INFECTION DIAGNOSTICS :: MICROBIOLOGY (A)
Nucleic Acid Extraction
DNA RNA Reverse
Transcripions cDNA
Library Construction Sequencing
Base Recognition Quality Control
Remove Host Sequence Background
Alignment to Reference Database
Species
Identification Report
Generation Sequencing: Over the past decade, a (B) Pre- Analytical
Analytical “Wet Lab”
Analytical “Dry Lab”
Post- Analytical Patient Presentation
• Clinical Syndrome
• Exposure History
• Timing
massive amount of commercially available sequencing platforms has emerged that offer high-throughput analysis. To gener- ate sufficient data for adequate sequenc- ing analysis, most platforms will pool libraries for sequencing. Quantification of the pooled libraries can be employed using several approaches, such as total DNA quantification, quantitative PCR normal- ization, and bead normalization. Within the clinical setting, several factors need to be taken into consideration when perform- ing or considering NGS sequencing. All sequencing platforms have an intrinsic error rate that needs to be considered for data analysis. Further considerations include the level of throughput for the number of total sequences obtained as well as their profile length; the number of base pairs obtained; the sequencing depth per sample; and the physical com- putational hardware for processing and storing large NGS data files.5 The Generations of Sequencing Plat- forms: Post the advent of first genera- tion of sequencing technology of Sanger Sequencing, second and third generation have emerged has the technology has advanced. The umbrella term of NGS includes second and third generation sequencing. Second generation requires template amplification prior to sequenc- ing, while third generation offer de novo assembly in real time without the need of template amplification.6 Platforms such as Ion Torrent, Pacific Biosciences, and Illumina are the current
Clinical/ Lab/
Radiographic Evaluation
• Initial Findings
• Empiric Treatment
Obtain Sample
• Site Location • Specimen Type
• Aseptic Technique
• Sample Quality
frontrunners of second generation sequencing technology.3
Ion Torrent is
unique in its detection method. Unlike other technologies that use fluorescence or chemiluminescence, Ion Torrent detects proton release during nucleotide incorporation of strand synthesis.3 Second generation sequencing has
significantly revolutionized and advanced the field, yet the technology is not without flaws. Second generation sequencing typi- cally has short sequence reads leading to sequencing gaps, alignment issues due to repetitive regions/pseudogenes, and PCR artifacts.3
As a means to overcome these
limitations, third generation sequencing, offering sequencing at the signal molecule level, was developed. PacBio SMRT and Oxford Nanopore Technologies are the current representatives of third-generation sequencing.3,6 PacBio SMRT has a similar library preparation except for specialized adapt- ers to circularize double-stranded DNA fragments. The circularized DNA and DNA polymerase are immobilized and analyzed on a chip. The signal from the incorpora- tion of fluorescently labeled nucleotides is measured via a CCD camera.3 Oxford Nanopore uses a novel technol-
ogy called nanopores. Nanopores are tiny bio-pores with nanoscale diameter, capable of measuring current changes. Each of the 4 types of nucleotides will pass through the nanopore, altering the channel voltage, and lead to a distinct current change that is measured by the platform. Nanopore technology is advantageous of short turn-
Transport to Lab
• Container Type
• Temperature
• Transportaion Time
Extract Nucleic Acid
• Pre-treatment • Centrifugation • Cell Lysis • DNA or RNA
Pathogen Enrichment
• +/- DNase
• +/-methyl-DNA Depletion
• Reverse Transcription
Library Preparation
• Amplification • Barcoding • Adaptor Ligation • Normalization
• Loading
Generate Sequence Data
Concentration • Instrumentaion
• Sequence Length/Depth
• Q-Score
Qualify Filter/
Debarcode
Threashhold • PCP Duplicates
• Barcode Binning
Human Subtraction
• Human Database
• Alignment Stringency
Organism Database Alinment
• Organism Database
• Annotation Quality • Database Coverage • Alignment Stringency
• Global vs Local Alignment
Identify
Significant Findings
• Results Visualization
• Background Flora • Normalization
• Thresholding
Generate Report
• Clarification • Formatting • Transmission
Review Report
• Report Viewer • Report Clarity
Interpretation of Clinical Context
• Results Correlation • Pathogen vs. Flora • Contamination?
• Exposure Control
Determine Management
• Other Diagnostics?
• Treatment Options
Treatment
• Medical/ Surgical/Drug
• Compliance • Effectiveness
Figure 1: (A) General Flowchart of Next-Generation Sequencing. The general procedure displayed is representative of metagenomic next-generation sequencing. Adapted from Duan et al.1
(B) Clinical Workfl ow Considerations of Next-Generation Sequencing. The 4 major steps of Next-Generation Se-
quencing are Pre-analytical, Analytical (Wet Lab), Analytical (Dry Lab), and Postanalytical. General Considerations and variables signifi cant to clinicians are presented within the arrows. Adapted from Miller et al.5
MLO-ONLINE.COM JUNE 2022 19
Data Analysis
Sequencing Process
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