Sequencing

16s rRNA Sequencing

DNA Services

Identification based on the 16s rRNA gene sequencing has become the important tool for Researchers. We Yaazh Xenomics will do 16s rRNA sequencing for Bacteria and Actinomycetes identification; In all cells, rRNA Gene is most conserved region. For Bacteria and Actinomycetes 16S rRNA gene is amplified by using the universal primers 27F/1427R and sequencing with 518F/800R. To get ~1400 bps sequences, the forward and reverse sequence properly assembled, and contigs prepared.

DNA Services

Our 16s rRNA sequencing services provides complete solution (DNA extraction, PCR amplification, Gel verification, Purification, Sequencing, Sequence assembling, Bioinformatics analysis (BLAST in Various Databases) and Construction of Phylogenetic tree. We also accept DNA samples, PCR products (Purified/Unpurified) for Sequencing. According to the samples charges may vary. For better result submit direct microbial culture plate or slant.

We provide below contents documents as a 16s rRNA Result:

  • Sequence raw data (Forward and Reverse) in different file format.
    • .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 Phylogenetic tree & Customer specific gene analysis with free of cost.

The rRNA gene is the most conserved DNA in all cells. Portion of rDNA sequence from distantly related organisms are remarkably similar. This means that sequence from distantly related organisms can be precisely aligned, making the true differences easy to measure. For this reason, genes that encode the rRNA have been used extensively to determine the taxonomy, phylogeny, and to estimate rates of species divergence among bacteria. Thus the comparison of 16s rDNA sequence can show evolutionary relatedness among microorganisms. Various biotechnological techniques have been developed for identifying various bacterial species at the sequence level. DNA hybridization and 16s rRNA gene sequencing are the most common techniques used for this purpose.

Ribosomal RNA in prokaryotes

Name Size (Nucleotides) Location
5S 120 Large subunit of Ribosome
16S 1500 Small subunit of Ribosome
23S 2900 Large subunit of Ribosome

The extraordinary conservation of rRNA genes can be seen in the fragment of the small subunit (16S) rRNA gene sequences from organisms spanning the known diversity of life.

  • The 16s rRNA gene sequence show a high degree of conservation among species. This is assumed to result from the importance of the 16s rRNA as a critical component of cell function.
  • 16s rRNA is present in almost all bacteria, often existing as a multigene family or operons.
  • The 16s rRNA gene(1500bps) is large enough for informatics purposes.
  • 16s rRNA gene sequencing studies as opposed to the most cumbersome manipulations involving DNA-DNA hybridization investigations.
  • 16s rRNA gene of various bacteria are extensively studied and is present in many databases.

The extracted DNA is used as the template for PCR to amplify the 16s rRNA gene sequence. Universal primers (27F/1492R or 27F/1525R) complementary to a conserved region (16s rRNA) are used so that 16s rRNA region can be amplified from any bacteria. The PCR amplification produces multiple copies of the target DNA sequence. The resulting product is used as the template for the sequencing.

Since we know the length and terminal base of each fragment, the sequences of the bases can be determined.

DNA sequencing can be done by the common Sanger sequencing method.

The two strands of the DNA (3' to 5' and 5' to 3') are sequenced separately using the forward and reverse primers. To sequence the amplified product most of the time universal primers are used same which is used in the PCR. Some time to cover the entire region different internal primers also used. Common Sequencing primers for the 16s rRNA (518F/800R).

A Phylogenetic tree is a tree structured graph constructed using bioinformatic tools. It is Used to visualize the result of hierarchical clustering calculation.

The phylogeny tree is presented either as the distance or the similarity between the clustered rows and columns depending on the selected distance measure.

  • Identifying unidentified bacteria or isolates with ambiguous profiles.
  • Identification of Mycobacteria
  • Identification Actinomycetes.
  • Discovery and Description of Novel pathogens.

 

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