DNA sequencing is a means to determine the exact order of the building blocks of any genetic code. There are four subunits, also called nucleotide bases, that have been identified that make up all DNA. Those bases are: Adenine, Guanine, Cytosine, and Thymine. Knowledge pertaining to DNA sequences has become the next frontier for biological research. Being able to determine the sequence order can unlock vast amounts of information provided within a sample. Once this sequence information is gathered, it can be used to explore which stretches of DNA contain genes and which stretches carry regulatory instructions which can, for example, turn genes on and off. In addition, and most importantly, sequence data can highlight changes, routinely called mutations. Mutations in genes are mostly associated the precursors or predisposition to disease.
In recent years, as computer technology has rapidly evolved, so has the speed of DNA sequencing. Combined with major advances in molecular biology, modern DNA sequencing technology has been instrumental in the research and evolution of complete DNA sequences for numerous types and species of life, including the human genome and numerous animal, plant, and microbial species. What we do here at Fry Laboratories, L.L.C. is use this DNA sequence information to identify pathogens found in a clinical sample directly.
There are two major approaches to DNA sequencing: Whole genome sequencing and Targeted sequencing. “Whole” genome sequencing looks at the entire genome of an organism. This can be very complicated since the entire DNA genome first needs to be broken up into small fragments. All those fragments are first sequenced then get pieced back together during data analysis. On the other hand, “Targeted” DNA sequencing differs in that it can zoom in on a very specific area of a genome. There are many reasons to use targeted sequencing in a clinical laboratory. One of the more common uses for targeted sequencing is to find Single Nucleotide Polymorphisms, or SNPs. These SNPs are single nucleotide base changes where bases are wrongly incorporated into the DNA code. These changes vary from person to person, and can also lead to disease. Another use for targeted DNA sequencing is for exome sequencing. Exomes are the portions of the DNA genome which contain genes. Sequencing just the genes is much faster and more efficient than the whole genome. This is because only this portion contains the regulatory elements, which accounts for roughly only 2% of the entire genome.
Fry Laboratories, L.L.C., uses the targeted sequencing approach instead of whole genome when sequencing DNA. This allows us to look specifically for microbial and pathogenic DNA within clinical samples. A few years of in-house research and development has allowed us to now target multiple regions simultaneously from the DNA of microbes and pathogens. Equipped with this information, data analysis using high powered computers and advanced software allows for the identification of pathogens present in a clinical sample.
The steps required to perform a targeted DNA sequencing run requires in depth knowledge of laboratory equipment and procedures, as well as a lot of hands on preparation time by a highly trained and qualified technologist.
The first step is to extract the total genomic DNA from a given sample. Samples come to the lab in numerous forms: Blood, tissue, body fluids, or urine. Once high quality DNA, free from cellular protein and RNA, is extracted from the sample, a technique known as a Polymerase Chain Reaction, or PCR is performed. This is where specific DNA “targets” of interest are amplified (exponential self-copying) into tens of thousands of copies of each region. At the same time, tiny unique “bar codes” are incorporated during PCR to each clinical sample for identification and informational purposes.
The resulting amplified products, now known as amplicons, are then purified and measured to determine how much DNA is present (concentration). These patient samples then get pooled together to create what is known as a DNA library. This is done by adding equal molar amounts of the DNA amplicons from each bar-coded sample to create our “Pool”. This means that each sample library has the same amount of DNA available for sequencing. This pool is then loaded onto a sequencing chip and which allows sequencing on the Genomic Analyzer.