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. 2011 Nov 29;7:38.
doi: 10.1186/1746-4811-7-38.

An Efficient Procedure for Plant Organellar Genome Assembly, Based on Whole Genome Data From the 454 GS FLX Sequencing Platform

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Free PMC article

An Efficient Procedure for Plant Organellar Genome Assembly, Based on Whole Genome Data From the 454 GS FLX Sequencing Platform

Tongwu Zhang et al. Plant Methods. .
Free PMC article

Abstract

Motivation: Complete organellar genome sequences (chloroplasts and mitochondria) provide valuable resources and information for studying plant molecular ecology and evolution. As high-throughput sequencing technology advances, it becomes the norm that a shotgun approach is used to obtain complete genome sequences. Therefore, to assemble organellar sequences from the whole genome, shotgun reads are inevitable. However, associated techniques are often cumbersome, time-consuming, and difficult, because true organellar DNA is difficult to separate efficiently from nuclear copies, which have been transferred to the nucleus through the course of evolution.

Results: We report a new, rapid procedure for plant chloroplast and mitochondrial genome sequencing and assembly using the Roche/454 GS FLX platform. Plant cells can contain multiple copies of the organellar genomes, and there is a significant correlation between the depth of sequence reads in contigs and the number of copies of the genome. Without isolating organellar DNA from the mixture of nuclear and organellar DNA for sequencing, we retrospectively extracted assembled contigs of either chloroplast or mitochondrial sequences from the whole genome shotgun data. Moreover, the contig connection graph property of Newbler (a platform-specific sequence assembler) ensures an efficient final assembly. Using this procedure, we assembled both chloroplast and mitochondrial genomes of a resurrection plant, Boea hygrometrica, with high fidelity. We also present information and a minimal sequence dataset as a reference for the assembly of other plant organellar genomes.

Figures

Figure 1
Figure 1
Circular representation of an assembly of the B. hygrometrica cp and mt genomes. Circle display (from the outside): (1) physical map scale in kilobase pairs (LSC region in blue, SSC region in green, and IRs regions in red); (2) read depths of the cp sequencing in yellow (step size: 1000 bp; cp assembly: range 0-100; mt assembly: range 0-160); (3) SOLiD mate-pair read validation with the 1-kb insert library in purple (insert size 600-1000 bp and step size 100 bp in the cp assembly and 500 bp in the mt assembly); (4) SOLiD mate-pair read validation with 2-kb library in orange (insert size 800-1200 bp and step size 100 bp in the cp assembly and 1000 bp in the mt assembly). The high variance in read depth of the mt genome results from the regions of chloroplast-like sequences. This figure was generated by the Circos program [33].
Figure 2
Figure 2
Contig length and read depth distribution in a de novo assembly of raw data. The Y-axis is an exponential scale with powers of 2.
Figure 3
Figure 3
Average read depth of contigs in chloroplast, mitochondrial, and nuclear DNAs.
Figure 4
Figure 4
Pipeline for plant organellar genome assembly. Mt-contigs and cp-contigs include genes that are highly conserved among all plants. The conserved parts of sequence contigs can be identified first, and using SOLiD mate-pair libraries or PCR walks, we can confirm putative connections and close gaps among contigs empirically.

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