Sequence-based microbial identifications are based on sequencing all or a portion of the 16S rRNA gene. The 16S rRNA gene has been widely used as an identifier for several reasons. Ribosomal genes are universal, highly conserved, and contain variable regions allowing for the differentiation of organisms at the species level. The rate of evolution of this ribosomal gene is such that there is very little difference in the sequence of strains of the same species. rRNA gene sequences have been used for over 30 years to taxonomically classify and re-classify microorganisms.
How does it work?
There are several major steps involved in sequencing a sample for identification. First, it is necessary to isolate the organism’s genomic DNA from other cellular components. A pure sample is essential. A mixed culture will result in mixed sequence data which cannot be analyzed for identification. However, the DNA of an organism remains the same regardless of the organism’s health or growth conditions. Damaged samples, non-viable samples, samples with very little growth, and samples on any type of media can be accurately identified using DNA sequencing. A variety of methods are available for the extraction of genomic DNA from microbial samples. An appropriate method should produce DNA that is clean enough to be used in a polymerase chain reaction (PCR).
Once the genomic DNA has been isolated for a sample, the target region of the DNA (the ribosomal gene) is amplified. This is accomplished through PCR (polymerase chain reaction), a method through which many copies of a specific piece of DNA can be made. First, the DNA is denatured through heating. Double stranded DNA is split into two single stranded pieces of DNA. Next, the temperature is lowered to allow primers specific to areas flanking the target region to anneal to the ends of the DNA target. Finally, in the extension step, the temperature is raised slightly to allow the DNA Polymerase enzyme to lengthen the strand by adding complementary deoxynucleotides (dNTPs) to the end of the strand. This cycle is repeated many times, resulting in millions of copies of the target DNA.
The resulting amplified DNA is purified in order to remove any excess primers or dNTPs remaining at the end of PCR. Following purification, dye-terminator Cycle Sequencing is performed on the PCR product (the 16S gene) to fluorescently label each base of the product. This process is similar to PCR, but involves the incorporation of both dNTPs and ddNTPs (dideoxynucleotides). The ddNTPs are fluorescently labeled nucleotides to which it is not possible to add a subsequent nucleotide. Therefore, when a ddNTP is used as the next nucleotide in the strand, elongation of the strand is terminated. The end result of cycle sequencing is DNA fragments of every possible length (approximately 20 bases- 500 bases), in which the last base (A, G, C, or T) of each piece is fluorescently labeled.
The Cycle Sequencing product is run on an automated fluorescent sequencer which “reads” the sequence. The DNA is separated by size as it migrates through a polymer filled capillary, with the smallest pieces of DNA moving faster than larger pieces. As the DNA exits the capillary, the fluorescence of each DNA base is excited by a laser, and is captured by a CCD camera. Each base has a characteristic fluorescent wavelength that the software reads at the corresponding A, G, C, or T base. The end result is a string of bases that comprise the “sequence” for the sample.
The sample DNA sequence is then compared to an identification library of known sequences and an identification report is generated. A data analyst trained in microbial phylogeny interprets the identification report and assigns a confidence level to the identification.