Computer-Assisted Bacterial Identification Using 16S rRNA Sequence Data

Abstract

Most microorganisms cannot be cultured and are difficult to identify. One of the most widely used markers to help identify bacteria is the ribosomal RNA gene. A component of the small ribosomal subunit, 16S rRNA is composed of alternating evolutionarily conserved and hypervariable regions. One strategy is to exploit the length heterogeneity in the highly variable regions and to use it for microbial identification. Techniques based on the polymerase chain reaction (PCR) are ideally suited for studying length heterogeneity of the 16S rRNA hypervariable regions. PCR primers were designed using the conserved regions (V1 and V1_V2) of the gene and the lengths of the resulting amplicons were estimated in the laboratory. The aim of this project is to design a computer program that takes as input the amplicon lengths arising from a PCR experiment with a given pair of primers and to output the set of known bacteria that can result in those sequence lengths. However, there are thousands of microbial organisms that display the exact same amplicon length for a given pair of primers. If two or more pairs of primers are used and the amplicon lengths estimated using PCR, then there is a much better chance of correctly identifying the bacterial organisms present in a sample. AmpliQue is a BioPerl program that addresses this problem. AmpliQue receives as input two pairs of primers and the lengths of the amplicons from the PCR experiment. It then reports all the microbial organisms that would result in those amplicon lengths. It uses the 16S rRNA sequence database from the Ribosomal Database Project (URL: http://rdp.cme.msu.edu/). Each set of primers was run independently against the 16S rRNA database using BLAST. Results from the BLAST hits were then merged into a single table. This resulting table was then queried with the observed amplicon lengths from the PCR experiments.