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DNA Barcoding - Science topic

For DNA barcoders to discuss general and technical issues pertaining to DNA barcoding
Questions related to DNA Barcoding
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Hi,
Could anyone suggest free software or websites used to generate unique DNA barcode sequences [5-10nt] to label XXX genes for library screening?
Thank you in advance
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HI My dear
http://biorad-ads.com/DNABarcodeWeb/ this URL can you generate BarCode for your sequence , may be simple and more helpful for you .
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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?
Thanks!
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Well, I think the second method is more appropriate.You can prepare a list of plants fed according to the desired organism and then proceed to extract their DNA and then check the feces and match the extracted DNA. Then you will find out the amount of DNA of the desired plant by using quantification extraction methods. Of course, it is a time-consuming process.
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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...
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One of my first software pieces written in Python, which are describing error-resilient massive-parellel computations known from biology, wete used in this research
Make sure that you check the free, open-source software written in Python and watch animations of simulations. Robust computing known from biology started to draw interest of researchers in bio-computing, biologist, and medical theoreticians.
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I am just wondering since markers are usually used to measure genetic diversity.
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It depends on the level and what you mean by genetic diversity. Within a single organism? No. Within a population? No. Within a species? Normally not. Between species - yes, but that would be genetic diversity in the sense of species diversity.
Cheers
Konrad
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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?
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Would you like to answer my question exactly? I think we both have the same things.
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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
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Apply the species delimitation methods (ABGD, ASAP, mPTP, bPTP, ....)
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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.
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Thank you so much for your valuable comments.
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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.
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Yes DNA barcoding is a roadmap
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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?
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I want to calculate DNA barcoding gap between a few species, and export the result as image, which software and algorithm should I use? Must I use Linux?
Thanks
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Roberta Piredda Thanks for your answer. It's a useful tool for calculating barcoding gap actually. I'll try it more.
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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?
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It depends on the question you are asking. If you are interested in systematics, than sample size is not really that important (certainly not in terms of statistical significance, or whatever). What you need is a sample of each taxon large enough to ensure that the character states you are observing are representative of the taxon and not of individual organisms.
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"DNA barcoding sea cucumber"
"Sequencing sea cucumber"
using Boolean Operators, Truncation and Wildcard as examples,
( Sequencing OR ''DNA barcoding'' ) AND "sea cucumber''
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If you are going to do barcoding at all, historically most methods have used a very nice cytochrome oxidase I gene because the primers have readily available for decades (see early Chris Simon lit).
However, I reason that barcoding is foolish because it depends on so little sequence. These days you should be doing a phylogeny with whatever multiple gene set you can obtain or generate sequences from - some nuclear and plenty of mtDNA. The clades in a molecular tree will be best if you use maximum likelihood techniques and generate a full tree. Then the branch lengths (corrected distances) will give you a good sense for whether the taxa look like different species (always a subjective judgment about just how distant taxa should be to be considered separate species). Better yet compare the molecular tree to a morphologically based one to see if your results make biological sense.
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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?
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Dear Ayan,
You can simply concatenate your markers using software such as SequenceMatrix (http://www.ggvaidya.com/taxondna/), and then use this concatenated alignment for distance calculation with software such as MEGA X (https://www.megasoftware.net/).
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Hello everyone,
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,
Lauren
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Dear Lauren,
In this case it is better to say that identifications are ambiguous. Having multiple hits with the same (low?) similarity means that your sequences are not very similar to anything that is so far available in the database.
One way around this would be to download the most similar sequences (or what you think is taxonomically closer to your focal taxa) and build a phylogenetic tree. Such a tree would give you a more precise insight into the taxonomic affinity of your taxa than just a simple percentage threshold.
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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.
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Agreed with Nouh Saad
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Dear all,
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.
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Hi Trina:
I did not know if you were writing about different contigs of a same gene or fragments of different genes. Looking at your profile I saw that you are working with DNA barcodes of plants using multiple loci, so I assume your question is about using more than one gene to generate a single phylogeny.
If this is the case, yes, concatenation ("make one long stretch of sequence") of your gene sequences is one option. However, the most important step is to align each gene independently, and later concatenate the aligned sequences. The result of this concatenation of multiple genes will be a matrix that can be analyzed using your preferred phylogenetic method(s).
You can easily concatenate your sequences using the program Sequence Matrix, available here: http://www.ggvaidya.com/taxondna/
I hope it helps!
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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?
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Thanks Miguel for the linking message. Hi Lawrence, in my opinion, @miniPCR is a versatile, precise and low cost equipment, useful for any process related to PCR and thermo block functions.
We have used in remote field work and in lab. Highly recomendable for education and portable research.
We currently have the https://www.minipcr.com/product/minipcr-lab-in-a-box-2/ with a Gyro mini centrifuge
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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
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It is generally advisable to use the primer pair as it is and not interchange (like forward of ITS 1 and reverse of ITS 2).
However, if you really require so, I can suggest you to do an In-silico analysis of the forward primer from ITS 1 together with reverse primer from ITS 2.
If you need any assistance in the analysis, feel free to ask!
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Aloha,
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.
Cheers,
Brenden
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Dear Brenden S Holland thnak you so much for this collaboration proposition.
Good luck ... I am sorry to tell you that this species is not present in Med sea and this region the shark is very accesory Sp.
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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.
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Hello Malitha,
you can check this article. They used SSRs for genetic diversity analysis. After that they selected 5 polymorphic SSRs for barcoding and for QR-code generation of the sertain cultivars of tea plant (go to page 11 to se the figure there).
Good luck!
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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?
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If you mean bioinformatics resources /databases
NCBI
EMBL-EBI
DDBJ
BOLD (FOR, COI barcodes)
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According to paper ( ) several DNA barcoding markers such as rbcL, matK, trnH-psbA, and ITS; chloroplastic matK gene region; matK + rbcL were used for discriminating species. So, I was confused about what kind of markers to choose.
Please, help me. Thanks.
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Try mitochondrial markers for barcoding
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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.
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Hi Samitha,
Some thoughts on your sequences.
DA1TRNHPSBAfp
Starts with strong (>1000) well spaced base peaks dropping slowly from base 105 to about 330 bases with a signal/noise of 200. Quite good sequencing but this kit is capable of 900 bases of strong intensity signal. This is usually cause by insufficient primer or template. If this sample has primer dimer ( PD) then this will measure as DSdna so less template will go into the sequencing mix and the signal will die early and the remaining template will give a weak but readable signal
DA2TRNHPSBAFP
The signal strength is only 28 and s/n ratio is 4 so no readable sequence at all.The reaction has failed completely so possibly primer and template do not match
DI5MATKKIMM13FP
Clean strong base peaks up to base 53 ( strength 1600)then signal drops to zero.
This can happen with some secondary structure problems in the template dna but can also happen if you run out of primer. In this case I suggest that the sequencing primer has been used up sequencing PD
DI7MATKKIMM13FP
Good clean strong ( 3700) sequence up to 46 bases then dropping to baseline intensity. There is no huge unincorporated dye peak so the sequencing reaction is working but clearly no on the template dna so I think that we are sequencing a strong PD
De1TRNHPSBAFP
Very large dye peak ( mixed bases) at start of sequence (>4500) then no peaks. The sequencing reaction failed completely. Either no primer, no template or the wrong primer used in sequencing.
D13MATKKIMM13FP2
160 strong bases all Ns. We have 2 primers annealing in different places and producing 2 sequences. As it is unlikely that you supplied 2 templates so something is acting as a second primer or one primer is annealing in 2 places on the template dna ( unlikely), The sequence stops much too early. I do not know the cause for this one but if you had not told me that there was no PD I would say the pd has dissociated and caused F and R primers to anneal to the template and has used up the sequencing mix too early.
RecA C92f
Huge unincorporated dye peaks (17000 and 4500) at ends of sequence with low intensity clean sequence in between. I think that you have light PD which has sequences and used much of the sequencing primer and the rest has produced a weak but accurate sequence that has not been basecalled. The reason for not calling the bases is that the unincorporated base peaks are so high that the base calls are below the set cut off value of 5% of the highest peaks so they are ignored.
RECAC92F
Huge (>12000) unincorporated dye peak at start dropping very rapidly after 35 bases. Dye peaks at 85 and 120 bases indicate that the sequencing has failed. I think the primer was all used on the PD sequence and the unincorporate dbases are from the unused sequencing mix
Overall I feel that your problems are caused by PD using up all the sequencing primer not leaving enough to get a good signal from the template dna. Additionally pd is double stranded so will measure as DS dna when calculating the amount of dna to sequence so you are actually sequencing too little dna.
You may need to clean up your pcr ( use less primer for a start) or gel separate the template and clean it up before sequencing.
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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?
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Re amplification must be treated with care. In early stage pcr every 10 cycles is 1000 times more product so if you use too much template or too many cycles the amplimer will self anneal and form smears of lonmers. I would use 1ul of first round product...dilute it into 100ul and use 1ul of this dilution. Set up 6 tubes of pcr and set for 24 cycles and remove a tube at 12,14,16.....24 cycles and see which cycle number gives good clean amplification.
A cleaner way is to design an internal primer set just inside the first pcr primers. They can be quite poor as primers because the template is almost all the correct amplimer.. Run 20 cycles of a 1% dilution of the first round product ( 1ul of 1/100 dilution) and it should give a clean product. There is no need to clean up the first round product between amplifications
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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.
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I think, it is possible. So, you can try to do it as early as possible Dr @Mélodie Ollivier
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Hello Researchers,
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.
,
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Generally speaking, the scientific community's standards for species descriptions, at least for macroscopic organisms, include morphological or other non-molecular characters by which the species may be distinguished from its close relatives based on observation. One can describe species based only on fixed nucleotide differences, but doing so means that all future specimens may only be identified by sequencing them, as well. So in my opinion, you need to think about why you want to describe species based on barcodes before you start doing it. It is a slippery slope.
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For animals' DNA barcoding, what is the most suggested marker: COI, Cytb, 12S, or 16S?
Are there any universal standards or reference databases?
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thank you for your answer Siva sankar Anisetti
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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.
Thanks,
Mélodie
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Dear Mélodie,
I strongly recommend you take a look at the polymorphism in your group of taxa.
People have used, besides rbcl and ITS, the matK and trnH-psbA (Galimbert et al. 2014) genes as potential species-specific makers, and often more than one together. The trnH-psbA seems to be promising. However, that is a little bit different from animals where the COI has some clock-like passe of mutation across many taxa (it still challenging in cnidarians, though).
You might find out more about plant barcodes in Hollingswort et al. (2011) and Li et al. (2015). Especially for pollen identification, I've found some interesting reports and opinions in Bell et al. (2016), Galimbert et al. (2014) and Bruni et al. (2015).
My advice is to think about what criteria you are going to use for identification. Some of them are base on genetic distances (like K2P thresholds; ABGD), other on trees like the coalescent models (GMYC) and Poisson tree process (PTP) models, and some other on diagnostic characters (see review in Dellicour and Flot, 2018). The point is that all methods bring some bias and a consense of methods from different natures would lead to a more accurate delimitation.
If you are going to use a multi-locus approach, I would recommend the PHRAPL framework (See Jackson et al. 2017).
I hope you find this helpful.
Cheers
References:
Bruni et al. (2015)
Galimbert et al. (2014)
Hollingsworth et al. (2011)
Li et al. (2015)
Jackson et al. (2017)
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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
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Well, that "depends". There are several alternatives, depending on how your dataset looks like. Perhaps, it would be more effective if we ask ourselves what does the bootstrap means and how it's "calculated".
Considering that it is the number of times that a certain topology or node is recovered (or sort of...) we would conclude that you probably have too many equally likely topologies.
Then you can choose either tuning your analysis to "prefer" a solution to the detriment of any other or step back and think why you are getting this.
If you pick the first path, I would recommend this topic (https://www.researchgate.net/post/How_to_improve_low_bootstrap_values_in_phylogenetic_tree) where people are discussing something similar for amino-acids data, but it is not that different for nucleotide sequences. Strategies usually include increase the replication numbers; replicate terminal taxa (both new samples and artificially created ones); tuning up parameters; reconsidering your alignment and their algorithm; trim out the sequences edges; picking up a different tree estimation methodology, maybe maximum likelihood or Bayesian frameworks...
However, I would recommend you to think a little bit about it. Why are you getting such results? If you are not screwing things (I believe not), the phylogenetic signal may be too low (i.e. very conserved gene/low informative sequence), then any technical improvement or tuning would result in a methodological artifact, not the real meaning of the thing. If you realize that your data is too poor then the obvious thing is to get more info on that. If it is not possible then just report it. Keep one eye on that uncertainty and you should be fine.
Good luck
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I want to know that how specific barcode combinations (cpDNA) are choosen for DNA barcoding of eudicot plants and what the criteria is
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If you go through the previous plant barcoding papers, you would realize that barcoding, per se, did not work as well in plants as in animals. If you want to work on specific cpDNA regions of your desired family, I would suggest you to go through previous gene specific marker works on the specific family. Also, you can check the Barcode of Life websites for further information.
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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.
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The short answer would be no. COI is a single maternally inherited marker, so it may provide relevant information on the directionality of introgression, or give you an idea about the geographic origin or the number of independent hybridisation events (in relation to the parental species' diversity). But identying hybrids themselves critically depends on nuclear genetic markers (and then COI can provide the additionally useful information stated above). Morphology may or may not be a good advisor in any case. Hope this helps.
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WANTED
We are looking for a frozen or in ethanol-preserved Cerambyx paludivagus for barcoding
SCIENTIFIC REWARD
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Dear Derradj,
Thank you very much for your collaboration offer.
Since the last records of C paludivagus that I know date from several decades ago, I had even thought that this species was extinct.
From your address I suposse that the material will be from the mountains near Constantine.
Best
Luismi
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How would this appear on a tree if COI only resolved those closely related species and not more distantly related species? Thank you
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support values would be low for deep branches and/or they would be unresolved (as Artur already mentioned). And COI is NOT suited to resolve deep branches. For closely related species it is fine, but also for closely related species you always have the problem that if you analyze a single gene, you will only receive a gene tree. Not the underlying species tree and that might be quite different
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Histograms in many papers have "Frequency (%)" in Y-axis and "Genetic Distance" in X-axis. What does this "Frequency (%)" mean?
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shamelessly promoting my own tool for this here: https://github.com/cdoorenweerd/PyCOIStats
It is better to focus on Dmin_NN (minimum distance to nearest neighbor) and Dmax (maximum intraspecific distance) values, which PyCOIStats also provides. Hope you find it useful
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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?
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It is a very important step, if implemented in India. There are more duplicacy in the variety exist in the country.
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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.
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Sergey Viktorovich Pushkin right statements. It really depends on the age of the samples. During my PhD thesis, I was dealing with old alcohol arthropods with an age of 15 to 30 years and it was impossible to extract DNA from them as I was really eager to do that. Instead I used a DNA extraction method to clear these specimens to inspect them under microscope for accurate identification for further biodiversity calculations. Just check the link below:
Maybe it is interesting for you colleagues in soil zoology.
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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?
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Principal Component Analysis (PCA) is normally able to gather similar data and discriminate between different sets. We successfully did it with coffee aroma analysis using GC-MS.
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Can we describe a new species of fish based on the examination of morphometric data, meristic characters and comparative materials without DNA barcoding?
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We exactly CANNOT identity new species based on barcoding! Barcoding data could have a signal for existing of a new species, it's true. Barcodind identifies some BINs, nobody known what is it, and they change all the times after adding new sequences. Regading these strange products as something species-like is clearly contradicting to the main approach declared in the Preambule to the International Code of Zoological Nomenclature created for STABILITY.
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Can someone suggest a particular barcoding region for clinical yeasts?
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ITS works!
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Hi everyone,
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! 
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I am studying a similar technique and had same question.
A paper i read talks about adding about 5 different 10nt long barcode in 5' end of the primer. This is called compressed barcoding space. According to this concept, one can create few number of barcodes and use its permutations to barcode many primers. I was wondering if it will cause any issues during PCR.
Can you please tell me from your experience how feasible is such experiment.
Incase, you are wondering what i'm reading https://www.biorxiv.org/content/10.1101/2020.04.06.025635v1
it is wide scale pooled sequencing for covid testing.
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Will DNA barcoding take over the more conventional Taxonomy?
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Thanks a lot Dear Dr. Shahnawaz Gul , I really appreciate your contribution. Regards
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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?
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I agree. Look for example in GenBank which marker is most commonly used. Chloroplasts, Internal transcribed spacer 1 or 2 (ITS1, ITS2) and 18s ribosomal RNA gene are commonly used, but look up genes relevant to the genera you're interested in. For classification the same thing is relevant, but look for papers and see which markers the best ones used to estimate phylogenies.
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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.
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Use one script "filter_otus_from_otu_table.py" in QIIME pipeline. You can find the detailed description at http://qiime.org/scripts/filter_otus_from_otu_table.html.
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I want to use Wilcoxon Signed Rank test to asses the performance of different markers for plant barcoding analysis. 
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If data are not normally distributed, Wilcoxon Signed-Rank Test is used in place of Dependent t-test. See this link to perform Wilcoxon Signed-Rank Test with SPSS.
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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.
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How does fingerprints work with remote sensing and DNA Barcodes?
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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.
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i think this paper will be workable for you.
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I am looking for recommended universal markers to amplify COI gene to using for DNA barcoding studies for sarcophagidae and calliphoridae insect species.Thanks
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Hello Abdullah,
You may assess the potential success or failure of the universal primers described by Folmer et al. (1994) based on the results of the enclosed article.
Deleted research item The research item mentioned here has been deleted
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Do you know any database for searching about endemic species, their genomes, differences, DNA barcodes etc.?
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Exactly, since endemic species are not universal but variable based on the geography, there can not be a standard database to have the information in one place.
In you original question you did not mention about which species you are interested in, animals; plants etc. thus referring you to any database is not logical. Also, most of the databases contains genomic data and metadata about environmental parameters, genome content etc. But I have not seen any database which contains the barcodes/primer information, and secondary metabolites.
Ofcourse you can use universal primers to target the species in question, and there are many out there on innternet or in published papers.
I dont think you can find all at one place, what you want to find, as of today. However, it would be a good database to make and have all such information in one place. May be you can make one and publish.
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Which Illumina-compatible sequencing kits are recommended for the preparation of multiplexed libraries that cover the ITS region of fungi?
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You can use
  • ThruPlex DNA seq-kit, Takara
  • NxSeq kit, Lucigen
  • Nextera or TruSeq kits, Illumina
  • NEXTflex kit, Bioo scientific
  • KAPA kits, Roche
  • Custom primer with barcodes (Hugerth et al, 2014 or Elbrecht and Leese, 2015 or Elbrecht and Leese, 2017)
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Fecal samples collected during handling procedures. Prey remains will include insect parts, seed cases, and fruit fibres.
Access to freezer space is limited.
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This response may be a little late. However there are faecal preservation buffers that can protect nucleic acids for 2 years at room temperature and prevent microbial growth.
Depending on your sample amount, there are smaller or larger tubes available. The smaller tubes will also come with glass beads and are compatible with any isolation workflows requiring homogenization.
Attached is more information on preservation of the gut microbiome diversity with fresh, frozen, TE-buffer, or Norgen preserved samples.
Hope that helps.
JP
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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.
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It depends which group of gastropods are you talking about. Methods can slightly vary in small and mid- to large-sized species / groups.
As for large specimens (e.g., Neritidae, Thiaridae, large Cyclophoridae) you have to to made a small hole(s) in the shell wall to ensure ethanol is coming inside of it and conserving the tissue for DNA.
Smaller species (e.g., Diplommatinidae, Pupinidae, smaller Cyclophoridae) are not as easy - usually you have to breake shell of one of specimens or use juvenile individuals for DNA barcoding since their opercula are less developed than in adults and ethanol comes through.
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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:
From paper....
Synthetic data:
Real DNA Barcode datasets are simulated with Coalescent package in Mesquite version 2.75 (see the related work [8]). 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...
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Hey,
may I ask for what purpose you need this synthetic dataset and how shall it look like and what did you do with your "real" dataset (methodically)?
Regards,
André
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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.
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I have read the question carefully and what you have wrote in brackets does not mean singleton species.
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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?).
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Sorry I don't know if it will help you, although i send u.
matK-QR classifier: a patterns based approach for plant species identification
Ravi Prabhakar More, Rupali Chandrashekhar Mane, and Hemant J. Purohit
Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals
Hui Yao, Jingyuan Song, [...], and Shilin Chen
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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?
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This method sort sequences into putative species based on pairwise distance, you can work with distance matrix or you can use. the alignment that the ABDG web serve make the distance matrix before analyzes. The ABDG web serve separates the species based on a range of maximum intraspecific distance. I mean, the ABDG consider different value of maximum intraspecific divergence to separete species (graph below) .You can click each point (yellow or red) to see which species were delimited for each maximum intraspecific divergence. If you know the intraspecific divergence of the gene in your taxonomic group, you can modify the parameter on web serve and maybe you get better results. Other important point is: the ABDG works better if you use the sequences closer phylogenetic each others(for e.g use sequences from the same class or genus). Because the substitutions rates (consequently intraspecific distance ) can vary among taxonomy groups. For this reason, I recommend exclude the outgroup from alignment before analyses. Since the greater pairwise distance between ingroup and outgroup can "confuse " ABDG.
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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.
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Hi Ammar, there is a reason. Cox1 gene is pretty good barcode marker for most groups of animals. However, it does not work so well for other groups such as plants (where rbcL is preferred), fungi (ITS rDNA is preferred), not even speaking about various groups of protists! For many groups, it does not have sufficient resolution (i.e. it cannot distinguish closely related species), universality or/and practicality.
I don't consider it wise to use whatever barcode markers you wish. Your choice should be guided by research on potential barcode markers carried out in your taxonomic group. If you are working on animals, plants or fungi, you can begin with standard markers for these, which are cox1, rbcl, and ITS, respectively. However, specialists on many other groups (especially protists) still did not agree on standard markers for those particular groups.
For example, diatoms are extremely species-rich group (cca 100 000 spp.) so it is difficult or perhaps impossible to find single barcode marker which would be sufficient in terms of universality, practicality and resolution. Thus we are forced to switch to dual-locus barcode system (and we did not yet agreed on which two markers should be selected as standard barcode markers for diatoms).
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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
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Hi, DNA barcoding is a very cool method for species identification but also for detection of taxonomic inconsistencies at the species level. Hovever, if you want to work on higher taxonomic levels, you need to perform the phylogenetic analysis and for this the variability of the barcodes (COI) is definitely not sufficient (e.g. due to saturation of substitutions). Besides you need to use several independent (nuclear) markers that complement the mitochondrial markers (which are not independent as they are inherited as one locus). Hope I could help.
Best,
Michal
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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.
Thanking you.
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Most journals (and funding sources) require you to make sequence data publicly available when you publish in a peer-reviewed scientific journal. Part of the collaborative nature of science, adding to the known data collection, etc. It's unprofessional to not make your data available.
A thesis isn't technically a peer-reviewed publication so you don't have to publish your sequences. But you can't cite your unpublished thesis.
That's what's normal for my field of research. Other fields might have different conventions.
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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.
Thanks,
Emanuele
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Hi Nikolaus Szucsich , thanks a lot! I'll try to use it!
All the best to you,
Emanuele
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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.
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Yes. It looks like they have a great deal invested in mapping the taxonomy and control the release of mapping results by utilizing a close-ended API. But since their BOLDYSTEMS map results for each taxa are in geographic space, you can screen capture them and add them individually as image overlays in Google Earth. Otherwise contact them to gain access to their data through a collaborative effort.
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Please help me to understand this "chemotype identification and DNA barcoding of medicinal plants" what thats mean
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Hi there Abhishek!
DNA barcoding is a technique in Molecular Biology that allows you to identify a plant species (hopefully, but sometimes genus only) by simply isolating a small fragment of their DNA. As you may know, different genes are subject to a different mutation rates which means that different part of the genome (or different genes) may differ a lot or not at all between different species. By targeting some of the most variable gene region, you can have a sequence of DNA that is always different between different species but still the same between individuals of the same species. For insects, for example, the most used DNA barcode region is COI, and there are entire database storing this gene region allowing you to compare your sequences with those already indexed. For plants, ITS is used quite a lot.
Chemotype identification is a similar process but based on the most abundant chemical produced by the plant. Different plant can produce different chemical, despite having morphologically similar phenotypes.
In short, both DNA barcoding and Chemotype identification are two techniques that may allow you to distinguish different species of plant (or animals, or fungi) even when you would not notice morphological variation between them because they look extremely similar (cryptic variation).
Hope this helps!
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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.
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is one of these samples a no template control sample?
These smears may be contamination of the reaction mix with previously amplified material. This will then amplify very well and become more concentrated than the primers and will then join with each other to form randomly long concatomers of amplified product. Can we see apicture of the amplimer with at least one sample being water not dna in the reaction mix please?
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The species has been reported from other areas of the world. i have identified the species through molecular taxonomy; DNA Barcoding. I dont have much information about the morphology.
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In case you have any zooplankton species, we shall be glad to provide possible help in view of our expertise particularly on Rotifera and Cladocera.
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Hello Everyone. I am working on DNA barcoding of Himalayan Plants. To address my objectives I have used sanger sequencing with few target genes (mat K, Trnf., petA etc.).I would like to know that how I can authenticate my data with NGS platform, if possible. Which platform will be the best for this purpose and what will be the cost per sample. Thanks in advance and with regards
Arun
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Illumina would be a best and cost effective solution. But you must look into the size of your amplicon since it is limited to the max 300 bp size. With the choice of right strategy, you can multiplex upto several hundred samples.
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Sequence similarity values for DNA Barcode comparisons differ between BOLDsystem and BLAST. How is similarity calculated on BOLD? A point to a reference would be appreciated.
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I believe they use a 2-parameter kimura model to establish distances for neighbor-joining trees. PAUP* will do this. See http://paup.phylosolutions.com/
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Hello,
I am starting a new project with DNA barcoding and I am not sure how to codify sequence DNA. In this project we are using the following molecular markers: rps16-trnk, rpl32-trnl and trnl-trnf (chloroplast).
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When I asked this question maybe I was not so clear. I was referring to the correct way of dealing with the DNA sequence to identify barcode. For example, how to deal with insertions, deletions, duplicated regions and microsatellites.
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I just got a result from microgen that unknown insect species(probably some kind of gall midge) using mitochondria COI region after DNA sequencing, alignment and blasting.
It was recognized that unknown sequence is 85% similar to Rhopalomyia sp.
(BOLD:ACC5938 voucher BIOUG06875-F04 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial)
I don't know whether I can identify it as a Rhopalomyia sp.
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I think here we should not confuse the BLAST sequence identity percentage and similarity/genetic distances used in barcoding. They have completely different purposes and computation behind. Therefore I recommended uploading the barcode to the ID service of BOLD and look at the tree based visualisation as well. Of course its purpose is species identification, but for orientation to see what sequences are most close to the queried one is enough.
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Sequence Submission, BOLD, DNA Barcoding
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BOLD accepts sequences from more than 150 genetic markers including the 4 main DNA barcoding markers: COI-5P, ITS, matK, and rbcL as well as dozens of others. The following link describes procedures for producing high quality barcodes using BOLD. Building the DNA barcode library requires careful validation of both the DNA sequence and specimen data.
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I have been attempting to extract DNA for barcoding on various macrobenthos and have at present been unsuccessful. I can only make use of one/two legs per specimen e.g of an amphipod. I am using the CO1 gene. I have tried the Qiagen blood and tissue kit, chelex method and the zymo research tissue and insect DNA microprep.
Are there any other kits/methods which would be useful to try?
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Hi,
Maybe you want to try direct PCR without extraction. Check this paper,
Good luck.
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Universal ITS primer for DNA barcode in animals
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Thank you, your suggestion will be helpful for me....actually i want these ITS primers, COI and COX primer for DNA bar-coding of some Indian species tortoises.
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Hi,
I have used RDP classifier in my pipeline to assign 16S sequence to their corresponding taxonomy.
I'd like to know if a similar function would exist to assign CO1 (cytochrome oxydase 1) sequences to their corresponding taxonomy.
Another way to solve my problem would be to download a database containing only CO1 sequence files (wether from BOLD or Genbank) and use the Usearch function but I was unable to find such a database yet.
many thanx for your help!
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For anyone still interested in this question, you could also try the Metaxa2 classifier. A recent paper describes new software for training Metaxa2 on any genetic marker (https://doi.org/10.1093/bioinformatics/bty482). We recently used this software to create an arthropod COI database (https://peerj.com/articles/5126/).
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Taxonomists,
What do you think about alternative systems of identification of species? Is DNA barcoding going to replace Linnaean binomial nomenclature? What are the advantages of a numeric system? Nomenclature is the topic of my dissertation and subject of my further research so I am interested in your opinions.
Tanya Kelley
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Dear Kelly
Even if we get universal marker one day, DNA barcoding will still remain a tool to aid Linnaean binomial nomenclature or Alpha Taxonomy. Dont get offended if I say, DNA barcode is just like the commercial barcode on the back of a toothpaste packet or tube, but it can't replace the name of the toothpaste on the front of the packet or tube.
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DNA barcoding of species has been a success, and millions of sequences are already submitted to Genbank and Boldsystems. Several genuses are difficult to identify only based on morphology, e.g. Henricia and Leptasterias (within Asteroidea, Echinodermata), and DNA barcoding is a useful tool to distinguish different lineages. Is it not about time to take advantage of that in forecoming taxonomic work?
I still observe several published Scientific articles on difficult genuses, also With descriptions of New species, without any form of DNA analyses. At least when live, or ethanol preserved specimens are available, DNA barcoding should be conducted. Perhaps journals should go in front...
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In plants (NO comments on other groups) what is even worst to me is to see young systematics researchers which are very good on phylogenetics but without any clue about traditional taxonomy. Basically, they don't know in detail the group which they study and they think that using only phylogenetics they will be able to separate all the species.
Without question, in the future phylogenetics will make great advances which are beyond of our expectations, but not exactly today.
Maybe for some groups could be compulsory, but to request that barcoding must be COMPULSORY for all the groups, considering the current information gaps for some groups, is not reasonable
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Hi,
I have one question. Before that let me explain my current work. Basically I am working on DNA Barcoding of sharks. I have one species of shark namely Cephaloscyliium sarawakensis. When I’ve done the sequencing and blast from the genbank I get result other species which is Cephaloscyllium umbratile. Both blast result either genbank and from BOLD gave me the same result. Maybe this is due to no record on DNA barcoding or other gene sequences/whole genome from C sarawakensis. That is why the blast result gave me the possible or nearest species. Even when I aligned my sequence with C umbratile, there was a few of nucleotide that different.
So my next plan to design a primer for C sarawakensis. Now my question is, since I don’t have any reference/whole genome from C sarawakensis, can I use the nearest species (in this case C umbratile) for my primer design?
This study is very important for me because there is a paper on the difficulties in identifying these genus. And maybe this is one of the reason mis-identification happened to C umbratile and somehow the result from the genbank/BOLD lead to different species and unfortunately no photos available for C. umbratile from BOLD or even paper cited in the genbank.
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For designing a primer the species should not make any difference as long as you have the consensus sequence for the primer to attach to. If SARAWAKENSIS and UMBRATILE share sequence similarity at the primer sequence, it should solve your purpose.
Another solution is to clone part of the sarawakensis genome into a vector. Use a common vector primer to sequence it and once you know the sequence of the gene, use tht to design your primer.
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Hi,
Currently I am working on DNA Barcoding of sharks and rays. I had used same default primer for DNA barcoding for same species for sequencing batch by batch. What I am confuse now is why when I used the same PCR protocols for the same species but it gave me different result after sequencing? For example:
1. For first batch the sequence result gave me the good, clear chromatogram and when I blast the result gave me with what I am expecting.
2. But when I send for the second batch of samples for sequencing (sample species and same PCR protocols), it gave me completely different species result from the blast. I am confident with my species identification during sampling and even I have a fish taxonomist expert that time. Suppose the result would be same as batch 1 because I am using same PCR protocol and primer.
3. And some samples, the PCR does not working. And I also get faint or no band during gel view.
What am I suppose to do? Is it the DNA was not ok? Should I re-extract or change primer? I had tried all the primers but the result still fail. Even the sequencing company also had done the re-sequencing but the result is still same.
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Hi Noorul,
I am not an expert in this, but I have been doing some PCR and sequencing for my PhD to develop a phylogeny. If I understand your question correctly you have sent several samples from the same species (different individuals?) to an offsite lab to have the PCR and sequencing performed and you get variable results back. In my experience there are a number of things that could cause the inconsistent BLAST results. There could be contamination in your DNA extraction or in the original sample. Your samples might have gotten mixed up. The original ID could be incorrect (it happens - even to experts). If you already asked the lab to re-run the sample and it gave the same results, it seems unlikely to be an issue with the PCR conditions. You might try another DNA extraction. If the results match the first sample you sent off for sequencing (the one you got the expected BLAST result) it was probably contamination in the previous extraction. If you have a sample from another individual of the same species you could try extracting DNA from that one. If those results match your first one, then it seems like your second sample (The one with the unexpected results) probably is from another species. I can't think of any way you can really check from your end without sending away more samples.
The weak gel bands could indicate that there was not much PCR product. The primers may not be a good match for that particular species or the PCR conditions might not be optimised for that species. You could ask the lab to run a temperature gradient for your DNA sample if this hasn't already been done. Have the primers you