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. 2013 May 7;8(5):e63673.
doi: 10.1371/journal.pone.0063673. Print 2013.

Fine de novo sequencing of a fungal genome using only SOLiD short read data: verification on Aspergillus oryzae RIB40

Myco Umemura  1 , Yoshinori Koyama, Itaru Takeda, Hiroko Hagiwara, Tsutomu Ikegami, Hideaki Koike, Masayuki Machida
Affiliations

Fine de novo sequencing of a fungal genome using only SOLiD short read data: verification on Aspergillus oryzae RIB40

Myco Umemura et al. PLoS One. .

Abstract

The development of next-generation sequencing (NGS) technologies has dramatically increased the throughput, speed, and efficiency of genome sequencing. The short read data generated from NGS platforms, such as SOLiD and Illumina, are quite useful for mapping analysis. However, the SOLiD read data with lengths of <60 bp have been considered to be too short for de novo genome sequencing. Here, to investigate whether de novo sequencing of fungal genomes is possible using only SOLiD short read sequence data, we performed de novo assembly of the Aspergillus oryzae RIB40 genome using only SOLiD read data of 50 bp generated from mate-paired libraries with 2.8- or 1.9-kb insert sizes. The assembled scaffolds showed an N50 value of 1.6 Mb, a 22-fold increase than those obtained using only SOLiD short read in other published reports. In addition, almost 99% of the reference genome was accurately aligned by the assembled scaffold fragments in long lengths. The sequences of secondary metabolite biosynthetic genes and clusters, whose products are of considerable interest in fungal studies due to their potential medicinal, agricultural, and cosmetic properties, were also highly reconstructed in the assembled scaffolds. Based on these findings, we concluded that de novo genome sequencing using only SOLiD short reads is feasible and practical for molecular biological study of fungi. We also investigated the effect of filtering low quality data, library insert size, and k-mer size on the assembly performance, and recommend for the assembly use of mild filtered read data where the N50 was not so degraded and the library has an insert size of ∼2.0 kb, and k-mer size 33.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Overview of our de novo genome assembly pipeline.
The assembly block is performed using SOLiD De Novo Accessory Tools 2.0 developed by Life Technologies. The data filtering and analysis blocks are written in shell, Ruby, or Perl languages.
Figure 2
Figure 2. Coverage of the reference genome sequence by the assembled scaffolds.
(a) Dot-plot alignments of assembled scaffolds vs the reference genome sequence of Aspergillus oryzae RIB40. (b) Reference genome sequences aligned by assembled scaffold fragments with lengths of ≤10 kb (yellow), >10 kb (green), and >50 kb (red). The Roman numerals I-VIII indicate the chromosome index of the RIB40 genome.
Figure 3
Figure 3. Reconstruction of gene regions in the assembled scaffolds.
(a) Plot showing the number of known Aspergillus oryzae RIB40 genes that were not found in the assembled genome. (b) Percentages of high-scoring segment pair (HSP; solid) and identical bases (open) in the gene regions. The graph includes the results of the assemblies using lib2.8 and lib1.9 with either unfiltered (nofilter), no undetermined bases (nodot), or QV >10 data. For lib2.8.qv10 and lib1.9.qv10, the results using k-mers of 25 to 35 are included.
Figure 4
Figure 4. Proportion of assembled scaffold fragments aligned to the Aspergillus oryzae RIB40 reference genome.
The length of aligned fragments are indicated by color (bluegreen, >50 kb; purple, >10 kb; gray, ≤10 kb; and yellow, 0 or none). The graph includes the results of the assemblies using lib2.8 and lib1.9 with either unfiltered (nofilter), no undetermined bases (nodot), or QV >10 data. For lib2.8.qv10 and lib1.9.qv10, the results using k-mers of 25 to 35 are included.
Figure 5
Figure 5. Cumulative lengths of assembled scaffolds (>95 bp).
(a) The profiles in the lib2.8.nofilter.k31, lib2.8.nodot.k31, lib2.8.qv10.k31, lib1.9.nodot.k31, and lib1.9.qv10.k31 assemblies using lib2.8 and lib1.9 with either unfiltered (nofilter), no undetermined bases (nodot), or QV >10 data. (b) The profiles of lib2.8.qv10 and (c) lib1.9.qv10 with changing k-mers from 25 to 35. The dashed grey line at 37.2 Mb in each graph denotes the size of the reference genome.
Figure 6
Figure 6. R50 and N50 values with different k-mer sizes.
(a) The R50 and (b) the N50 values for lib2.8.qv10 (closed square) and lib1.9.qv10 (open square). The R50 value corresponds to N50 using sequence fragments of the reference genome covered by highly accurate sequences of assembled scaffolds.
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References

    1. Marroni F, Pinosio S, Morgante M (2012) The quest for rare variants: pooled multiplexed next generation sequencing in plants. Front Plant Sci 3: 133. - PMC - PubMed
    1. Lee SW, Markham PF, Markham JF, Petermann I, Noormohammadi AH, et al. (2011) First complete genome sequence of infectious laryngotracheitis virus. BMC Genomics 12: 197. - PubMed
    1. Cunningham C, Gatherer D, Hilfrich B, Baluchova K, Dargan DJ, et al. (2010) Sequences of complete human cytomegalovirus genomes from infected cell cultures and clinical specimens. J Gen Virol 91(Pt 3): 605–615. - PMC - PubMed
    1. Ghosh W, George A, Agarwal A, Raj P, Alam M, et al. (2011) Whole-genome shotgun sequencing of the sulfur-oxidizing chemoautotroph Tetrathiobacter kashmirensis . J Bacteriol 193(19): 5553–5554. - PMC - PubMed
    1. Perez Chaparro PJ, McCulloch JA, Cerdeira LT, Al-Dilaimi A, Canto de Sa LL, et al. (2011) Whole genome sequencing of environmental Vibrio cholerae O1 from 10 nanograms of DNA using short reads. J Microbiol Methods 87(2): 208–212. - PubMed

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