Sequencing of chloroplast genome using whole cellular DNA and solexa sequencing technology

Abstract

Sequencing of the chloroplast (cp) genome using traditional sequencing methods has been difficult because of its size (>120 kb) and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the cp genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassicarapa accessions with one lane per accession. In total, 246, 362, and 361 Mb sequence data were generated for the three accessions Chiifu-401-42, Z16, and FT, respectively. Micro-reads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7-99.8 or 95.5-99.7% of the B. rapa cp genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of cp genome.

Keywords: Brassica rapa; Solexa sequencing technology; chloroplast genome; sequencing; whole cellular DNA.

Figure 1

Schematic view of micro-reads assembly method for chloroplast (cp) genome. De novo assembled contigs (blue bars) are aligned to reference (red bar) to extract sequences generated from cp genome (thin pink bars). Draft consensus (thick pink bar) was constructed guided by reference. Gaps were filled by extending sequence and joining two contigs that overlapped (green bar) by 10 or more nt.

Figure 2

Plot showing sequencing depth by position for chloroplast genomes of three B. rapa accessions sequenced by Solexa Genome Analyzer with whole cellular DNA as template. Number of micro-reads per position (y-axis) is plotted against position in the assembly (x-axis, in kb) in a window size of 100 bp. Numbers above x-axis indicate boundary sites of large single copy (LSC) and small single copy (SSC), and two inverted repeats (IRa and IRb).

Figure 3

Comparison of assemblies for Chiifu-402-41 guided by chloroplast genome sequences of B. rapa (DQ231548), A. thaliana (NC000932), and N. tabacum (NC001879). Assembled consensuses are represented by three bars. For B. rapa, black parts of bar indicate LSC or SSC; red parts of bar indicate IRa or IRb. For A. thaliana and N. tabacum, silver parts indicate gaps. Blue block between bars of B. rapa and A. thaliana or N. tabacum indicates identity >95%.

Figure 4

Circular gene map of Brassica rapa chloroplast genome. The thick lines indicate the extent of the inverted repeats (IRa and IRb, 26,213 bp), which separate the genome into small (SSC, 17,777 bp) and large (LSC, 83,282 bp) single copy regions. Genes on the outside of the map are transcribed in the clockwise direction and genes on the inside of the map are transcribed in the counterclockwise direction.