Introduction to 16s r rna sequencing cd genomics
- 2. What is 16S rRNA gene? What is 16S rRNA sequencing? Workflow of 16S rRNA sequencing CONTENT 1 2 3
- 3. 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)
- 4. 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.
- 5. 16S rRNA Sequencing Species Annotation Phylogeny Diversity Analysis
- 6. 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
- 7. 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.
- 8. Workflow of 16S rRNA Sequencing
- 9. 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.
- 10. Table 1. Primer sets for the amplification of 16S rRNA. F=forward,R=reverse Name of primer Sequence Name of primer Sequence 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
- 11. The suitable primer sets for various sequencing system
- 12. Sequencing For example: Illumina MiSeq V3 and V4 region Purification Quantification Pool Sequencing Platforms Sanger sequencing Illumina MiSeq 454 pyrosequencing PacBio SMRT sequencing
- 13. 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.
- 14. 45-1RamseyRoad,Shirley,NY11967,USA We are more than happy to be of assistance! 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