Questions related to DNA Barcoding
Currently, I am working on the genus Abraxas, and their ID's are based on the morphology, genitalia structure and DNA Barcode. But I have come across a problem, the species which have been identified as same species based on genitalia structure and DNA Barcode and also shows same species in the phylogenetic tree, but BOLDSystems has assigned them in different BIN's. Is it possible to have two or more different BIN's for the same species?
Abraxas pusilla (IJ3561) - BOLD:AAJ4317
Abraxas pusilla (IJ3566) - BOLD:ABX6196
Abraxas pusilla (MF052436) - BOLD:AEE2493 - https://www.boldsystems.org/index.php/Public_RecordView?processid=GBMNC53061-20
I will use the Oxford Nanopore MinIon.
I will just access the COI gene region.
I am studying DNA barcode (COI) on amphipods but so far, I can´t achieve product PCR+. I have used three primer (Folmer/HCO2198 LCO1490 – Zplank/ZplankF1 ZplankR1- JG/jgHCO2198 jgLCO1490), parameter different of annealing temperature, +/- DNA concentration, + Mg+2, Trehalosa (potent PCR enhancer) to 5%. I used two extraction method DNA: 1) Hot-shot and 2) Wizard SV Genomic DNA. The amphipods are marine (Stenothoidae, podoceridae, maeridae and caprellidae). The concentration of DNA is between 5 ug/uL to 50 ug/uL for Method Hot-shot, and 1 ug/uL to 10 ug/uL for Method Wizard SV Genomic DNA.
Something idea about How I can achieve product PCR+?.
I would be very grateful for his assistance.
We had sent some phytoplankton samples for sequencing. And we had just received the generated sequences, and the next step was to do BLAST to identify what the phytoplankton that we sent is. Basically DNA Barcoding.
To give some context, when we send our samples for sequencing to the sequencing facility, they send us back two files, one for the forward sequence and another for the reverse sequence, based on the primers (forward and reverse) we gave.
So, the initial step involves us checking the quality of the sequences, specifically looking for any signs of low quality, ambiguity, or overlapping signals in the chromatograph.
Now, I'm a bit uncertain about the next steps.
The following step would be sequence trimming. To do this, I need to identify the start of each sequence by locating the primer sequence. This means finding the forward primer sequence in the generated forward sequence and doing the same for the reverse primer in the reverse sequence.
Afterward, I perform reverse complementation on the reverse sequence.
Following that, I conduct a pairwise alignment between the generated forward and reverse sequences and subsequently generate the consensus sequence.
My questions are, as I am a bit stumped with this (I apologize in advance, I'm a bit new with bioinformatics), (1) what if neither of the generated sequences have the primer sequences? Would that mean the sequences generated were of bad/low quality? and (2) Is this approach correct, or have I missed a crucial step?
I just want to know if someone can provide me an example input data for microbial association network analysis using SPIEC- EASI. Also, if possible, an R script how to do this network analysis will be very helpful. Thank you very much in advance.
I generally fix these nematodes in fomaldehyde-alchohol solution.
Shall I have to use different solution to preserve them for molecular study? And how to select amplification primers for an unknown (or new) species in PCR?
I am working on DNA barcoding of herbal plants and dried herbs. I have tried several protocols like CTAB, SDS, and phenol chloroform extraction methods but the OD is always less then 1.5. i have also used qiagen DNA extraction kit but the results were same.
Using the MEGA11 program to find the relationship for my dataset using DNA barcoding, how can I calculate and draw the Barcoding gap between intra- & interspecific genetic distances?
I think it is from computing within & between groups distances, but how can I convert these distances to a chart with frequencies and (k2p) percentage?
I am working on a final project related to DNA Barcoding using the matK gene. The sequencing results I have show a length of 1530 bp. Meanwhile, I read some literature that the ideal DNA Barcoding DNA sequence ranges from 400 - 800 bp. I have permission to ask: Can a DNA sequence with a length of 1530 bp be used for DNA Barcoding analysis? If not, what is the reason?
Please help. Thank you
I would like to extract DNA from dried insect museum samples and try to amplify a short mitochondrial gene fragment (short portion of cox1). Reading several articles on this topic, I notice that it is quite common to use high specificity Taq enzyme. However, appears not to be convergence on a specific product or type of enzyme. So I would like to ask for advice, based on your experience, on what might be the best Taq DNA Polymerase and/or best practice for trying to amplify cox1 short fragment from low concentrations and degraded DNA from museum sample extraction (in particular insects).
Just for knowledge, the products that I have seen several times in the newspapers are:
- Platinum™ Taq DNA Polymerase Catalog number: 10966018,
- AmpliTaq Gold™ DNA Polymerase with Buffer II and MgCl2 Catalog number: N8080241
- HotStarTaq DNA Polymerase (250 U) Catalog number: 203203
- TAQKB Roche KAPA Taq PCR KitCatalog number: KK1014
I would like to ask about the depth of analysis for producing a paper on identification of species using DNA barcoding technique. For example, I have sampled 3 species of spiders in cave and extracted their gDNA, design COI primers, run PCR and managed to obtain their respective barcodes for species identification. The similarity precentage of all the sequences was between 99-100%. If the species could be determined after BLASTn search, do I still need to proceed for phylogenetic analysis or other relevant analysis? Is the data enough to produce a paper because many papers that I have referred include a number of in depth analysis. Please enlighten on this. Thank you.
There are different plant barcoding regions present in plants like RbcL which stands for Ribulose 1,5-biphosphate carboxylase and MatK which is Maturase K.
So what are these regions stands for which include; trnH-psbA, trn L, rpoC1, rpoC2, rpoB, accD, psb k1 and atpF-F?
The QIAmp Fast DNA Stool Mini Kit has two main protocols listed in the handbook, accompanied by some simple notes:
1) Isolation of DNA from Stool for Human DNA Analysis
Lysis conditions in this protocol are optimized to increase the ratio of human DNA to nonhuman DNA.
2) Isolation of DNA from Stool for Pathogen Detection
Lysis conditions in this protocol are optimized to increase the ratio of nonhuman DNA to human DNA.
I'm interested in extracting and amplifying plant DNA from mammalian faecal samples to explore dietary habits. From my reading, it seems that both of these protocols have been utilized interchangeably in various papers to investigate diet, however, I'd appreciate any thoughts on whether one of these protocols might be better than the other for increasing plant DNA yield?
I am extracting DNA of fish samples for DNA Barcoding. The protocol involves an incubation step at 56C. How to do that? Can a Thermostatic Drying Oven be used?
Hello folks, I am planning to carry out research on DNA Barcoding of animal species from my area. I want good suggestions from you about my research.
Hi. I am working with arthropods (decapods, stomatopods, maxillopods...) using COI standard genetic marker for the identification. I am trying to assign my sequences to reference sequences from GenBank (via MIDORI). But what percent identity do I have to choose ? I don't find any resource mentioning it. Your kind response would be highly appreciated. Thanks Regards Lisa Loze
Given that the DNA barcoding is not an effective tool to delineate interspecific divergence but intraspecific divergence between coral species (Shearer and Koffroth,2008), how accurate is the species level identification of corals based on DNA barcoding along with morphological identifications.
I know that the 'Target capture bait set' approach is highly efficient in delineating corals with very close relatedness, specially the Family Acroporidae (Cowman et al,. 2020)
That being said, can you state new records of coral species were identified (let's assume 5new records of Acropora corals in the vicinity of local waters) based on the DNA barcoding results?
Kindly requesting some expert knowledge on this topic.
Can we use just only universal primer for invertebrates or do we need to develop or design primer for certain classes or group for the amplification method to be successful?
I have look for research paper about sample size of DNA Barcoding must not be less than 11 - 15. Is this apply for spider study as well since the spider genitalia are species-specific. How many samples should i do for this study?
"DNA barcoding sea cucumber"
"Sequencing sea cucumber"
using Boolean Operators, Truncation and Wildcard as examples,
( Sequencing OR ''DNA barcoding'' ) AND "sea cucumber''
I am a Botany student with Taxonomy specialization. I am a beginner of molecular biology. I want to make a DNA barcode of a species. I have inter and intraspecific genetic distances of single gene. But I want to find genetic distances of combined gene like matK+rbcL etc. How I do this work?
I have some questions in regards to using BLAST and sequencing data to give a species I.D. for fungal isolates.
We have created pure isolates of fungal colonies and then sequenced a gene region (shown in previous research to be a suitable DNA barcoding region for this genus) and ran this region (after sequencing both the forwards and reverse, creating a consensus sequence and trimming) through the BLAST database. While most isolates come back with mainly one species in the top search results, some have come back with various species in the top search results.
Therefore, how do researchers determine which species is the more appropriate I.D. using the BLAST database, or would you just say it is ambiguous based on the results?
For example, a general rule of thumb I was told in my undergraduate degree was you need an e-value below 10-4 and a percentage identity above 70%, however, if there are multiple species with similar values in the search results, how do you then decide which is most appropriate?
Is it purely based on the numbers, or would you look at the source of the result and preferably use isolate collections? Or, is it perhaps that newer species will have fewer entries in the database, so other species are likely to appear in the search results?
Thank you for your time,
I have been trying to extract DNA of mushrooms but I think my CTAB protocol is not correct. Can I please get some help and a right protocol for extraction of DNA.
I would like to know how to combine two or more DNA barcode sequences in one phylogenetic tree. Do you combine all sequences first to make one long stretch of sequence and align it with others (that you make in the same way?). Thank you.
I am tempted to purchase a miniPCR® Lab Starter Pack and use it for nematode analysis and reverse taxonomy (
I am wondering if anyone has used these for PCR analysis and how did the miniPCR® fair in comparison to alternatives? I thinking it may be fun to explore the wonderful world of PCR analysis!
Below is a link to a miniPCR® Lab Starter Pack for further information about the lab kit:
Or can someone can recommend a basic PCR functional lab setup?
I want to sequence a specific dicot plant genus with ITS marker for DNA Barcoding test. I've found in some literature that ITS marker is suitable for this specific genus, on which I'm working. Now, I want to know that if I target this ITS region, then is it mandatory to use forward and reverse sequences of both ITS1 and ITS2, or forward primer of ITS1 and reverse primer of ITS2? Please give me suggestions with the primer sequence.
Thanks in advance
My research group is embarking on a DNA barcoding effort focusing primarily on COI based phylogeography of Pacific basin species of the genus Apristurus, of which there are around 40 congeneric species! We have downloaded all of relevant COI sequence fragments from Genbank, and are interested in accurate placement of a species collected here in the Hawaiian islands. Happy to chat about potential co-authorship and collaboration with interested researchers.
I just want to know whether we can utilize the DNA barcoding approach to check genetic variation among species, eg: Sorghum bicolor
I am planning to use SSR markers to check the variability among the population, apart from that I would like to conduct phylogenetic analysis with DNA barcoding. Would it be possible? Has anybody conducted both approaches?
Your valuable comments are highly appreciated.
DNA barcoding is used to obtain taxonomic information about unidentified organisms. Apart from that what other types of Bioinformatics analysis might be performed with the DNA barcode data? What are the Bioinformatics Resources for DNA barcoding data analysis?
According to paper (
Please, help me. Thanks.
I am currently conducting a study about molecular characterization of Dioscorea spp. in Sri Lanka. I have prepared PCR products using both matK kim m13 and Trnh-psba primers.Clear bands were obtained through agarose gel electrophoresis but when sequencing only few base pairs were sequenced.
Does the reamplification of the PCR product need any alterations in the original PCR components and procedure to get better and reliable amplicons? Also how best the issue of insufficient quantity of PCR product can be solved by reamplification?
Is it possible to run out BINs analyses on BOLD based on 16S sequences (wild bees)?
My fear is that BINs analysis is only possible for large dataset (such as CO1 sequences) but would not be relevant for 16S as insect species are not well represented on BOLD for this marker.
Any advice for diversity analysis of 16S for approx 100 species (up to 3 replicates each)?
I was thinking about looking at K2P distances, compare 16S and CO1 trees to validate our new barcodes.
As far as species identification based on the molecular DNA barcoding is concerned, what factors would be the best description to be considered for a particular organism?
Eg. Baysean Inference Probability , bootstrap support, p distance, k2p distance.
Does transition and transversion mutation play any role in species description?
I have a little knowledge regarding interpretation.
My aim is to reconstruct plant-arthropods interactions using molecular gut content analysis but first attemps using rbcl and ITS2 did not allow to have accurate level of identifications (family level only).
Any paper related to that? Could aslo applies to plant-pollinator relationship, analysing pollen DNA.
the bootstrap value is relatively low in my phylogenetic tree, i have choose the different group and species, also some similar species, build a NJ tree with default index, but the bootstrap value is very low at the root of the tree? I don't know how to optimise this
I want to know that how specific barcode combinations (cpDNA) are choosen for DNA barcoding of eudicot plants and what the criteria is
DNA barcoding is mostly being performed using the mitochondrial COI gene. As this gene is maternal, can it be used for the DNA identification of interspecific hybrid fish?
Thank you in anticipation.
How would this appear on a tree if COI only resolved those closely related species and not more distantly related species? Thank you
Histograms in many papers have "Frequency (%)" in Y-axis and "Genetic Distance" in X-axis. What does this "Frequency (%)" mean?
Most of the current existing plants are the result of an adaptation of plants to various factors, especially the environment, of course, there are small portions of species or cultivar plants produced by humans. This evolutionary and adaptation process results in various species of plants growing only in certain geographic environments, where could be owned by certain countries based on the territory. A country where certain plants have grown may claim that this particular endemic plant belongs to the concerned country. Please give an opinion, whether this claim can be justified or not? Is DNA barcoding needed to be legal evidence to support the claim concerning those certain plants?
I am an student of entomological field, I am interested to work on DNA barconding of beetles, I have some fresh collection and some Museum specimens as well. I am not much clear that whether Museum specimens are suitable for DNA extraction or not. I will appreciate your kind and valuable suggestions.
I have three dataset of quantitative and qualitative liquid chromatography, GC-MS and DNA barcoding of few samples.
What would be the best way to discriminate and visualize the data?
Can we describe a new species of fish based on the examination of morphometric data, meristic characters and comparative materials without DNA barcoding?
I want to add some barcode sequences to primers then PCR them from different samples. Just want to know if there are any principles behind the barcode? I mean the length or the sequence features.
I know there are some commercial barcodes. But I need to add barcode to primers not the PCR product.
Hope for your reply!
I am working on a study of many species of local plants. My questions: 1: how can I choose the best genetic marker to make DNA barcoding? 2: What is the best technique to classify plants using DNA barcoding?
Hello everyone. I'm working in a metagenomic study using a short rbcL marker as barcode. I used Illumina Miseq for sequencing and I made the routine processes for denoising and filtering Illumina reads. I picked OTUs using a 97% of cutoff value. I've got approximately 8000 OTUs in all my dataset. Each OTU has an read value, and I filtered OTUs by read number, removing OTUs with one single read and keeping the rest. I think that I could remove more OTUs taking into account the number of reads. Anyone knows if is there a cutoff value for filter OTUs.
Thank you very much for your answers.
Do you think, one day the technology will be developed by which each and every single human being can be monitored continuously without any device with them; just sensing their DNA barcode? Their location, activities, temperature, wellbeing, etc.
Taxonomic identification of plants can be done by gene barcoding. There are several reported gene markers to be used to barcode unknown species. My question is "Is it possible to identify the taxonomy of hybrid plants using gene barcoding ?"'. Does the same gene markers will be used for hybrid plants as well ? I need research article references answering this question. An example of hybrid plants is given in the following link.
I am looking for recommended universal markers to amplify COI gene to using for DNA barcoding studies for sarcophagidae and calliphoridae insect species.Thanks
Which Illumina-compatible sequencing kits are recommended for the preparation of multiplexed libraries that cover the ITS region of fungi?
Where can we get best web-course beginning from basic Python programming and using Kernels till analyzing data with Machine learning algorithms.
I think most of the Biologist don't need much to know on how to write programs or design Kernels but should be able to write essential codes to analyze data through already available programs.
I found some online courses, one is: pythonforbiologists.com that appears instantly when searched with the concerned keywords, but was not able to get detailed reviews.
Another is Python for Genomic Data Science by Coursera, the course content looks good, but the reviews say that it lacks later materials (Weeks 3 and 4) in uses/applications that makes virtually impossible to finish the course.
Suggestions appreciated for recommending efficient web-course on Python for Biologist...
Fecal samples collected during handling procedures. Prey remains will include insect parts, seed cases, and fruit fibres.
Access to freezer space is limited.
Can we remove a gastropod animal from its shell before preservation? many times it happens that when we preserve an animal then after few days it get decay. So I am wondering for removing the animal from shell without damaging tissue and without breaking the shell. especially for DNA barcoding when the specimen exposed to ethanol it covers the body inside by operculum so the proper amount don't get inside the shell and after sometime the body of organism will be decayed. please give an appropriate answer for how to remove the animal from the shell for best preservation result.
Help appreciated!! We are having Empirical data sets of plant barcodes (matK, ITS and rbcL), and want to compare analytical methodology used for Empirical data sets with the Synthetic data sets.
How can we generate such kind of Synthetic datasets? I have found relevant papers but was not able to understand methodology. I would appreciate if any one could provide detailed procedure or provide any tutorial.
REFERENCE: Supervised DNA barcodes paper:
Real DNA Barcode datasets are simulated with Coalescent package in Mesquite version 2.75 (see the related work ). The data are simulated considering time of species divergence and the effective population size (Ne), i.e., the number of individuals in a population (of a species) that are contributing genes to the succeeding generations.
Another similar paper...
Conference Paper Species Identification using DNA Barcode Sequences through S...
I am having many singleton Species (species representing only one DNA sequence). These singletons lack resolution potential as they are single, when applied machine learning classifiers singletons are not considered as there is no reference for it... Even using other relevant methods, we cannot resolve singleton species confidently.
Please suggest any method/program that could simulate or generate reference sequences by taking those singletone species into consideration. Further this reference sequence could be used to resolve singleton species in the multiple species sequence dataset.
I have NCBI GenBank mined DNA sequences belonging to matK and ITS2 regions. I would appreciate if anyone suggests alignment free tools to calculate K-mer frequencies OR Genetic distances in order to use them for phylogeny construction OR for classifying using machine learning classifiers (I use WEKA... any suggestions?).
I am working to associate a leaf mining caterpillar with a species of adult microlepidoptera. Does anyone have suggestions on approaches to DNA extraction for microleps? I have COI-5P primers from BoLD http://v3.boldsystems.org/index.php/Public_RecordView?processid=GMLC413-11
I would like to ask you for help with interpretation of abgd results. According to morphology and phylogenetical tree clades, I should have here 6 species. ABGD gives me some weird results, with no barcoding gap, moreover I don't know how to connect the plot with partitions with the histogram of distances, because the scales are in different orders of magnitude. Anyone has some ideas what's wrong here?
i have read about DNA barcoding and it states that it involves CO1 gene to undergoes sequencing and is compare to the gene library. however, there are a lot of researches that uses other kind of primer like cox1, cox2, ITS1, 16S or 12S. im wondering if there is a reason behind it or is it some kind of a test primer or we can actually use these primers to do DNA barcoding too? hope the question is clear.
Many genera have been established either on highweight characters like Complexity, Constancy and consistency or low weight characters like Variability, Regressive, Monogenic and Narrow specializations. so DNA barcoding would be enough to distinct these type of characters
Suppose someone is working on molecular characterization of particular fungus using ITS region. He obtained the DNA and amplified using specific primer pairs and went for sequencing. However, without submitting the sequence to GenBank or any other related databases, is it possible to publish in journal or PhD thesis. How authentic/ scientific is such type of work?
Anticipating a positive response.
HI, I would like to know if there is an easy way to unify sequences downloaded from BOLD and from NCBI in a single dataset avoiding repetitions.
I would like to know the distribution of the speciemens of a species present on BOLD system. I notice that on the single sample code it's possible to see the position on Google map. So I would ask if it's possible to map on Google earth the position (if it's present) of the all samples of a species present on BOLD. Thanks.
I am working with fungal DNA amplification from plate culture at ITS and 28s rRNA regions by using ITS1/4 and 5.8SR/LR7 primers. from previously, I have done to amplified the fungal DNA from both regions before and now I got the problem with my pcr amplicon from ITS region. Because at the same sample and same times, I got smeary bands from ITS unlike the results from 28s. I attach a photo of an agarose gel in order you could visualize those weird smeary bands! Thank you in advance.