2022

16S rRNA Sequencing.

Assignment No.02
Introduction to microbiology Lab
BS1141-S2
AHMED MUSTAFA
BBT211023
Submitted To:

Mam Sumayya
Ijaz

Submission Date:
30/04/2022.

1
 16S rRNA Sequencing

Introduction
16S ribosomal RNA (or 16S rRNA) is the RNA component of the 30S subunit of a prokaryotic ribosome
(SSU rRNA). It binds to the Shine-Dalgarno sequence and provides most of the SSU structure. Since the
advent of high-throughput sequencing, PCR-amplified 16S sequences have typically been clustered based
on similarity to generate operational taxonomic units (OTUs) and representative OTU sequences compared
with reference databases to infer likely taxonomy. Although convenient and powerful, such usage of 16S
has necessitated certain assumptions, e.g., the now historic assumption that sequences of > 95% identity
represent the same genus, whereas sequences of > 97% identity represent the same species.
16S sequences have also been exploited using low-throughput methods to distinguish strains (sometimes
called subspecies) based on polymorphisms within the gene. Single-nucleotide polymorphisms (SNPs)
have been used to track strains of clinical relevance or, when they are stably linked to other parts of the
bacterial haplotype, to predict phenotypic characteristics. Thus, accurate and complete 16S sequences are
of high utility in many applications. Until recently, however, accurate, full-length 16S sequences have been
beyond the scope of high-throughput sequencing platforms.
Availability of third-generation technologies means that high-throughput sequencing of the full 16S gene is
becoming commonplace. Circular consensus sequencing (CCS) combined with sophisticated denoising
algorithms to remove PCR and sequencing error, mean it is now possible to discriminate between millions
of sequence reads that differ by as little as one nucleotide across the entire gene. Together, these
technological and methodological advances mean that for the first time it is becoming possible to exploit
the full discriminatory potential of 16S in a high-throughput manner.

What is 16S rRNA Sequencing

16s rRNA sequencing refers to sequencing the 16s rRNA gene that codes for the small subunit (SSU) of the
ribosome found in prokaryotes such as Bacteria and Archaea. There are several factors that make the 16s
rRNA gene the perfect target to complete your taxonomy or phylogeny studies. 16s rRNA sequencing has
become one of the leading methods for phylogenetic studies. The popularization of 16s sequencing
methods has been due in large part to the wide availability of PCR and Next-generation sequencing
facilities, such as MRDNA. Because the 16s gene codes for the SSU of the prokaryotic ribosome,
researchers can rely on the fact that their target gene will be present in every cell.
The 16s gene contains both highly conserved regions as well as hypervariable regions.
 The presence of the highly conserved regions allow researchers to design primer pairs that will
accurately and reliably amplify the 16s hypervariable region of their choice.
 The presence of the hypervariable regions affords researchers the ability to differentiate between
closely related genera or species detected in their samples.

2
The overall size of the 16s rRNA gene is relatively short ~1500bp. While sequencing the entire 16s gene is
difficult due to read length restrictions of many NGS platforms, sequencing one or more hypervariable
regions is relatively quick and affordable. Two of our most requested assays for 16s rRNA sequencing are
27F-519R (V1-V3 region) and 515F-806R (V4 region).

Significance
16S rRNA gene sequencing is commonly used for identification, classification and quantitation of microbes
within complex biological mixtures such as environmental samples (ex marine water) and gut samples (ex
human gut microbiome). The 16S rRNA gene is a highly conserved component of the transcriptional
machinery of all DNA-based life forms and thus is highly suited as a target gene for sequencing DNA in
samples containing up to thousands of different species. The 16S rRNA is the central structural component
of the bacterial and archaeal 30S ribosomal subunit and is required for the initiation of protein synthesis
and the stabilization of correct codon-anticodon pairing in the A site of the ribosome during mRNA
translation.
16S rRNA gene (16S rDNA)-based identification of bacteria offers a possible alternative when phenotypic
tests fail. Therefore, we evaluated the reliability of 16S rDNA sequencing for the species-specific
identification of campylobacters. Sequence analyses were performed by using almost 94% of the complete
16S rRNA genes of 135 phenotypically characterized Campylobacter strains, including all known taxa of
this genus. It was shown that 16S rDNA analysis enables specific identification of most Campylobacter
species. The exception was a lack of discrimination among the taxa Campylobacter jejuni and C. coli and
atypical C. lari strains, which shared identical or nearly identical 16S rDNA sequences. Subsequently, it
was investigated whether partial 16S rDNA sequences are sufficient to determine species identity.
Sequence alignments led to the identification of four 16S rDNA regions with high degrees of interspecies
variation but with highly conserved sequence patterns within the respective species. A simple protocol
3
based on the analysis of these sequence patterns was developed, which enabled the unambiguous
identification of the majority of Campylobacter species. We recommend 16S rDNA sequence analysis as an
effective, rapid procedure for the specific identification of campylobacters.
Various primers targeting conserved regions within the 16S rRNA gene have been developed as a result of
the widespread use of 16S rRNA gene sequences in evolutionary and phylogenetic studies of prokaryotes;
the most widely used. Innumerable full-length and partial regions of 16S rRNA genes have been sequenced
using the Sanger method and more recently with next-generation sequencing platforms. The deposition of
ever-increasing numbers of 16S rRNA gene sequences in public databases without quality control filters
inevitably leads to the accumulation of many poor quality sequences. Uncertainties during PCR and
sequencing processes always create a certain level of inherent errors which are exacerbated when
sequencing is carried out only once in a single direction. Furthermore, low sequencing depths of coverage
can also result in erroneous sequences. When the Sanger method is used, four to five combinations of
sequencing reactions with different primers are generally necessary to generate high-quality full-length 16S
rRNA gene sequences. All bases should be sequenced at least twice in both directions. In addition, primer
regions should not be included in the final sequence as primer binding regions are not sequenced during the
Sanger sequencing process.

Bacterial identification virtual Lab

Following steps:
Sample Preparation
The process was initiated by preparation of sample in which a bacterial colony is moved to centrifuge tube
by the aid of a wire-loop. After this transfer has been done we added some essential enzymes in the
centrifuge tube via the help of a micropipette. After this we had to wait off approximately 3 hours for the
denaturation of the enzymes in the sample prepared. In the result of first step DNA occurred as a
supernatant.

PCR Amplification
The second step involved the preparation of PCR master mix solution which is then added to all tubes by
positive control DNA. These tubes are then loaded on the PCR machine. In the result of this step PCR
product purification is obtained.

PCR Purification
The third step includes steps that are required for PCR purification. In these steps we add PCR buffer
solution into PCR product purification. To trap DNA plus columns are centrifuged for 15 minutes. Then it
is too loosened to the new tube, inverting column and buffer solution is added, first column is then
discarded after this assembly is centrifuged to collect DNA in collection tube while the column is discarded
again, in the result of this step PCR product is purified.

Sequencing Preparation
This step is all about sequencing preparation, for which the PCR product is diluted to increase its volume.

4
DNA Sequencing
This is the second last step of the whole process in which DNA sequencing is done by loading all products
in tray then this tray is loaded on DNA Sequencing Machine.

Sequencing Analysis
The last step is about the SEQUENCE ANALYSIS in which the sequence from the DNA sequencing
machine 4 appears on the computer screen for identification, and by the sequence of DNA, a bacteria /
tissue cells can be easily identified.

What have you learn from this virtual lab?

This procedure was extremely useful in figuring out the distinguishing proof of microscopic organisms.
With the assistance of this virtual lab explore, I concentrated on the methods engaged with the sequencing
of a quality too as scholarly the significance of 16S ribosomal RNA in the recognizable proof of
microscopic organisms. We can distinguish a species based on its DNA sequence. It was very captivating
on the grounds that each stage was obviously characterized and all of the data was promptly accessible.
Labels were placed on equipment and tools. It shows that each progression is significant in disciplines, for
example, microorganism research, criminal recognizable proof, etc. Understood that in an examination,
each progression is similarly huge and that results can change regardless of whether any of the progression
is ignored. This lab taught me about DNA sequencing and the requirements for doing as such with the end
goal of DNA sequencing I had the option to fathom how DNA sequencing aids species ID.