A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing
| Autor: | Robert J. Meagher, Kelly P. Williams, Kunal Poorey, Annette E. LaBauve, Rachelle Y. Hamblin, Zachary W. Bent |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Gene Expression lcsh:Medicine Pathology and Laboratory Medicine Biochemistry Chromatography Affinity Klebsiella Pneumoniae Mice Klebsiella Medicine and Health Sciences Genomic library lcsh:Science Pathogen Multidisciplinary High-Throughput Nucleotide Sequencing Nucleic Acid Hybridization Genomics Complementary DNA Bacterial Pathogens Nucleic acids RNA Bacterial Medical Microbiology Biotinylation Host-Pathogen Interactions RNA extraction Pathogens DNA Probes Transcriptome Analysis Research Article Forms of DNA 030106 microbiology Molecular Probe Techniques Virulence Biology Research and Analysis Methods Microbiology 03 medical and health sciences Extraction techniques Genetics Animals Molecular Biology Techniques Microbial Pathogens Molecular Biology Gene Gene Library Bacteria Sequence Analysis RNA cDNA library Macrophages lcsh:R Organisms Biology and Life Sciences Computational Biology DNA Avidin Genome Analysis Genomic Libraries Molecular biology Probe Hybridization lcsh:Q Transcriptome |
| Zdroj: | PLoS ONE, Vol 11, Iss 12, p e0168788 (2016) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs. |
| Databáze: | OpenAIRE |
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