Introduction To 16s RRNA Sequencing-CD Genomics
Introduction To 16s RRNA Sequencing-CD Genomics
Introduction To 16s RRNA Sequencing-CD Genomics
www.cd-genomics.com
CONTENT
rRNA——molecular clock:
1. Universality
2. Activity in cellular functions
3. Extremely conserved structure
and sequence
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
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
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