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    16s RNA, 18s RNA

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    lun tin
    This document summarizes 16S rRNA sequencing. It discusses that 16S rRNA sequencing targets the 16S rRNA gene, which is a phylogenetic marker that is universally distributed and abundant. It describes the workflow of 16S rRNA sequencing, which involves DNA isolation, PCR amplification of the 16S rRNA gene using universal primers, sequencing on platforms like Illumina MiSeq, and bioinformatics analysis including OTU clustering and taxonomic annotation. The document also notes some advantages and disadvantages of 16S rRNA sequencing compared to other metagenomic approaches.

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    16s RNA, 18s RNA

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    lun tin
    64 views16 pages
    This document summarizes 16S rRNA sequencing. It discusses that 16S rRNA sequencing targets the 16S rRNA gene, which is a phylogenetic marker that is universally distributed and abundant. It describes the workflow of 16S rRNA sequencing, which involves DNA isolation, PCR amplification of the 16S rRNA gene using universal primers, sequencing on platforms like Illumina MiSeq, and bioinformatics analysis including OTU clustering and taxonomic annotation. The document also notes some advantages and disadvantages of 16S rRNA sequencing compared to other metagenomic approaches.

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    This document summarizes 16S rRNA sequencing. It discusses that 16S rRNA sequencing targets the 16S rRNA gene, which is a phylogenetic marker that is universally distributed and abundant. It describes the workflow of 16S rRNA sequencing, which involves DNA isolation, PCR amplification of the 16S rRNA gene using universal primers, sequencing on platforms like Illumina MiSeq, and bioinformatics analysis including OTU clustering and taxonomic annotation. The document also notes some advantages and disadvantages of 16S rRNA sequencing compared to other metagenomic approaches.

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    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    64 views16 pages

    16s RNA, 18s RNA

    Uploaded by

    lun tin
    This document summarizes 16S rRNA sequencing. It discusses that 16S rRNA sequencing targets the 16S rRNA gene, which is a phylogenetic marker that is universally distributed and abundant. It describes the workflow of 16S rRNA sequencing, which involves DNA isolation, PCR amplification of the 16S rRNA gene using universal primers, sequencing on platforms like Illumina MiSeq, and bioinformatics analysis including OTU clustering and taxonomic annotation. The document also notes some advantages and disadvantages of 16S rRNA sequencing compared to other metagenomic approaches.

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    16S rRNA Sequencing

    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 bet
    ween hypervariable regions, with more conserved regions correlating to higher-lev
    el taxonomy and less conserved regions to lower levels, such as genus and specie
    s.
    16S rRNA Sequencing

    Species Annotation

    Phylogeny

    Diversity Analysis
    Advantages

    i. Universally distributed
    ii. Abundance
    iii. Capability to measuring phylogenetic relationships acro
    ss 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 pri Sequence Name of p Sequence
    mer rimer
    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 clustere
    d 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 deter
    mination by OTU analysis.
    18 rRNA Sequencing
    75%

    7%

    55%
    75%

    7%

    55%

    Fish apicomplexan 18S rRNA gene map and diagnostic primers. Avian apicomplexan 18S rDNA
    primer set (EF/ER) amplified a region of an approximate 1,500-base pair (bp) fragment from whole
    blood of the redlip blenny (Ophioblennius macclurei). Within this large segment, internal primers
    were designed to target variable regions. Variable regions (V1-V8) were established by aligning all
    query 1,500-bp fragments generated with the 18S rDNA fragment from Toxoplasma gondii
    (GenBank L37415). Based on alignment, a series of fish apicomplexan diagnostic primers were
    developed and tested including Primer set A (PsA), Primer set B (PsB), and Primer set C (PsC)
    amplifying regions of 500 bp, 550 bp, and 1,100 bp, respectively. 

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