Christopher R. Robles

Interactions with diverse bacteria species can be helpful or harmful to human health.   These diverse bacteria are able to survive in many conditions due to regulation of gene expression.  Gene expression can be examined by studying the total RNA transcribed, or transcriptome, of these bacterial organisms. Recent advances in High Throughput Sequencing (HTS) technologies have greatly aided transcriptome analysis in both eukaryotic and prokaryotic organisms. However, investigation of bacterial transcriptomes using these approaches have been limited by several challenges, including the difficulty in enriching bacterial cDNA libraries for non-abundant transcripts and for contending with the great differences in the genome sequences of diverse bacterial species.

The central objective of this project is to develop improved experimental and analytical approaches for using high throughput sequencing to define the transcriptomes of diverse bacteria. Our first aim was to develop a faster and less work intensive protocol for constructing cDNA libraries representing bacterial transcriptomes.  In order to do this we developed improved methods for extracting and fragmenting total RNA, allowing capture of small regulatory RNAs and potentially eliminating the need for size selection using gel electrophoresis.  

Our second aim was to enrich for non-abundant transcripts in cDNA libraries, by employing adaptations of Not-So-Random Primers (NSR), a method for depletion of abundant transcripts such as rRNAs and tRNAs that has been successfully used in studies of gene expression in eukaryotes. In doing so we discovered and tested a trend between rRNA transcripts and doubling time by using computational tools to determine proportions of different bacterial rRNA transcripts to their genome size and doubling time.  By employing these new methodologies and analyzing data set, we hope to overcome the current challenges of examining transcriptomes of different bacterial species with HTS.

 

PROJECT: Developing and Implementing Improved Methodologies for Exploring Bacterial Transcriptomes Using High-throughput Sequencing

Mentor: Jonathan Livny, Infectious Disease Initiative

Christopher R. Robles

"My summer at the Broad Institute not only taught me excellent research skills I will be able to utilize in the future, but also gave me insight into the life and mind of a research scientist.  The scientific communication classes also helped to develop the way I exchange ideas clearly with both the scientific and non-scientific community.  Working at the Broad provided me with a unique environment and experience that enlightened me to the various paths I can pursue in the future."