rRNA Genes - Science topic
Genes, found in both prokaryotes and eukaryotes, which are transcribed to produce the RNA which is incorporated into RIBOSOMES. Prokaryotic rRNA genes are usually found in OPERONS dispersed throughout the GENOME, whereas eukaryotic rRNA genes are clustered, multicistronic transcriptional units.
Questions related to rRNA Genes
What are the possible genotyping methods for Acinetobacter spp.? In other words, what are the possible interspecies genotyping methods for different Acinetobacter spp.?
I'm not aiming for identification at this point but only for typing isolates into species-specific groups.
One that I know is the amplification of the 16S rRNA genes by PCR followed by RFLP.
Are there other methods with better discriminatory Power?
We are trying to quantify 16S rRNA gene of total bacteria in wastewater samples by using 341f/534r primers in qPCR assay. We got okay standard curves despite the efficiency was unsatisfactory (<85%). However, we got strange amplification curves, low Ct values (2.00) but no melting peaks for all wastewater samples. The gel electrophoresis confirmed no expected amplicons (194 bp) in these samples except smeared bands (Lane 2: standard; Lane 6, 7, 8: wastewater samples). It will be greatly appreciated if somebody can provide us with insightful opinions.
To give some background, I was instructed by my supervisor to analyse the relationship between the bacteria using the measurements listed in the title and to see whether they were in the same genus using 16S rRNA, ANI, AF and DDH value measurements. So I have had no problems finding the percentage genus cutoff for the 16S rRNA, but I can't find anything for the ANI, AF and DDH and this is particularly problematic because from my understanding, you can't just rely on 16S rRNA to determine whether they're in a different genus, because the 16S rRNA gene is only one gene and very short and so I would imagine you'd need other measurements to back up the idea that they're in different genera. What would be the best way to go about doing this?
For example, when a 16S rRNA gene sequence is searched against the database by BLAST with the max target sequence being set at 500, the total hits (root/bacteria/etc.) are much higher than 500. How?
Hi dear researchers,
While two organisms sharing ≥97% identical 16S rRNA genes may or may not belong to the same species, so why many researchers using 16S rRNA gene for their studies? For example, the 16S rRNA genes of three Bacillus species (B. anthracis, B.thuringiensis, and B. subtilis) are >99% identical, yet key features of their physiologies differ greatly, which is why they are treated as separate species.
I want to construct bacterial phylogenetic tree based on 4 house keeping genes (16S rRNA, gyrB, rpoD and rpoB genes).
Is there any online tools where I can upload the my novel bacterial genome sequence and where I can select the above 4 genes to construct phylogenetic tree?
Note that: I have my novel bacterial genome sequence and I know well to construction of phylogenic tree (NJ, ML etc) using 16s rRNA gene. And also I can construct MLSA tree using whole genome sequence.
I want suggestions or link from expert to construct bacterial phylogenetic tree based on 4 house keeping genes (16S rRNA, gyrB, rpoD and rpoB genes).
Thanks in advance.
I want to make a phylo tree using the 16S rRNA gene of the complete Aeromonas hydrophila genomes/strains available from GenBank. But each genome has several 16S rRNA genes (as many as 10) and they may have different sequences.
Do I just select one and omit the rest? Do them one by one (but takes a lot of time as there are many existing genomes)?
References or manuals list to analyze 16S rRNA rumen bacteria data
Would appreciate any advice/suggestions on the following task:
The sample was Illumina NGS sequenced for 16S amplicon and by WGS approach (I have fastq files for both). From 16S amplicon data, I have 400 nuc piece of 16S rRNA gene of bacteria which need to be analyzed by qPCR from the original sample. In the original sample these OTU is quite abundant - 15-20%. It would be nice to have a sequence of the whole 16S rRNA gene to be able to design several specific qPCR primers.
What could be the best strategy/software (is it possible at all?) to extract/re-construct the sequence information of the whole 16S rRNA gene from fastq files of WGS data? Any comments on a strategy of opening the fastq file in notepad and trying to do alignment and assembling starting from the known 400 nuc piece?
Many research articles have removed the 3rd codon positions in PCGs and rRNA genes for the Phylogenetic tree construction.
I'm optimising a protocol for coral mucus sampling and DNA extraction for 16S rRNA gene sequencing. There are many different methods described in papers, mainly by syringes or swabs and different extraction kits being used. Which method minimizes coral tissue contamination and gives the highest DNA yield?
I am doing 16S rRNA gene sequencing on the Illumina MiSeq platform. In several papers, I found that they have targeted different variable regions for this purpose. Although there might not be a concrete answer to this, I want to make sure that I target the best variable region during 16s library preparation. In this case, I am positive about targeting variable region V3-V4. Does anyone have a good suggestion regarding this? Help will be appreciated. Thank you!
I want to extract a bacterial complete 16S rRNA gene sequence from WGS (Whole-genome sequence). I have a bacterial WGS (FASTA file=assembled scaffold's FASTA) with 6857405 bp that was divided into 22 Scaffolds.
Can you suggest to me that how can I easily know/extract the complete 16S rRNA gene sequence from WGS ? Is there any software or online tools?
Thank you so much.
I am designing a PCR to detect the presence of Proteus vulgaris. Unfortunately, all the literature I have come across use either the 16S rRNA gene or the 16S-23S ITS. Does anyone know of a virulent or structural gene specific only to Proteus vulgaris?
What would be the cause of a smeared bacterial 16s rRNA band?
I add 3ul of DNA in the PCR reaction.
I load 5ul of the PCR reaction and 2ul of the losing dye into gel electrophoresis .
1 % gel 100 V for 60 minutes 1kb ladder.
If I have the genomic sequence of a bacterium and know the region where a 16S ribosomal RNA gene is located, How do I determine 5' and 3' ends of the 16S ribosomal RNA gene? Or, how do I figure out the 16S rRNA sequence from this gene?
I got the Illumina paired-end 16S sequences results. I tried to analysis them with QIIME2, but unfortunately, I found myself stuck many times. Can anyone help me,and how much will it cost? Please inbox me
I have whole genome sequence of bacteria (which I want to prove as a novel spp. and 99% novel). I want to extract the 16s rRNA gene sequence from it digitally. What is the easiest method to do that?
P.S:In the past, I did you Truebac app from Ezbiocloud and it was pretty easy but I guess, they do not allow more than two genomes to evaluate freely? (Perhaps, i need to pay after analzing two new genomes)? In short, I am not able to use Trueback to analyze another new genome.
I am confused with NADs (Nucleolus associated chromatin domains) and NORs (Nucleolus organizer regions). NOR contain multiple copies of ribosomal RNA genes (rDNA) around which nucleoli form. They are located on the short arms of the acrocentric chromosomes (13, 14, 15, 21 and 22 in humans). These regions code for 5.8S, 18S, and 28S ribosomal RNA. NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. if you see the figure 3 of this paper "Genome organization in and around the nucleolus" or in this linkhttp://www.ur.de/laengst/anemeth.html The NADs found in all chromosome but NOR found only in acrocentric chromosome. Overall nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. So my doubt is nucleolus forms due to NOR regions basically acrocentric chromosome then how NAD associated with all chromosome? Also, does anyone know the gtf file of chromosome position of NOR and NAD? Thanks.
Please I will like to know the hypervariable region of bacteria gene that will reveal abundant taxonomic composition from metagenomic DNA.
I'd like to ask for advices on how to combine the results from two different variables of 16S rRNA (V3 and V6, respectively) of human gut microbiome sequences. Would you combine their raw fastq reads and process them (I'm using LotuS here), or would you process them separately and then merge their taxonomic results together?
I've searched a lot and found only this paper (
18S rRNA gene has high level of amplification in candida genome (from 20 to 180 copy number)
is there any database who can approximate an average number?
I was trying to amplify 16S rRNA gene of my Lactic acid bacteria (LAB) isolates. However, some commonly used primers failed to amplify some of my LAB isolates. Can anybody suggest the best primer pair for this purpose?
At the current moment I've come across 2 studies: Castelino et al. 2017 and Parnell et al. 2017 who compared different regions regarding amplification of low biomass samples and the comparison with negative controls (Parnell). Perhaps anyone could suggest any other study covering this topic?
Hello, everyone. At present, I plan to study the diversity and environmental distribution of bacteria belonged to the same order. Therefore, I am eager for someone to show me how to design a pair primers (16S rRNA gene or housekeeping gene) for specific amplification of bacteria within the same order.
I am looking forward to receiving the kind replys, no matter to the detailed protocal, the similar researches or references.
I am working on a project aimed to determine the influence of long-term fertilization on soil microbial communities. I am sampling both the rhizosphere and the bulk soil and hope to use the current best choice of primers for targeting bacterial and archaeal 16S rRNA genes. Initially I planned to use the primer pair 341F/785R, which targets the V3-V4 region of 16S and is reported to have good domain coverage for both bacteria and archaea. However, I now also have the option to use separate, archaea (956F/1401R)- or bacteria (969F/1406R) -specific primers, which target the V6-V8 region of 16S. The benefits of the separate primers are better coverage for archaea, and less eukaryotic sequence contamination, but the V3-V4 primers are the standard tool typically used in similar research. I am confused with which set should I proceed with or if there are any other primer sets I should be considering?
What dna extraction kit do I need to use for fecal sample to prepare them for a Swift Amplicon 16s +ITS panel? The panel provides a primer pool for 16S rRNA genes (V1-V9) and the Fungal ITS1 and ITS 2 genes.
Hello, I want to construct 16s rRNA gene phylogentic tree. I have retrieved the similar sequences from NCBI and alligend that sequences through clustalx . I have installed Paup4 to my PC what should I do know to construct a tree with bootstrap values and also how will I save that tree?
I have downloaded a sequence of 16S rRNA gene of Staphylococcus aureus from online database SILVA (https://www.arb-silva.de). While the DNA sequence of any gene is supposed to have 'Ts' instead of 'Us' the sequence of 16S rRNA gene is showing Us instead of Ts. I am not able to figure this out. Can someone please guide?
I need to perform this real time PCR in order to obtain an estimation of total bacteria in my samples by amplifying the 16s rRNA gene of each microrganism. The problem is that my melting curve looks as you can see, very steep with sharp and matching peaks for replicates but the amplification in my samples is noisy and with chunky peaks. The samples consists of DNA coming from wastewater microorganisms. The point is, how can i improve the preparation or conditions to have more reliable data? Do you have any experience in the field of qPCR with 16s of bacteria?
Hi, I am doubtful about quality of 16s rRNA reverse sequence. I want to know how to confirm sequence quality. Please find attached file below to ascertain whether it is good enough or not.
We would like to study the microalgae diversity in environmental samples (being mostly constituted by microalgae) using culture-independent studies: a first approach using DGGE followed by NGS, Illumina. Which would be the most specific primers for microalgae amplification from environmental samples?
In several studies, 18S rRNA gene is the target for the study of microbial eukariotic diversity for both DGGE and NGS. The primers Euk1A and Euk 516r (Diez et al 2001) are used in several recent studies. Also the primer pair 3NDF (5′-GGCAAGTCTGGTGCCAG-3′)/V4_euk_R2 (5′-ACGGTATCT(AG)ATC(AG)TCTTCG-3′).
For cyanobacteria the primers described in the literature are Cya-b-F37/Cya-R783 and
GC-CYA353f/CYA781rA (Zwart et al 2005; Nubel et al. 1997) but always with simultaneous amplification of bacteria and plastid DNA.
Which primers you recommend for this type of experiments?
Thanks in advance!
My questions concern the Silva and Greengenes databases to assign taxonomy in 16S rRNA gene sequencing studies. I recognize that each database has its merits and drawbacks and as such I am evaluating which of these two databases is best suited for my data. Therefore, I used both to assign taxonomy and analyzed for which percentage of reads each database was unable to assign taxonomy to each of the taxonomic ranks (from domain to species). In other words, for Greengenes, I counted the blank cells at the domain, phylum, class, etc. rank. For Silva, I counted not only the blank cells, but also cells containing any of the following: ambiguous taxa, uncultured bacterium, and uncultured. The output yielded the following, which includes the absolute number of unassigned cells as well as their respective relative proportions:
Rank | Silva_absolute_unassigned | gg_absolute_unassigned
Domain | 0 | 0
Phylum | 446 | 447
Class | 936 | 682
Order | 2637 | 1778
Family | 4398 | 7969
Genus | 10573 | 14200
Species | 18044 | 17734
Rank | Silva_relative_unassigned | gg_relative_unassigned
Domain | 0.000000 | 0.000000
Phylum | 2.460282 | 2.467160
Class | 5.163283 | 3.764212
Order | 14.546558 | 9.813445
Family | 24.260812 | 43.983883
Genus | 58.324139 | 78.375097
Species | 99.536628 | 97.880561
Here, Silva and Greengenes assigned similar proportions of features at the phylum level (note that domain is 0 because I filtered the reads to obtain bacteria only), then Greengenes assigned more for the class and order ranks, after which Silva assigned a greater proportion of features at the family and genus ranks. Since Silva does not curate its database to include the species level, it makes sense why Greengenes assigned more features here. Has anyone else observed this pattern that Greengenes assigns more features than Silva at the class and order ranks? This seems somewhat counterintuitive considering that Silva is the larger of the two databases.
I have looked into the publication SILVA, RDP, Greengenes, NCBI and OTT — how do these taxonomies compare? by Monika Balvočiūtė and Daniel Huson (2017) for answers and noticed that in the Venn diagrams depicted in Figure 3, the amount of unique taxa in the Greengenes database increases until the order rank and begins to decrease from family onwards, mirroring my observations. However, I am not sure if there is a concrete reason for this similarity or if I'm seeing this pattern simply because I am searching for an answer. So, in short, my questions are: Why does Greengenes assign more features at the class and order ranks than Silva? Based on the table provided, which database is better suited for my analysis?
Many thanks in advance!
I need step by step guidance on how to analyse 16s rRNA gene. I received sequencing results containing Forward and Reverse primers along with a chromatogram file.
I need the primer sequence for reverse transcription of 18S rRNA gene, to be used as internal standard while working with plants.
like a general threshold of 97 or 99 percent at species level, or 90% at genus level, although there are exceptions. Hence there will be some percentage that will be shared by every bacterial species . I need to know that, thank You very much in advance.
Hi! I am trying to build a phylogeny using a mixture of mitochondrial and nuclear protein-coding genes and rRNA genes. Is it possible to specify the specific type of gene in the partition file? I am using RAxML through the CIPRES portal to create the phylogeny. Many thanks in advance!
The universal 16S rRNA gene and universal 16S rRNA primers are admittedly used to identify both bacteria and archaea domains at the same time. I would like to learn more about its feature, and compare bacterial and archaeal 16S rRNA genes each other as well as their application in molecular level. It is actually required papers or book to reach my answer.
I am working with qPCR on Lactobacillus plantarum WCFS1 and there is a high expression level of 16S rRNA gene comparing to the other genes. Therefore, I am looking for the other housekeeping genes that have lower expression level. If anyone knows, please tell me. Thank you very much!
I intend to use illumina 2 X 150 bp sequencing kit to sequence the V4 region of 16s rRNA gene. The DNA samples are from different soils. I was wondering what is the recommended sequencing depth per sample.
Greetings.I am aware of tools like Vikodak, PiCrust,Tax4Fun etc for prediction of functional potential from 16S rRNA genes of bacterial communities.In my current study, I also checked for fungal community structure and diversity targeting internal transcribed spacer (ITS) region using ITS3 and ITS4 primer combination.
So, I am interested to know is there any similar tool for analyzing functional potential for Fungi?
Looking forward for some suggestions.
Does using High fidelity polymerase helps in the amplification of 16S rRNA gene from the total community DNA (sediment, water) if the normal Taq polymerase don't work ?
Best software for amplicon seq data analysis for malaria parasite. Edit
I know that there are many software (QIIME,mothur, and others ) to analysis amplicon sequence data for microbial (SPECIALLY FOR BACTERIA). I am currently doing amplicon sequencing using 200 loci specific primers for malaria parasites (Vivax). 200 primers were designed for each locus and multiplex at least 10 primers in primary PCR. I,e a total of 20 PCR reactions. 2nd secondary PCR was done to add sample specific barcodes and Illumina index primers for library preparations and sequenced using MiSeq Illumina. So what would be the best software to analyze this data for detecting different malaria parasite clones from different patients and downstream Population genetics analysis. Can I use QIIME or mothur for my data analysis assuming different parasite clones as OTU???
I am limited on funds so a kit is out. I need to extract gDNA from pure culture GN rod bacteria that is clean and not sheared. I am trying to PCR up the 16S rRNA gene and potential toxin genes as well.
Everything I have tried to date will extract the gDNA but none of my PCRs will work. My positive control gDNA is working fine so nothing is wrong with my primers, buffers, Taq, or thermocycling conditions.
Thanks in advance!
it is well-known that the 18s rRNA lacks Poly-A tail. I was wondering whether, why 18s rRNA gene can be amplified when cDNA is synthesized by oligo-dT?
I am working on microbial diversity and planing to send samples for Next Generation Sequencing (NGS); Which hypervariable region of 16S rRNA gene will appropriate to target most of bacterial taxa present in seawater.
I already have the ones from beiresources, ATCC and Zymo.
Any help would be appreciated!
I designed synthetic DNA template containing 4 targets of 16s rRNA gene of bacteria in Bacteroidales group for using as DNA standard in real time PCR (standard curve generation). The synthetic DNA template has already validated in all 4 target assays. I would like to know that does it have any database that I can submit my synthetic DNA template for publication?
I want to compare bacterial communities using 16s rRNA sequences. I used Illumina Miseq and I has big differences in the number of OTUs and reads in all the samples. I use Primer-6 software to get diversity indexes and comparative analysis (ie. MDS, CAP). The question is: Is it necessary to normalize the total number of reads for these analyses and comparisons? If it is true, Which methodology is recommended?
Mitochondrial DNA s are getting integrated into nuclear DNA as NUMTS. I want to know how many copies of 16 S r_RNA genes are integrated into human and chicken nuclear DNA /
I have a bacteria for which 16S rRNA gene was sequenced and BLASTn showed ~98% similarity with organism A. However, when WGS was done for my bacteria, whole genome comparison using ANI shows very low similarity ~70% with not only with organism A but all the top 10 hits in my 16S rRNA BLASTn result. What does this mean?
I'm working with Oomycetes sequences, and I need to compare them (from 454 pyrosequencing and after ITS extraction) with ITS1 sequences from GenBank for the genus Pythiogeton, to understand which is the closest species.
Which is the similarity treshold I should use? Can I set it in MEGA 7?
I am using universal 16S primers in a qPCR on total DNA extracted from stool samples. Have repeated the qPCR several times and have tried other universal 16S primer pairs, but am still consistently getting melting peaks showing multiple amplicons.
Could this be because I am using DNA from a mix of bacteria, resulting in amplicons of varying length?
If not, what could be the reason?
And, if so, are there any other techniques for determining relative concentration of bacteria from DNA, e.g. a gene conserved across all/majority of bacterial phyla with the same/similar copy number in each species? I understand there is varying gene copy number of 16S so it may not be the best gene to quantify bacterial load.
the best protocol for analyzing 16S rRNA gene to identify bacterial strain
I am looking for Universal primers sequences, protocols, articles and so on to identify bacterials strains isolated from the nature. I would like from researchers with experience in this topic.
I am looking forward to analyze changes in soil bacterial community by 16S amplicon sequencing.
Previously there were more reports on targeting V1-V2 region with primer sets of 27F/514R(or something similar). But recent reports e.g. Klindworth et al 2012 and Thijis et al 2017 suggest usage of V3-V4 region preferably with primer sets 341F/785R(or 805R). Is here any specific reason for change in targeting regions?
Reports suggest this region(primer set) gives better coverage and high phyla spectra but somehow the aforementioned primer set wasn't included in the comparative studies.
May be i am missing something. All help will be appreciated.
Hi, pls can any one help me on how i can prepare master mix for a PCR reactionto for identification Acinetobacter species
I have recently come across some papers that suggest 16S RNA sequencing as a technique to know the active microbial population in metagenome and require some views about it. Also if this experiment is effective, I would like to know how to specifically isolate rRNA as most kits offer specific isolations of mRNA only.
I am using ARB silva software to create a 16Sr Phylogenetic tree. I created the tree by using the Neighbor-joining method. The general stopping criteria typically stop computations after 550-800 replicates
Q2. I have 651 bacterial 16Sr sequences and when I create tree then I always get only 130 nodes in my tree. ...why?
I recently decided to opt for the ITS3_KYO2/ITS4 primer combination in the Toju et al., (2012) High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples. PLos One. In the paper it specifies and tests a mock community composed of Ascomycetes and Basidiomycetes, but I wonder if anyone has used these primer sets and got a wider coverage of the fungal community from environmental samples?
The paper by Balint et al., (2014) An Illumina metabarcoding pipeline for fungi. Ecology and Evolution that used ITS3_KYO2/ITS4_KYO3 says that they lost a number of reads during the clean up and only managed to assign 33% of OTUs to fungi.
I also then found a paper by Asemaninejad et al., (2016) New Primers for Discovering Fungal Diversity Using Nuclear Large Ribosomal DNA. PLos ONE that compared a new primer set with the Toju et al (2012) primer set. Their new primer set target the LSU and while they performed better than the Toju primer set, is it better to target the ITS region where a lot of the research has previously been performed so that we have a larger database to assign taxonomy?