7. Secuenciación de Genoma Completo: El proceso experimental

Introduction to Whole Genome Sequencing

Overview of Molecular Biology Techniques

• The previous module covered the fundamentals of molecular biology techniques for obtaining DNA from organisms and assessing its quality and integrity.

• PCR was discussed as a tool for analyzing specific antimicrobial resistance genes, alongside the Sanger method for sequencing up to 800 base pairs.

Transition to Whole Genome Sequencing

• This module introduces whole genome sequencing (WGS), also known as second-generation sequencing, aimed at identifying all antibiotic resistance genes in bacteria.

• WGS is currently the most relevant technology for DNA analysis, allowing identification and characterization of resistance genes, virulence factors, and pathogenicity islands.

Importance of Whole Genome Sequencing

Epidemiological Studies

• WGS provides context on resistance genes and evaluates their phylogenetic relationships locally and globally.

• The information gathered through WGS is crucial for epidemiological studies that track the dissemination of antimicrobial resistance.

Bacterial Genomes: General Characteristics

Structure of Bacterial Genomes

• A bacterial genome consists of total DNA; it comprises four nucleotide units (adenine, thymine, guanine, cytosine).

• Bacterial genomes are relatively small compared to human genomes; they range from 160,000 to 14 million base pairs. For example, Salmonella has about 5 million base pairs.

Chromosomal Features

• Most bacteria have a single circular DNA molecule called a chromosome containing essential functional genes.

• Additionally, many bacteria possess extrachromosomal DNA known as plasmids which carry non-essential functions including some resistance genes.

Challenges in Genome Sequencing

Technical Complications

• Sequencing requires fragmenting the DNA into smaller pieces for easier analysis; computational programs then reconstruct the original sequence like a molecular puzzle.

Methods of Sequencing

• Various platforms exist for whole genome sequencing; this video focuses on synthesis-based sequencing methods.

Synthesis-Based Sequencing Process

Mechanism Overview

• Synthesis-based sequencing involves adding bases to an elongating DNA strand with fluorescent tags that emit distinct light signals upon addition.

Data Generation

• Each optical signal is recorded and translated into letters representing sequences. The volume of data generated is substantial due to high throughput capabilities.

Considerations Before Sequencing

Key Factors

• Important considerations include genome size, chosen sequencer type, sample number, desired read length, and depth—defined as how many times each nucleotide position is sequenced.

Advancements in Sequencing Technology

Third Generation Sequencing

Process of Whole Genome Sequencing

Introduction to Experimental Process

• The discussion begins with the transition to the experimental process of whole genome sequencing, specifically focusing on genomic characterization of bacteria causing foodborne illnesses at CENASICA.

Steps in Whole Genome Sequencing

Step 1: DNA Extraction

• After isolating the bacteria, DNA extraction is performed using a specific kit. Quality metrics for DNA include A260/A280 ratios between 1.8 and 2.1, and A260/A230 ratios between 1.9 and 2.2.

Step 2: Library Preparation

• The sequencing process consists of two main phases: library preparation and sequencing correction. Libraries represent a random collection of DNA fragments from the organism.

Key Steps in Library Preparation

Conditioning of DNA

• The first step involves conditioning extracted DNA to a specific concentration using fluorometry, which measures fluorescence intensity as an indicator of concentration.

Fragmentation and Tagging

• In this phase, enzymes fragment the DNA into smaller pieces while adding short sequences (tags) that will help identify samples later in the process.

Indexing

• Unique index sequences are added to each sample's fragments for identification throughout the sequencing process, ensuring distinct combinations for each bacterial genome.

Amplification

• PCR is conducted on indexed samples to create sufficient copies of each fragment, forming libraries ready for further processing.

Step 3: Purification of Libraries

• This step cleans up libraries by removing unused components from amplification reactions (like adapters and salts), ensuring uniform sizes across all prepared libraries through magnetic bead purification methods.

Step 4: Quality Assessment

• Quality control checks involve capillary electrophoresis to verify library size distribution against expected ranges based on previous steps' parameters.

Final Steps in Normalization

Normalization and Sequencing Process

Normalization of Libraries

• The lowest acceptable normalization concentration is selected and diluted in libraries requiring resuspension buffer. Libraries with concentrations below the established normalization must be repeated.

Preparation of PUL

• After normalizing all libraries, an equal volume mixture called PUL is created, combining normalized libraries from all master samples. This marks the final stage before sequencing begins.

Denaturation and Dilution

• Denaturation involves opening the double-stranded DNA into single strands by adding a base. Following this, dilution occurs to achieve the desired loading concentration for PUL, which is then loaded into a cartridge for sequencing.

Cluster Formation in Sequencer

• Upon entering the sequencer, cluster formation occurs through PCR amplification. Each bristle on a brush represents complementary sequences that anchor the libraries during this process.

Sequencing Output and Analysis

• An enzyme copies the libraries while adding nucleotides marked with specific colors. The optical system records signals translating them into bases, resulting in numerous files for bioinformatics analysis to understand bacterial genomes.

Advantages of Whole Genome Sequencing

Enhanced Genetic Information

• Whole genome sequencing provides extensive genetic information with unprecedented precision compared to traditional techniques, aiding in pathogen detection and characterization while reducing public health risks.

Resistance Gene Identification

• It allows identification of antimicrobial resistance genes along with their genetic context and variants, facilitating outbreak analysis—an advantage over conventional molecular methods like pulsed-field gel electrophoresis.

Data Accessibility for Future Research

• Once generated, sequencing data can be stored in databases for future research opportunities without strain on strain collisions, enabling repeated analyses and discovery of new genes.

Limitations of Whole Genome Sequencing

Challenges with Unknown Genes

• Limitations include unknown or newly generated genes not present in databases that may go undetected during sequencing efforts aimed at identifying microbial resistance genes.

Expression Level Evidence Gaps

• There’s often no evidence regarding expression levels of identified resistance genes; they could be overexpressed or truncated proteins making it difficult to determine actual resistance levels in bacteria.

Complementary Techniques and Future Outlook

Integrating Other Techniques

• Complementing genomic data with other techniques such as transcriptome studies can provide more comprehensive biological insights despite computational demands for analysis and storage.

Growing Importance in Public Health

• While whole genome sequencing has limitations, its utility is increasing rapidly; it is expected to become a significant technique across various public health domains including antimicrobial resistance monitoring.

Sequencing Process Overview

Steps Involved in Second Generation Sequencing