Science method
Metagenomics - Science method
The genomic analysis of assemblages of organisms.
Questions related to Metagenomics
Hi,
I'm new to shotgun metagenomics and just realized that there are multitudes of databases available to analyze the data, however no clear way to differentiate which one is better suited for which environment..
Which one to use for human gut shallow shotgun metagenomics data? (not looking to do de novo genomes, just to map to existing curated genomes..)
Looking at taxonomy databases for now, like GTDB, Web of Life, UHGG catalog....
Thank you for any advice!
Dear researchers
In my subject, I work on the characterization of the Moroccan marine microbiota by metagenomics under the discipline of microbial ecology.
During the sampling, we could not have all the in situ measurements of the Physico-chemical parameters of the studied microbiome. That's why we had recourse to the extraction of spatial data from the NASA website to complete.
my question is: do we have the right to combine our own data with those of a database to analyze them? ethical and copyright aspects. since these data are submitted to the public with open access.
I am planning to run16S metagenomic sequencing on libraries prepared from colon content of C57BL/6 mice to understand the gut microbiota diversity of control group and DSS induced colitis group. I am having problem of very low library concertation of some samples from DSS groups after quantifying by qPCR. However, the qubit showed considerable amount of library concentration. I have repeated the library preparation on same samples by increasing DNA input and cycle numbers for PCR. But the result is still same. Can I diluted the libraries based on qubit concertation for further sequencing? Can I use fecal samples instead of colon samples for those samples for preparing libraries? Any information would be greatly appreciated. I have used following kits for library preparation and quantification.
16S Library preparation kit : Ion 16S™ Metagenomics Kit, A26216
Library quantification kit : Ion Universal Library Quantitation Kit, A26217
Dear researchers
In my subject, I work on the characterization of the Moroccan marine microbiota by metagenomics under the discipline of microbial ecology.
During the sampling, we could not have all the in situ measurements of the Physico-chemical parameters of the studied microbiome. That's why we had recourse to the extraction of spatial data from the NASA website to complete.
my question is: do we have the right to combine our own data with those of a database to analyze them? ethical and copyright aspects. since these data are submitted to the public with open access.
Basically, the circular plasmid is more specific to identify its completeness. However, when we gain the metagenomic sequencing result from bacterial community or whole-genome sequencing result from bacterial isolates, after assembly of contigs, it is not clearly to confirm the completeness of linear plasmid.
I am trying to analyze the diversity of the bacteriome at different taxonomic levels
Hi,
I have biological replicates: they come from the same raw sample (tube of sediment) but from different DNA extraction & sequencing experiments. These are metagenomes and I used whole genome sequencing.
If I treat them separately, the abundances are consistent, some of them cluster together on a PCoA but not so well on hclust.
Question:
1) Should I concatenate the raw fastq files together beforehand?
2) Or should I treat them separately until after the alignment to a db step and then sum the counts of each replicates together?
What is the best procedure?
Thanks
It's about RNA viruses metagenome.
I am looking for packages to predict functional profiles from metagenomic 16S rRNA data in R language, as an alternative to PICRUst in Python. I found the Tax4Fun, but there are only a few examples of how to apply it. Does anyone know other similar and well documented packages?
I would bé gratful if you could send me a protocol to extracted metagenomic Dna from soil. The aime of work is shotgun metagenomics sequencing,so i'm looking for a good yield ans concentration. I already try with the power max kit of quiagen comptant and the concentration is very low.
Thanks
What are the best webserver/online tools for Downstream Analysis of 16S amplicon Metagenomic dataset besides Microbiomeanalyst ??
Thanks in advance...
as we submit genomic DNA sequences in various repositories (ex. NCBI). so is there any specific repository to submit metagenomic analysis results of 16s rDNA sequencing.
I have completed taxonomy assignment of the assembled contig from raw data but I cannot get the relative abundance of the species present. Is there any tool that can do that?
Hello,
I want to determine the gut microbiome composition from my mice feces by using DNA metabarcoding sequencing of V3-V4 region from 16S rRNA.
I have contacted Novogene for doing it, and they offer 150.000 reads/sample and 30K Raw Tag.
Do you have any experience in using Novogene services?
Do you think this numer of reads for sample and Raw Tag is enough for species level discrimination?
Thanks in advance
I have a set of metagenomic sequence data from aquatic eDNA samples, and have been able to analyze the the bacterial/16S aspects of the samples but the program I use cannot analyze eukaryotic data. Does anyone have recommendations for programs that can be used to analyze eukaryotic metagenomic data?
Hi everyone! I'm trying to figure out how to process metagenomic data (obtained using qiime2 and picrust2 for qiime) to do some network analysis using igraph or similar (do you have any recommendations?).
Now, unfortunately I can't find a good tutorial about this passage, so do any of you have something which could help me?
which extraction kit could be used for studies on metagenomics in bees?
Hello.
I am trying to find differentially abundant microbes between two conditions. I have the relative abundance data but not the absolute read counts.
Is there any method that considers relative abundance data as input?
or any way to transform this data before use?
Regards,
Pratyay
I have metagenomic data of gut bacteria of tribal population of Himachal Pradesh, can anyone help me regarding analysis of same for writing a research article. we will share authorship for the same.
I am currently analysing Illumina sequences for a metabarcoding project. The primers used in the process have NOT been removed from the raw data. But after exploring the data, I found that ~5% of my raw data has no primers.
What could explain this 5% ? Should I discard these primerless sequences?
PS: The data was prepared according to the following protocole : 16 Metagenomic Sequencing Library Preparation Part #15044223 Rev. B (copy paste on internet to find it)
Thank
I work on investigating the microbiome of the grapevines Rhizosphere using Shotgun-metagenome sequencing-Illumina. DNA was extracted using PowerSoil® DNA Isolation Kit - QIAGEN and further purified by Sodium acetate. Metagenomic DNA libraries (50 ng input) were prepared using NEBNext® Ultra™ II Prep Kit for Illumina®. The readouts of Bioanalyzer, Qubit™ dsDNA HS, or 16s PCR look perfect. However, the sequencing run gets always underclustered in case of my samples. Nevertheless, it disrupts the clustering of the other samples, too.
I look forward to any help!!
I have done both full-length 16S metagenomic microbiome and cultured microbiome identified with sanger sequencing. The results turned out greatly different from the metagenomics and cultured one, which should be usual. However, many microbes I cultured do not have the corresponding OTUs/ASVs in the metagenomic microbiomes. Regardless of the possibility of contamination, is there any other possible factor affecting the results?
Thanks in advance!
The key difference in data (structure and function), effectiveness for which analysis, and which one gives more clarity for gene identification.
Good morning,
Please I have an issue with my run and I need some help.
We did a metagenomic 96 sample run on Minion Mk1B (short-read 16S 400bp amplicons). The run lasted for 72 hours. Output was 9.7 gigabases (FAST5 files are 241GB). After this base-calling was initiated.
After 24 hours, the base calling was only 17% at which we aborted the base calling to do it on our server.
After 4 days now and only less than 20% is done. Why is it taking so long? We are using guppy v5.
What is the expected output size for such runs?
Does analysis usually take this long?
Is there a time limit by which we should stop the runs?
Thank you in advance.
Hello, I am trying to get a metagenomic analysis and found Novogene whose prices are pretty cheap (almost 1/3 of our university core). Does anyone have any experience with this company? about the data quality or reliability?
Please let me know,
Thank you,
For 16s rRNA profiling/ metagenomic analysis.
Please suggest a tool that can provide alpha diversity and beta diversity of microbes from shotgun metagenomic data either from raw sequences or assembled contigs.
I need to extract DNA from the vaginal flora of cows for metagenomic studies. Does anyone know any extraction technique?
Hi,
Has someone experience in building a custom db using Kraken2?
I have downloaded the fasta files for some taxa and build the new db but it produced an unmapped.txt file with a long list of accession numbers.
What does this file mean? How can I deal with it? Can I overcome this issue?
How can I found out which taxa have been successfully included in the db that I have created?
Thanks
Any body working on metagenomic DNA analysis in my connections? I need some help as regards the said analysis.
I extract environmental DNA from a soil sample and sequenced using Illumina's next-generation sequencing technology.
Hi,
This may not be the forum to ask this question, but we are looking for someone to analyse the raw metagenomic data. That person can become a part of the publication/s we aim to produce. All analysis will be done remotely.
I can give more specific details if anyone is interested.
Thank you.
Thilini
I am planning to go for metagenomics microbiome analysis. I have selected 4 primers for the same which are:
520F: 5'- AYT GGG YDT AAA GNG -3'
802R: 5'- TAC NVG GGT ATC TAA TCC -3'
338F: 5'-CCTACGGGNGGCWGCAG-3'
806R: 5′-GACTACHVGGGTATCTAATCC-3′
However there are some ambiguous sequences like, N,H,W,V,Y in my primers. Even though I know the coding standards like N stands for any of the 4 nucleotides(A,C,T or G) but i am confused which nucleotides should i put in to replace ambiguous letter. If anybody has any information. Please help
I am working on sequence based metagenomics analysis, and I am having difficulty choosing which tool to use for binning of the contigs.
I know that Shi et al 2016 inferred hosts for novel RNA virus genomes by searching their genes across cellular genome databases, so linking them with endogenous virus elements (EVEs).
Are you aware of other methods infer hosts for RNA virus contigs?
Thank you,
Guillermo
16s rRNA gene sequencing/metagenomics.
Hi everyone
I'm processing some leaf samples collected in different rivers for shotgun metagenomic sequencing (Illumina NovaSeq 6000).
The company that will sequence my DNA samples (Novogene in UK) requires a 260/280 ratio =1.8-2.0 (no degradation or RNA contamination).
(Novogene webpage:
But I've sent samples in the past (to the same company but for amplicon sequencing) with 260/280 = 1.7-1.8 and they passed the quality control and went full analysis.
Regarding the 260/230 ratio, they do not refer any requirement but my 260/230 ratios are even lower (the lowest is 0.6).
I'm using the DNeasy PowerSoil kit (Quiagen).
So my question is, how low can these ratios be for shotgun metagenomic sequencing?
What's the limit?
I know that I will probably have to clean some samples but I just want to have an idea to help me select the ones I definitely have to clean.
Thanks a lot!!!
Previously, I colllected the oral swab samples in Zymo DNA/RNA sheild solution. I would like to perform the saponin based host depletion before doing the shotgun metagenomic sequencing.
I have concerned that DNA/RNA sheild could break the bacterial cell prior to host depletion. I have no idea about the component of this reagent. Do anyone have suggestion about this concern?
How can I best analyze the 16s amplicon sequencing data obtained from multiple runs? Do I have to run all the sequencing files in Qiime again? Are there any tutorials available?
I am performing a metagenomics based experiment, where I need to send the sample to an external facility for NGS. The transit is of approximately 7 days. What can be the extraction and transfer protocol that can be followed?
Hello all Professors
What are the kits used in 16S rRNA Metagenomic sequencing (MiSeq Illumina) after the DNA extraction from human tissues (biopsy)?
Thank you in advance
I have microbiome sequencing data and I have analysed it to determine which taxa are up or down-regulated with my treatment, however, this doesn't really tell me much about the metabolic changes.
Is there anything I can use that is similar to the Qiagen IPA where I can place my metagenomics data and it can tell me if there are any changes in metabolic pathways?
Any links and tutorials would be greatly appreciated!
I was wondering which databases would be best for a scoping review on how drugs effect the microbiome. Not sure if this falls into pharmacology, microbiology or metagenomics, but I would probably want to search a database that contains all of them!
I am interested in doing microbial diversity analysis associated with a plant. I was looking for PNA clamps suppliers who can provide me mitochondrial PNA and plastid PNAs. Let me know if anyone has used it in their experiments or if someone can provide me as a courtesy.
Thanks in advance.
Hello,
I made index from my reference file and run command to align my metagenomic data by bowtie2. command is
bowtie2 -x <index_referance> -1 <paired_end_read_path_1.fastq> -2 <paired_end_read_path_2.fastq> -s <outputname.sam> and i got this result on screen without getting sam file in output folder.
16190304 reads; of these: 16190304 (100.00%) were paired; of these: 16189692 (100.00%) aligned concordantly 0 times 612 (0.00%) aligned concordantly exactly 1 time
0 (0.00%) aligned concordantly >1 times
----
16189692 pairs aligned concordantly 0 times; of these:
59 (0.00%) aligned discordantly 1 time
----
16189633 pairs aligned 0 times concordantly or discordantly; of these:
32379266 mates make up the pairs; of these:
32379200 (100.00%) aligned 0 times
66 (0.00%) aligned exactly 1 time
0 (0.00%) aligned >1 times
0.00% overall alignment rate
Can anybody tell me what are these results and what's wrong with this??
I'm working on ARGs analysis from shotgun metagenomic data sets. I want to learn how to analyze mobile genetic elements by using ACLAME database. Is there any guidelines ?
I am looking to use the Illumina DNA prep kit 96 well edition for metagenomics on stool extractions. I've heard that you can get many more samples then just 96 out of a single kit, how many can you actually get out based on the amount of buffers, beads, etc? Looking to see if anyone has empirical experience with this. Obviously the indexes will have to be on a second plate if there are more than 96. Thank you!
We are collecting human samples and thinking to use some storage buffer to ensure optimal preservation of RNA and DNA, among other molecules.
We saw several papers, and authors mentioned Allprotect Tissue Reagent (QIAGEN) as storage buffer, but we did not find anything about its composition. Then, I decide to ask if anyone knows whether Allprotect Tissue Reagent (QIAGEN) was a buffered salt solution such as RNAlater (Life technologies) is, since we we need to take this into account for the subsequent treatment of the samples.
Thank you so much.
Nerea
For my metagenomic shotgun analysis, I extracted total DNA from my food sample. I checked the concentration through nanodrop; there I got the good concentration of my samples but while running the gel, I did not receive any prominent band. I used 1 microliter of 6X loading dye and 3 microliter of my sample. what could be the possible reasons?
Hello,
I am interested in ordering universal hybridisation capture probes that can bind and identify as much animals species as possible. And same for plants and fish.
Later this captured (enriched) region can be sequenced on Illumina platform.
Since DNA barcoding for animals is done via COI region, is there a universal probe that can capture and purify this region for sequencing later?
Thanks
There are several protocols available to perform sampling for metagenomics, such as Splash freezing, freezing, EDTA etc. Each method has its own pros and cons. Which sampling and sample preservation protocol is the best to reduce biasnesss as well as the taxonomy consistent?
We are doing a Metagenomics study to find a possible link between gut microbiomes (with different diets) and activation of immune response. Considering that the DNA amount in faecal samples might be low, I'll need an expert advice on choosing the right DNA extraction kit which someone has used during their Metagenomics research. Thanks!!
Hi All,
I'm currently working on identifying lignin-degrading enzymes from metagenomic sequence data. I have come across several papers and articles where the FOLy (Fungal Oxidative Lignin enzymes) database has been mentioned and described but I have not been able to find a link or address where I can access this database.
Can anyone with useful information help me out, please!
I am soon starting a project, in which I aim at isolating bacteria from the muccus of Aiptasia ( a model organism for corals). I do not have a lot of experience in this field and I hope to get some technical tips and tricks. I will have three different strains of Aiptasia and try to obtain bacterial isolates from all of the three. The obtained colonies will be subjected to whole genome sequencing in order to determine the exact strain that was isolated (16S rRNA sequencing would not be enough for that). Is there a robust control, to exclude that the bacteria originated from the surrounding water and not from the muccus of Aiptasia? Thank you in advance.
I have carried bacterial metagenomic analysis between multiple groups (five in total). The groups represent daywise changes in metagenomic contribution. To compare the data taking into account the sequencing depth and also gene length, the nearest normalization method I came across is TPM (Transcripts per million).
Is it ok to use TPM values to compare metagenomic groups and percent TPM contribution (by calculating the percentage of TPM values) of each gene/function?
Similarly, is there any better statistical method (other than TPM conversion) to compare gene abundance change by normalizing the data (Not include DE analysis)?
Thanks:)
I am currently carrying out the analysis of effluent samples from agricultural processing factory, and among others, I will be needing a lab where I can carry out the metagenomic analysis to reveal the bacteria, fungi and archaea present. Kindly send me a mail on kolafasina@com or leave a message here for me. Thanks
Hi,
I have been trying to isolate metagenomic DNA using the conventional method of DNA extraction by using SDS for cell lysis and Isopropanol for precipitating the DNA followed by 70% ethanol wash. The resultant DNA contained humic acid contamination.
To avoid such contamination I was suggested to use PEG 6000, because of the non-availability of PEG 6000 can I use PEG 4000. If so how can I compensate PEG 6000 with PEG 4000?
Kindly help me in this regard.
Thank you in advance for your answers.
Hello, research community,
I am looking for some open problems in bioinformatics specifically in the area of, but not limited to, proteomics, and genomics. Since I am new to this area, any useful suggestions, a discussion on open problems and relevant resources are welcome.
Thanks.
Rahul
I have assembled "bins" from metagenomic samples. But is there any tool that can tell me which species do these bins/putative genomes belong to?
After metagenomically assembling bins, how do I check their quality? is there any tool to improve their quality? Are there any tool to validate the genomes?
Dear Colleagues,
Is it possible/feasible to assign Gram-positive and Gram-negative bacteria in a sample purely based on 16S or metagenomic sequences without gram staining?
Thank you very much.
Regards,
Nathanael
This is my first time of collecting water samples from marine environment. Could somebody please tell me the technique? I mean what kind of equipments (I usually used plankton net for freshwater) and procedures, in terms of collecting samples for metagenomic analyses. I could read from articles (and I did) but I need more practical quidance. Thanks a lot!
I want to extract bacteria DNA present in blood. Could somebody recommend me a protocol.
Any certificate course or institution where I can learn the basics and advanced level of metabolomics and metagenomics data analysis?
I am working on a urine metagenomics project, and I was wondering if there is a manual urine microbial DNA extraction protocol that provides DNA from gram positive and gram negative bacteria along with fungal DNA too, so I can run a pretty general, inclusive metagenomics test on urine?
I am working on developing metagenomics as a tool for diagnostics, but I am stuck at providing a significant value or amount corresponding to the number of reads obtained from sequencing.
Which DNA extraction kit or protocol would you recommend for studying the fish gut microbiome (16S and metagenomics)? I noticed that not every well-established protocol for human stool samples works well for fish-feces too.
Thanks for sharing your thoughts!
I am looking for ideal configuration details for a workstation to perform a metagenomic, transcriptomic and whole genomic analysis.
STAMP(Statistical analysis of metagenomic profile) can create a stamp file from MG-RAST profile. The present version of MG_RAST is providing TSV file or TSV detailed file. So, I am unable to create a stamp file from the MG-RAST web server data. Can someone help me in creating the stamp file from the present version of the MG-RAST web server data output.
I have used eztaxon database (http://eztaxon-e.ezbiocloud.net) to find the taxonomic classification 16S rRNA sequences. This tool analyzes only for one sequence at once. There are thousands of sequences in metagenomics data. So I want to analyze for multiple sequences.
Hello, I have a few hundred trimmed 16S metagenomic sequences from the Human Microbiome Project online database that I would like to search for the presence of several strains of bacteria via pairwise sequence similarity. Does anyone have any recommendations for software that can achieve this?
I am not a bioinformatician by any means, so something that has a user-friendly UI and can run on Windows would be much appreciated (although I can make my way around a command-line interface if need-be)!
As an aside, can I make a reasonable claim for species-level identity solely using 16S sequence identity? If so, is the >97% threshold generally accepted or is there another metric (e.g., score-based) that is more reliable?
I am preparing a metagenomics experiment from the soil sample. I will use Oxford Nanopore technology, which has only limited throughput (approx. 8 GB), so I need to discard all eukaryotic cells before the sequencing (because their genomes are really large, and would saturate the device quickly). So I am interested in the simple and reliable method, how to obtain only the prokaryotic and viral species. Is it a good idea to use some syringe filters? E.g. with the pore size 1 micron? So the eukaryotic cells will be stuck on the filter surface, and bacteria, archaea, and viruses will go through?
Thanks for all suggestions,
Martin
I've successfully uploaded my metadata file and assembled, shotgun metagenomic dataet to my MG-RAST inbox. The metadata passed the QC-like step, my sequwnce data uploaded just fine and I could happily move on to the submission step. Everything works beautifully until I get to the tab where I submit my sequence data, which is there but in red text and I can't select it.
Has anyone else had this issue and knows what the problem is?
I have deleted and re-uploaded both the sequence and metadata files but with no success. Any help/advice would be hugely appreciated!!
Thanks in advance.
Hi
I am extracting genomic DNA from dust samples and the 260/280 ratio is 1.4 whereas 260/230 is 1.35. I need to perform the metagenomic sequencing and for the same, the recommended 260/280 ratio should be greater than 1.8. Can anyone help me with how can I improve this. I have tried ethanol precipitation but it causes a significant reduction in the yield. As these are environmental samples the yield of genomic DNA is already low.
Thanks
I am working on some soil samples acquired from 16S RNA sequencing. In order to see which environmental factors influence community composition, I am using RDA(because dca() axis length <3.0).
I am confused on how to interpret the result from RDA and the subsequent anova.cca().
Q1: after running ·mod <- rda(otu_data ~ pH + T + N, env_factor)·, we get constrained eigenvalues of ·RDA1 RDA2 RDA2·, so is RDA1 means pH ?? If not, what does RDA1 represent?
Q2. using · anova(mod, by='term')·, we get a permutation test result like
` pH p-value=0.001; T p-value=0.03, N p-value=0.002 `. p-value of environmental factor pH is significant for what? It means pH is significantly responsible for community difference? This anova() function permute what(otu_data or env_factor) to refit the model?
In Jari Oksanen's vegan tutorial, ·“The test is sequential, and the order of terms will influence the results“, does that mean pH will have smaller p-values in 'pH + T + N' than it in 'N + T + pH'?
Q3. Do we need to process environmental factor of different range. factor1 may in range 1~ 10, while factor 2 may in range -50 ~ 50. z-score not work, because it raise error in rda.
Thanks to you all in advance.
Hi, does anyone know of a method to remove, as possible, plant DNA by conserving as much fungal and protozoan DNA as possible, for metagenomic analysis?
Next year I will start teaching metagenomics to master students (mainly focused on bioinformatics). Can anyone recommend a text book related to this topic? I am also interested in other related material such as video seminars. Thanks
I am trying to work with my first data set of metagenomic analysis. I have a data set of gene abundance (normalized) organized in two categories. first categorie is more general: Aceton metabolism, Aminoacids and Derivatives, Carbohidrates, ....And a second categorie with more specific groups: Acetone carboxylase subunits 2, Alanine biosynthesis, ....
I only have numerical values , corresponding to normalized relative abundance. The other information I have is not numerical, but categories of genes ( like I explain above) How I can calculate a functional diversity index with this type of data? Can someone give me any hint or tell me some paper where I can get information? thanks marta
Dear Friends and Colleagues,
To date very few journals are available to accept video articles for submission/publication in our field, although these types of articles are of paramount importance in microbial ecology in order to share new techniques and approaches.
For this reason, I decided to open a Collection titled “Multi-targeted approaches to evaluate microbial interactions and ecosystem assemblages in diverse environments” on the Journal of Visualized Experiments (JoVE): https://www.jove.com/methods-collections/919.
The purpose of the Collection is to offer a comprehensive overview of wet- and dry-labs perspectives in the field of microbial ecology.
The most recent Impact Factor for JoVE (ISSN 1940-087X) is 1.163, according to the 2019 Journal Citation Reports released by Clarivate Analytics in 2020, and the Journal is currently indexed in the major databases, including PubMed, EMBASE, Scopus and Web of Science.
After submission, each manuscript will be editorially and peer reviewed, which is typically a 1-2 month process. Once a text article passes review, a script will be generated. Generally, a filming date will be scheduled within 4-8 weeks after acceptance. A videographer will be sent to the authors’ site to film the procedure.
If you are interested, either message me or submit an abstract here:(https://www.jove.com/methods-collections/submit-an-abstract?collection_id=919).
Best Regards,
Prof. Elaine CP De Martinis, Ph.D – Full Professor, University of São Paulo, Brazil
Otávio GG Almeida, B.Sc. – Ph.D. candidate, University of São Paulo, Brazil
Guest editors.
I am currently in the phase a deep analysis of metagenomic data present in a water sample. There are multiple micro organisms that are only presented with one read in the data. Does that indicate that it is present in the sample, or are they just traces of the pathogen ?