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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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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.
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pone-0063673-g005: 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.

Mentions: First, we compared the assembly results between lib2.8.nofilter.k31 and lib2.8.nodot.k31. The lib2.8.nofilter.k31 and lib2.8.nodot.k31 assemblies displayed nearly the same pattern of assembled scaffold cumulative lengths; reaching a plateau at ∼37.5 Mb with ∼1000 scaffolds (Fig. 5a). The number and maximum size of assembled scaffolds (Table 2), in addition to the reconstruction percentage of the total genes and genome sequence (Fig. 3 and 4), were also similar for the two assemblies. Therefore, although the N50 value (1.36 Mb) of lib2.8.nodot.k31 was lower than that (1.68 Mb) of lib2.8.nofilter.k31, the overall assembly performance was not significantly affected by unfiltering reads including undetermined bases. The SOLiD De Novo Accessory Tools can handle reads including undetermined bases, but it is considered better to exclude such reads because undetermined ‘N’ bases are automatically converted to ‘A’ by the Velvet program.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646829&req=5

pone-0063673-g005: 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.
Mentions: First, we compared the assembly results between lib2.8.nofilter.k31 and lib2.8.nodot.k31. The lib2.8.nofilter.k31 and lib2.8.nodot.k31 assemblies displayed nearly the same pattern of assembled scaffold cumulative lengths; reaching a plateau at ∼37.5 Mb with ∼1000 scaffolds (Fig. 5a). The number and maximum size of assembled scaffolds (Table 2), in addition to the reconstruction percentage of the total genes and genome sequence (Fig. 3 and 4), were also similar for the two assemblies. Therefore, although the N50 value (1.36 Mb) of lib2.8.nodot.k31 was lower than that (1.68 Mb) of lib2.8.nofilter.k31, the overall assembly performance was not significantly affected by unfiltering reads including undetermined bases. The SOLiD De Novo Accessory Tools can handle reads including undetermined bases, but it is considered better to exclude such reads because undetermined ‘N’ bases are automatically converted to ‘A’ by the Velvet program.

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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