Decoding the rRNA Gene

Fragments of rDNA remain remarkably similar in organisms that are distantly related. Therefore, sequencing and comparing it can show evolutionary relatedness. It also helps measure the difference between organisms.

There are many ways to study it; 16s rRNA gene sequencing is the most common. At Yaazh, a true whole-genome sequencing lab, we help you understand rRNA to determine taxonomy and phylogeny. Our accurate results can help estimate the divergence rates in bacteria.

We give you access to one of the most important tools for both actinomycetes and bacteria using universal primers 27F/1427R. We sequence with 518F/800R. We sequence, assemble and analyse. As part of our genomic services, we also construct the phylogenetic tree.

Better and More Comprehensive Results

Our next-generation sequencing services are full-package, including DNA extraction and PCR amplification followed by gel verification and purification.

Sequence raw data (Forward and Reverse) in different file formats.

.ab1 - Sequence with chromatogram

.pdf - Sequence with chromatogram

.txt - Only sequence

.fasta - Only Sequence

Assembled sequence (Contig) - ~ 1400bp or more

Blast result generated from all GenBank + NCBI+EMBL+DDBJ+PDB sequences.

Summary report comprises

Methodology for DNA Extraction, PCR, Sequencing and Bioinformatic analysis.

Sequence analysis report contains sequence read normal length, Q16 & Q20 read length, GC content details.

Proper contigs sequence with coverage image

Final result

Phylogeny tree.

We provide all the bioinformatics analysis, including the phylogenetic tree & customer-specific gene analysis with free of cost.

Customer-specific gene analysis and a phylogenetic tree are provided free of cost.

Why is Sequencing Coverage Important in 16S rRNA Sequencing, and How is it Determined?
Sequencing coverage is crucial in 16S rRNA sequencing because it directly influences the precision and detail of the analysis. High sequencing coverage ensures that there are enough reads—both in number and length—to accurately identify and classify the diverse microorganisms within a sample.
Impact on Accuracy:
Detailed Resolution: Adequate coverage provides a more detailed resolution, enabling the detection of even the less abundant species in the sample.
Accurate Classification: Especially for complex or diverse microbial communities, sufficient coverage is necessary to differentiate closely related strains.
Optimization of Coverage:
Evaluate Rarefaction Curves: Assessing rarefaction curves from your sequencing data helps determine if enough sequences have been generated to comprehensively capture the microbial diversity present.
Adjust According to Sample Complexity: More complex samples may require increased sequencing depth to achieve accurate classification, which means generating more data.
Balance with Cost: While greater coverage enhances accuracy, it also increases costs. Therefore, it’s important to find a balance based on the complexity of the sample and the budget available.
By effectively managing sequencing coverage, researchers can achieve a high degree of accuracy in microbiome studies, leading to reliable and insightful results.

How Contamination Affects 16S rRNA Sequencing and Ways to Mitigate It

Contamination in 16S rRNA sequencing poses a significant challenge, particularly because it involves amplifying bacterial and archaeal DNA from minimal sample volumes. This makes the process susceptible to interference from unwanted environmental bacteria. Such contamination can lead to inaccurate sequencing results, misrepresenting the actual microbial composition you're trying to study.

Strategies to Mitigate Contamination

Adhere to Sterilization Techniques
Employ stringent sterilization methods and strict handling protocols. Utilizing sterile equipment and maintaining a clean work environment can reduce the risk of contamination. This is especially crucial when working with low microbial biomass samples, like skin swabs or biopsies, which are naturally more vulnerable to contamination.
Employ Control Measures
- Negative Controls: Implement negative controls to check for external contamination in your sequencing results. This helps in identifying contaminants that may arise from sources like DNA extraction kits and laboratory reagents, allowing you to adjust or eliminate them during analysis.
- Positive Controls: Use positive controls which incorporate known mixtures and amounts of microorganisms. This practice helps validate if your DNA extraction and library preparation methods are effective and reliable.

Following these practices not only minimizes contamination but also ensures the accuracy and reliability of your 16S rRNA sequencing data.

Exploring the Pros and Cons of 16S rRNA Sequencing
16S rRNA sequencing stands as a vital method for studying microbial communities, offering several advantages and facing particular challenges. Here's a breakdown of its strengths and limitations:
Strengths of 16S rRNA Sequencing
Bacterial and Archaeal Identification: This technique excels in identifying bacteria and archaea, making it a go-to for microbiology researchers focused on these groups.
Cost-Effectiveness: Compared to methods like whole metagenome shotgun sequencing, 16S rRNA sequencing is more budget-friendly, allowing wider access and application in various research scenarios.
Extensive Databases: The method benefits from well-curated databases developed over years of research, enhancing the reliability of bacterial and archaeal identification.
Limitations of 16S rRNA Sequencing
Limited Scope: Its focus restricts it primarily to bacteria and archaea. For fungi, viruses, or other microorganisms, alternative sequencing techniques such as qPCR or whole genome approaches are necessary.
Taxonomic Resolution: While effective at broader levels, 16S rRNA sequencing often struggles with distinguishing between closely related microbial strains. In contrast, shotgun sequencing can provide a more detailed taxonomic breakdown.
Functional Insight Constraints: This sequencing targets a single gene, lacking the ability to detail the functional capabilities of microbial communities. To achieve insight into microbial functions, researchers must turn to comprehensive methods like shotgun sequencing.
Risk of Contamination: The sensitivity of 16S rRNA sequencing to contamination is noteworthy, particularly in samples with low microbial presence, requiring meticulous handling to avoid skewed results.
Balancing these factors, 16S rRNA sequencing remains a crucial tool in microbiome research, though understanding its boundaries is key to maximizing its efficacy in scientific explorations.

Key Considerations for DNA Extraction in 16S rRNA Sequencing

16S rRNA sequencing is a powerful tool for studying microbial communities, but the accuracy of the results heavily depends on the quality of DNA extraction. Here are the essential considerations to ensure optimal DNA extraction for 16S rRNA sequencing:

1. Sample Type and Preparation

The first step involves choosing the right sample type, as different samples (e.g., soil, water, or human tissues) may require distinct handling procedures.
Effective sample preparation is crucial—it involves thorough homogenization to ensure representative DNA extraction from the entire sample.

2. DNA Extraction Kit Selection

Select a DNA extraction kit that suits the specific sample type and the objectives of your analysis. Different kits vary in their efficacy depending on the composition of the microbial community.
Kits often utilize both chemical and physical methods to lyse cells, so ensure it is compatible with your sample's characteristics.

3. Lysis Efficiency

Lysis, or the breakdown of cell walls to release DNA, includes both chemical reagents (such as enzymes) and mechanical methods (like bead-beating).
It's crucial to achieve complete lysis to prevent biased representations of the microbial community.

4. Contaminant Removal

Effective purification is essential to eliminate contaminants that can interfere with sequencing accuracy. Look for a process that involves precipitating impurities using solutions like salt and alcohol, followed by a washing step.

5. DNA Purity and Concentration

Measure the purity of extracted DNA, usually indicated by the A260/A280 ratio. The ideal range is around 1.8–2.0 for pure DNA.
Also, ensure DNA is concentrated to a level suitable for downstream processes without excess dilution.

6. Protocol Customization

Tailor the extraction protocol to the unique aspects of your sample and research goal. Adjust the steps if necessary, especially in challenging samples like those with high organic content.

By carefully considering these factors, researchers can improve the reliability and accuracy of their 16S rRNA sequencing results, providing deeper insights into microbial ecosystems.

The Technique- A Complete Solution

PCR amplification

After the bacteria is isolated and DNA extracted, we amplify the 16s rRNA sequence. We rely on 27F/1492R or 27F/1525R, the universal primers that complement the region. Once we have multiple copies of the target DNA, we utilise them as sequence templates.

DNA sequencing

This step uses the common Sanger sequence method. Each strand - 3' to 5' and 5' to 3' – undergoes separate sequencing. We use forward and reverse primers. Often they are the same universal primers from amplification. Internal primers are used when entire regions need to be sequenced. The common primers Yaazh wields for 16s rRNA are 518F/800R.

Sequence comparison

The last stage is creating a phylogenetic tree using bioinformatic tools to visualise the result. The tree shows either the similarity or distance between the clustered calculation. It also helps compare sequences.

Power of Functional Genomics

The only whole-genome sequencing lab you will ever need for 16s rRNA sequencing services!

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