16S rRNA Sequencing

www.cd-genomics.com
 CONTENT

1 What is 16S rRNA gene?

2 What is 16S rRNA sequencing?

3 Workflow of 16S rRNA sequencing

 16S rRNA

rRNA——molecular clock:
1. Universality
2. Activity in cellular functions
3. Extremely conserved structure
and sequence

Three types of rRNA in prokaryotic ribosomes:

• 23S (3300 bp)
• 16S (1550 bp) —— a standard in bacterial
taxonomic classification
• 5S (120 bp)
 16S rRNA:
 Risosomal RNA
 Phylogenetic markers
 1542 bp

The 16S rRNA gene consists of eight highly conserved regions and nine variable
regions across the bacterial domain. The degree of conservation varies widely
between hypervariable regions, with more conserved regions correlating to higher-
level taxonomy and less conserved regions to lower levels, such as genus and
species.
16S rRNA Sequencing

Species Annotation

Phylogeny

Diversity Analysis
Advantages

i. Universally distributed
ii. Abundance
iii. Capability to measuring phylogenetic relationships
across different taxa
iv. Horizontal gene transfer isn't a big problem
v. Low costs
Disadvantages

i. Copy numbers per genome can vary

ii. Relative abundance measurements are unreliable
iii. Diversity of the gene tends to overinflate diversity estimates
iv. Unable to differentiate between closely related species
v. Limited information: as sequencing costs drop, microbiome research
is moving from 16S rRNA gene sequencing to more comprehensive
functional representations via whole genome or shotgun metagenomics
sequencing.
Workflow of 16S rRNA Sequencing
 Library Preparation

After DNA isolation, the DNA is selectively PCR-amplified using primers targeting the 16S rRNA gene.
Common next-generation sequencing platforms cover 100–600 base pairs per single read with varying
degrees of accuracy, but the full-length 16S rRNA gene consists of approximately 1,500 base pairs.
Therefore, primers are chosen to cover only a portion of the 16S rRNA gene.
 Table 1. Primer sets for the amplification of 16S rRNA. F=forward,R=reverse
Name of Sequence Name of Sequence
primer primer
8F AGAGTTTGATCCTGGCTCAG 785F GGATTAGATACCCTGGTA
27F AGAGTTTGATCMTGGCTCAG 805R GACTACHVGGGTATCTAATCC
336R ACTGCTGCSYCCCGTAGGAGTCT 806RB GGACTACNVGGGTWTCTAAT
337F GACTCCTACGGGAGGCWGCAG 907R CCGTCAATTCCTTTRAGTTT
337F GACTCCTACGGGAGGCWGCAG 928F TAAAACTYAAAKGAATTGACGGG
341F CCTACGGGNGGCWGCAG 1100F YAACGAGCGCAACCC
515FB GTGYCAGCMGCCGCGGTAA 1100R GGGTTGCGCTCGTTG
518R GTATTACCGCGGCTGCTGG 1492R CGGTTACCTTGTTACGACTT
533F GTGCCAGCMGCCGCGGTAA
The suitable primer sets for various sequencing system
 Sequencing
Sequencing Platforms
 Sanger sequencing
 Illumina MiSeq
 454 pyrosequencing
 PacBio SMRT sequencing

 For example: Illumina MiSeq

V3 and V4 region

Purification

Quantification

Pool
Bioinformatics

75%

7%

55%

After high-throughput sequencing, filtered and trimmed sequences of high quality are then clustered
into Operational Taxonomic Units (OTUs) commonly based on 97% identity of the reads. Species
annotation, OTU phylogeny, diversity analysis, and other studies can be performed after OTU
determination by OTU analysis.
Contact US for 16S rRNA amplicon sequencing

www.cd-genomics.com

info@cd-genomics.com

1-631-275-3058
45-1 Ramsey Road, Shirley, NY 11967, USA

45-1 Ramsey Road, Shirley, NY

11967, USA

We are more than happy

to be of assistance!