics. May 28, 2013; 14 357.
Efficient high-throughput sequencing of a laser microdissected chromosome arm.
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. 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. 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.
PubMed ID: 23714049
PMC ID: PMC3701504
Article link: BMC Genomics.
Grant support: R01 DC011901 NIDCD NIH HHS , U117560482 Medical Research Council , U117597137 Medical Research Council , MC_U117560482 Medical Research Council , MC_U117597137 Medical Research Council , MRC_MC_U117597137 Medical Research Council , MRC_MC_U117560482 Medical Research Council
Genes referenced: acp2 adamts1 adgra1 adh1b agmat ap2m1 asmtl atp13a1 atp6ap1.1 atp6v1h atp7a b4galt5 babam2 cabin1 camk2d cbl ccdc40 cdk16 cfp chd3 crim1 csrnp1 cyp1b1 dab2ip dmap1 dusp9 e2f4 elk1 epb41 exoc1 ext2 ezh1 f9 fbxl7 fgfr4 fignl1 fut1 gata1 gemin5 glipr2 got1 grin1 gyg2 hoxb3 kcnd1 kidins220 lacc1 map2k4 mast2 mast3 mat1a mef2d mfn2 mgc145260 mpp7 myo6 naif1 nmt2 nop2 nr6a1 nsd2 nufip1 olig3 otud5 pan3 pcsk9 pfkfb1 pias2 piga plce1 ppp4r2 prepl ptprg rasgrp2 rhbg rps6ka6 rybp slc12a3 smarcal1 sox6 sp2 src srpx stat4 sts syn1 taf4 tbcel tbr1 tram2 ttc27 ubqln4 was zfp36l1 znf142 znf423
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|Figure 1. Chromosome dissection and sequencing workflow. (A) Dissociated Xenopus tropicalis froglet testes were cultured in colchicine, and 15 Chromosome 7 short arms were laser-dissected, collected and (B) amplified using Sigma WGA3/WGA4 systems. (C) Sequencing libraries were prepared by Nextera transposome-mediated simultaneous fragmentation and adaptor ligation to minimize resequencing WGA adaptors, and (D) sequenced on an Illumina GAII. (E) The resulting reads were trimmed and mapped to X. tropicalis genome assemblies v4.1 and v7.1 using Bowtie, visualized on scaffolds/chromosomes, and selected positions were validated by FISH-TSA. 1- Centromere, 2- secondary constriction.|
|Figure 2. Uniquely-localized read distribution on physically mapped scaffolds. Schematic of X. tropicalis karyotype showing genes mapped by FISH-TSA [8,10,20] and cognate v4.1 scaffolds (scaff_number). High read density (17�80 hits/kb) is seen for scaffolds localized to the microdissected region (red region). Genes that physically localized to non-7p regions were all contained by scaffolds with <1 hit/kb (black regions).|
|Figure 3. FISH-TSA analysis of three hybrid scaffolds. Selected genes from read-rich and read-absent areas of v4.1 scaffolds_75, _266, and _270 were physically mapped to X. tropicalis chromosomes using specific cDNA probes. Top line shows probe gene location on scaffold, second line shows distribution of uniquely-mapping reads, third line shows distribution of repetitive sequence (similar in hit-rich and hit-absent areas).|
|Figure 4. Sequence of dissected chromosome arm identifies misassembled regions. Pileup of uniquely-mapping reads from dissected chromosome 7p on X. tropicalis v7.1 assembly superscaffolds/chromosomes 2 (top), 3 (middle), and 7 (bottom). Reads mapping uniquely to typical non-7p regions exemplified by chromosomes 2 and 3 are rare, with the exception of a misassembled region at 37–38 Mb on chromosome 3. Most of the short arm (left side) of chromosome 7 is heavily decorated by reads, but gaps to the left of centromere (arrow) and read-dense regions to the right identify misassembled regions.|