DNA Sequencing Services:
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.
TYPES OF SEQUENCING FOR DIAGNOSTICS:
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.
REFERENCES FOR NGS, INFECTIOUS DISEASES, AND CLINICAL MICROBIOLOGY:
- Clavel. 2017. Deciphering interactions between the gut microbiota and the immune system via microbial cultivation and minimal microbiomes. Immunol Rev.
- Dehingia. 2015. Gut bacterial diversity of the tribes of India and comparison with the worldwide data. Sci Rep.
- Jovel. 2016. Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics. Frontiers in Microbiology.
- Joensen. 2017. Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease. Eur J Clin Microbiol Infect Dis.
- Kasai. 2016. Comparison of human gut microbiota in control subjects and patients with colorectal carcinoma in adenoma: Terminal restriction fragment length polymorphism and next-generation sequencing analyses. Oncol Rep.
- Liang. 2017. Diversity and enterotype in gut bacterial community of adults in Taiwan. BMC Genomics.
- Liu. 2017. Starter Feeding Supplementation Alters Colonic Mucosal Bacterial Communities and Modulates Mucosal Immune Homeostasis in Newborn Lambs. Front Microbiol.
- Petrukhina. 2017. [Microbiocenosis of subgingival biofilm and intestinal content in chronic periodontal disease in patients with metabolic syndrome]. Stomatologiia (Mosk).
- Petrukhina. 2016. [Study of mutual dependence of periodontal and colonic microbiome in health and pathology using NSG-sequencing]. Stomatologiia (Mosk).
- Rintala. 2017. Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor. J Biomol Tech.
- Schulz. 2016. RiMINI – the influence of rifaximin on minimal hepatic encephalopathy (MHE) and on the intestinal microbiome in patients with liver cirrhosis: study protocol for a randomized controlled trial. Trials.
- Adami. 2015. The microbiome at the pulmonary alveolar niche and its role in Mycobacterium tuberculosis infection. Tuberculosis (Edinb).
- Aho. 2015. The microbiome of the human lower airways: a next generation sequencing perspective. World Allergy Organization Journal.
- Al-Momani. 2016. Microbiological profiles of sputum and gastric juice aspirates in Cystic Fibrosis patients. Sci Rep.
- Bacci. 2017. A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease. Int J Mol.
- Boutin. 2017. Acquisition and adaptation of the airway microbiota in the early life of cystic fibrosis patients. Mol Cell Pediatr.
- Huebinger. 2013. Examination with next-generation sequencing technology of the bacterial microbiota in bronchoalveolar lavage samples after traumatic injury. Surg Infect (Larchmt).
- Mizrahi. 2017. Comparison of sputum microbiome of legionellosis-associated patients and other pneumonia patients: indications for polybacterial infections. Scientific Reports.
- Toma. 2014. Single-molecule long-read 16S sequencing to characterize the lung microbiome from mechanically ventilated patients with suspected pneumonia. J Clin Microbiol.
- Yoshida. 2015. Invasive pulmonary aspergillosis due to Aspergillus lentulus: Successful treatment of a liver transplant patient. J Infect Chemother.
- Kong. 2017. Performing Skin Microbiome Research: A Method to the Madness. J Invest Dermatol.
- Rhoads. 2012. Clinical identification of bacteria in human chronic wound infections: culturing vs. 16S ribosomal DNA sequencing. BMC Infectious Diseases.
- Wolcott. 2016. Analysis of the chronic wound microbiota of 2,963 patients by 16S rDNA pyrosequencing. Wound Rep and Reg.
- Aralaguppe. 2016. Multiplexed next-generation sequencing and de novo assembly to obtain near full-length HIV-1 genome from plasma virus. J Virol Methods.
- Brenner. 2018. Next-generation sequencing diagnostics of bacteremia in sepsis (Next GeneSiS-Trial): Study protocol of a prospective, observational, noninterventional, multicenter, clinical trial. Medicine (Baltimore).
- Decker. 2017. Immune-Response Patterns and Next Generation Sequencing Diagnostics for the Detection of Mycoses in Patients with Septic Shock-Results of a Combined Clinical and Experimental Investigation. Int J Mol Sci.
- Dirani. 2018. A novel next generation sequencing assay as an alternative to currently available methods for hepatitis C virus genotyping. J Virol Methods.
- Dubourg. 2016. Emerging methodologies for pathogen identification in positive blood culture testing. Expert Rev Mol Diagn.
- Païssé. 2016. Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing. Transfusion.
- Gosiewski. 2017. Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method – the observation of DNAemia. Eur J Clin Microbiol Infect Dis.
- Long. 2016. Diagnosis of Sepsis with Cell-free DNA by Next-Generation Sequencing Technology in ICU Patients. Arch Med Res.
- Mwaigwisya. 2015. Emerging commercial molecular tests for the diagnosis of bloodstream infection. Expert Rev Mol Diagn.
- Paisse. 2016. Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing. Transfusion.
- Ring. 2018. Moderate to severe hidradenitis suppurativa patients do not have an altered bacterial composition in peripheral blood compared to healthy controls. J Eur Acad Dermatol Venereol.
- Sabat. 2017. Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification – increased discrimination of closely related species. Sci Rep.
- Song. 2014. Profiling of the bacteria responsible for pyogenic liver abscess by 16S rRNA gene pyrosequencing. J Microbiol.
- Suzuki. 2017. Comprehensive detection of viruses in pediatric patients with acute liver failure using next-generation sequencing. J Clin Virol.
- Xu. 2014. RNA CoMPASS: a dual approach for pathogen and host transcriptome analysis of RNA-seq datasets. PLoS One.
- Bereza. 2016. Comparison of cultures and 16S rRNA sequencing for identification of bacteria in two-stage revision arthroplasties: preliminary report. BMC Musculoskeletal Disorders.
- Gaviria-Agudelo. 2015. Genomic Heterogeneity of Methicillin Resistant Staphylococcus aureus Associated with Variation in Severity of Illness among Children with Acute Hematogenous Osteomyelitis. PLoS One.
- Sabat. 2017. ‘Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification – increased discrimination of closely related species’, Sci Rep.
- Street. 2017. Molecular diagnosis of orthopaedic device infection direct from sonication fluid by metagenomic sequencing. Journal of Clinical Microbiology.