Application of Genome De novo Assembly and Reference-based Transcriptome Assembly Strategy for Studying Lactobacilli in the VBNC State
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Abstract:
A new reference-based assembly strategy was applied for the transcriptome analysis of a Lactobacillus strain in two physiological states, isolated from beer. First, the genomic information was collected using the Illumina HiSeq 2500 sequencing system and de novo assembly. Next, this Lactobacillus strain was induced into the viable but nonculturable (VBNC) state and total RNA were extracted in the normal and VBNC states. The quality of total RNA was evaluated and RNA with acceptable quality was reverse-transcribed into cDNA. A library was constructed and Solexa sequencing was performed. Based on five relevant indices, the sequencing quality was evaluated. Subsequently, the whole genome sequence obtained previously was used as a control for sequence alignment between the transcriptome and genome, as well as to analyze differences in gene expression. The results showed that the total mapping ratio was greater than 95% after sequence comparison, and 21 significantly up-regulated and seven down-regulated genes were detected. Compared to conventional de novo RNA-Seq technology, this application of genome de novo assembly and reference-based transcriptome assembly strategy can help avoid the tedious steps required in de novo assembly, shows high accuracy, and provides an important foundation for studying the molecular regulation mechanisms of food microorganisms in different physiological states.
