Gain Confidence and Achieve Accuracy
The cGMP compliant DNA sequencing process we utilize at Accugenix has been evaluated for accuracy, repeatability and robustness. It has also been streamlined so that a same-day sample can go from culture to identification in as little as 6 hours. There are several major steps involved in sequencing a sample, bacterial or fungal, for identification.
Culture Conditions and DNA Isolation
First, it is necessary to isolate the organism’s genomic DNA from other cellular components. A pure sample is essential for identification. A mixed culture will result in mixed sequence data, which cannot be analyzed for identification and would necessitate subculturing for purity and isolation. Damaged samples, non-viable samples, samples with very little growth and samples on any type of media can be accurately identified using DNA sequencing. Additionally, the DNA of an organism remains unchanged regardless of the organism’s health or growth conditions. As a result, samples can be submitted for sequencing via a variety of preparation methods. Genomic DNA is extracted from the microbial samples resulting in DNA that is then amplified and sequenced by the polymerase chain reaction (PCR).
DNA Amplification and Sequencing
Once the genomic DNA has been isolated from the sample, the target region of the DNA (the ribosomal 16S or ITS2 region) is amplified through polymerase chain reaction. PCR is a method that results in millions of copies of a specific piece of DNA. First, double stranded DNA is split into two single stranded pieces of DNA, then primers specific to areas flanking the target region anneal to the ends of the target DNA. These primers are different for bacteria (BacSEQ) and fungi (FunITS). Finally, the DNA replication enzyme lengthens the strand by adding complementary deoxynucleotides (dNTPs) to the end. This cycle is repeated many times, resulting in the amplicon, or PCR product. Occasionally, this process is not completely successful. Some compounds can inhibit PCR, including blood, pigments, high DNA concentrations, phenolic compounds and humic acids. Accugenix has refined our process to make the PCR amplification step more robust to accommodate these conditions.
The resulting amplified DNA is purified to remove excess primers and dNTPs remaining at the end of the PCR reaction. Following purification, cycle sequencing using dye- terminators is performed on the 16S or ITS2 PCR product. This process is similar to PCR, but involves the incorporation of both dNTPs and fluorescently labeled ddNTPs (dideoxynucleotides). The dNTPs will lengthen the DNA strands, and ddNTPs will be incorporated into growing DNA strands at different times, terminating the elongation. 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 as the corresponding A, G, C, or T base. The end result is a string of bases that comprise the “sequence” for the sample that results in the production of an electropherogram of the sample.
Manual Analysis and Interpretation is the Key
A Molecular Phylogeneticist then evaluates the DNA sequence, compares it to our extensive, curated, validated proprietary library of known sequences and an identification report is generated. The data analyst interprets the identification report and assigns a confidence level which is reviewed by a Quality Assurance technologist prior to release.
