De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.
De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.
复制标题
DOI：
10.1371/journal.pgen.1000891
复制
发表时间：
2010-04-08
期刊：
影响因子：
4.5
通讯作者：
Freitag M
中科院分区：
文献类型：
--
作者：
Nowrousian M;Stajich JE;Chu M;Engh I;Espagne E;Halliday K;Kamerewerd J;Kempken F;Knab B;Kuo HC;Osiewacz HD;Pöggeler S;Read ND;Seiler S;Smith KM;Zickler D;Kück U;Freitag M
丝状真菌在生态学、农业、医学和生物技术中具有重要意义。因此，超过 100 种丝状真菌的基因组已被测序（其中大部分是通过桑格测序完成）也就不足为奇了。虽然下一代测序技术彻底改变了基因组重测序，例如对于菌株比较、遗传图谱或转录组和 ChIP 分析，真核基因组的从头组装仍然存在重大障碍，因为它们的尺寸较大且重复序列延伸。丝状真菌在其 30-90 Mb 基因组中包含很少的重复区域，因此是测试短序列读取的从头基因组组装的合适候选者。在这里，我们展示了通过 Illumina/Solexa 和 Roche/454 测序相结合获得的大孢子菌基因组的高质量草图序列。基因组 DNA 的双端 Solexa 测序达到 85 倍覆盖率，并通过单端 454 测序额外获得 10 倍覆盖率，得到约 4 Gb 的 DNA 序列。使用 Velvet 汇编器将读数组装成 40 Mb 草稿版本（N50 为 117 kb）。与脉孢菌基因组的比较分析将 N50 增加至 498 kb。大孢子菌基因组比其测序最接近的粗糙链孢子菌基因组包含的重复区域甚至更少。与其他真菌的基因组比较表明，形态发生和减数分裂的模式生物大孢子菌含有多个与自我/非自我识别相关的基因的重复。此外，大孢子虫比粗糙猪笼草含有更多的聚酮化合物生物合成基因。系统发育分析表明，其中一些基因可能是通过水平基因转移从远缘子囊菌群获得的。我们的研究表明，对于典型的丝状真菌，仅从短序列读取进行基因组从头组装是可行的，Solexa 和 454 测序的混合显着改善了组装，并且所得数据可用于比较研究，以解决真菌生物学的基本问题。真菌对生态系统具有巨大影响，并影响社会的许多方面。它们被用作基础研究的便利生物体，因为它们典型的单倍体遗传学能够直接进行突变表型分析，并且因为大多数真菌细胞可以分化整个生物体。真菌具有紧凑的基因组，几乎没有重复序列，并且它们的基因组应该比哺乳动物或高等植物的基因组更容易从短序列读取组装。为了测试这个想法，我们使用 Solexa 和 454 测序从丝状真菌 Sordaria macrospora 生成约 4 Gb 的原始序列数据。从头组装产生 5,097 个重叠群。通过与三个密切相关的脉孢菌属物种的参考基因组进行比较，该组装得到了改进，从而将约 40 Mb 的基因组序列放置在 152 个支架中。通过比较预测的蛋白质，我们得出结论，大孢子虫携带一组用于信号传导和发育的保守基因，这应该鼓励其进一步用作形态发生和减数分裂的模式生物。我们证明，通过短读段从头组装真菌基因组既便宜又高效。传统上不被视为“模式生物”但等待基因组测序以进行比较和功能基因组学分析的物种最终可以进行深入的全基因组分析。
Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology. Fungi have immense impacts on ecosystems and affect many aspects of society. They are used as convenient organisms for fundamental research because their typically haploid genetics enable straightforward phenotyping of mutations and because most fungal cells can differentiate the entire organism. Fungi have compact genomes with few repetitive sequences, and their genomes should be much easier to assemble from short sequence reads than genomes of mammals or higher plants. To test this idea, we used Solexa and 454 sequencing to generate ∼4 Gb of raw sequence data from the filamentous fungus Sordaria macrospora. De novo assembly yielded 5,097 contigs. This assembly was improved by comparison with reference genomes of three closely related Neurospora species, resulting in placement of ∼40 Mb of genome sequence in 152 scaffolds. From comparisons of predicted proteins we conclude that S. macrospora carries a conserved set of genes for signaling and development, which should encourage its further use as a model organism for morphogenesis and meiosis. We demonstrate that de novo assembly of fungal genomes from short reads is cheap and efficient. Species that are not traditionally considered “model organisms” but await genome sequencing for comparative and functional genomics analyses are at last amenable to in-depth genome-wide analyses.
登录
查看更多内容
DOI：
10.1007/bf00299131
发表时间：
1992-03-01
期刊：
MOLECULAR & GENERAL GENETICS
影响因子：
--
作者：
DABOUSSI, MJ;LANGIN, T;BRYGOO, Y
通讯作者：
BRYGOO, Y
影响因子：
4.5
作者：
Coleman JJ;Rounsley SD;Rodriguez-Carres M;Kuo A;Wasmann CC;Grimwood J;Schmutz J;Taga M;White GJ;Zhou S;Schwartz DC;Freitag M;Ma LJ;Danchin EG;Henrissat B;Coutinho PM;Nelson DR;Straney D;Napoli CA;Barker BM;Gribskov M;Rep M;Kroken S;Molnár I;Rensing C;Kennell JC;Zamora J;Farman ML;Selker EU;Salamov A;Shapiro H;Pangilinan J;Lindquist E;Lamers C;Grigoriev IV;Geiser DM;Covert SF;Temporini E;Vanetten HD
通讯作者：
Vanetten HD
影响因子：
11.4
作者：
Ballario, P;Vittorioso, P;Macino, G
通讯作者：
Macino, G
影响因子：
5.8
作者：
Conesa, A;Götz, S;Robles, M
通讯作者：
Robles, M
影响因子：
2.5
作者：
Averbeck, NB;Jensen, ON;Osiewacz, HD
通讯作者：
Osiewacz, HD