Survey of the Applications of NGS to Whole-Genome Sequencing and Expression Profiling

Abstract

Recently, the technologies of DNA sequence variation and gene expression profiling have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454 and Illumina/Solexa systems, along with bioinformation analysis technologies of whole-genome de novo assembly, expression profiling, DNA variation discovery, and genotyping. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2× and 30× depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through de novo assembly in any whole-genome sequenced species. The 20× and 50× coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively, is effective to create novel expressed reference sequences. However, only an average 30× coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence.

ChIP‐seq reveals a role for CrzA in the Aspergillus fumigatus high‐osmolarity glycerol response (HOG) signalling pathway

“…The protein–DNA complexes were cross‐linked and then sonicated into short fragments, after which the CrzA‐bound DNA fragments were immunoprecipitated using the GPF antibody. Enriched ChIP DNA fragments were selected by size (about 300 bp) and were sequenced on an Illumina sequencer (Lim et al ., ). Several negative controls were designed to confirm the accuracy of the technique.…”

FgHtf1 Regulates Global Gene Expression towards Aerial Mycelium and Conidiophore Formation in the Cereal Fungal Pathogen Fusarium graminearum

“…The harvested mycelia were either immediately frozen in liquid nitrogen for RNA isolation or subjected to DNA extraction for ChIP-seq. Two Illumina ChIP-seq libraries of the ΔFghtf1-Com and ΔFghtf1 strains precipitated with GFP antibody were prepared for sequencing (28), followed by 76-bp end sequencing (29) and data analysis with the CLC Genomics Workbench.…”

Genome‐wide survey of transcriptional initiation in the pathogenic fungus, Candida glabrata

“…Furthermore, NGSs provide more accurate digital‐quantification by counting the sequencing reads obtained from CAGE and/or SAGE experiments (Lim et al . ).…”

“…More recently, use of the new generation sequencers (NGSs) with high-throughput (Mardis 2008;Ansorge 2009), resulted in more in depth CAGE sequencing from a single experiment (de Hoon & Hayashizaki 2008). Furthermore, NGSs provide more accurate digital-quantification by counting the sequencing reads obtained from CAGE and/or SAGE experiments (Lim et al 2012).…”