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

Umemura M, Koyama Y, Takeda I, Hagiwara H, Ikegami T, Koike H, Machida M - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan.

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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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.
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pone-0063673-g002: 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.

Mentions: To determine the feasibility of de novo sequencing the RIB40 genome using only SOLiD short reads, performance of the genome assembly was evaluated based on the N50 values, maximum size, and total coverage of the reference sequence by the assembled sequences (Table 2). The assemblies were named by including the library number (lib2.8 or lib1.9), data filtering (nofilter, nodot, or qv10), and k-mer size (k25-k35, restricted to odd integers). The assembly lib2.8.nofilter.k31, which used unfiltered read data, was treated as the standard assembly. The standard assembly had an N50 of 1.7 Mb and a maximum scaffold size of 3.4 Mb without continuous undefined nucleotides. The coverage of the reference sequence by the assembled scaffolds reached 98.87% (Table 2). As shown in Figure 2, most regions of the reference sequence were covered by the assembled scaffolds that consisted of >10-kb fragments.


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

Umemura M, Koyama Y, Takeda I, Hagiwara H, Ikegami T, Koike H, Machida M - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646829&req=5

pone-0063673-g002: 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.
Mentions: To determine the feasibility of de novo sequencing the RIB40 genome using only SOLiD short reads, performance of the genome assembly was evaluated based on the N50 values, maximum size, and total coverage of the reference sequence by the assembled sequences (Table 2). The assemblies were named by including the library number (lib2.8 or lib1.9), data filtering (nofilter, nodot, or qv10), and k-mer size (k25-k35, restricted to odd integers). The assembly lib2.8.nofilter.k31, which used unfiltered read data, was treated as the standard assembly. The standard assembly had an N50 of 1.7 Mb and a maximum scaffold size of 3.4 Mb without continuous undefined nucleotides. The coverage of the reference sequence by the assembled scaffolds reached 98.87% (Table 2). As shown in Figure 2, most regions of the reference sequence were covered by the assembled scaffolds that consisted of >10-kb fragments.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan.

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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