Sorry my English!

Question and answer:

1- What is the percentage of acceptable similarity that might suggest

a new body?

The metrics provided by the blast are not recommended to infer species (s) new (s)

it just shows you the similarity of sequence with pre-deposited sequences.

2- Is there any special conditions for fungal nucleotide sequences

as against bacterial?

This question can not answer, because I work with mosquitoes.

3- What other software can use to establish the phylogeny given some concerns

raised by some authorities about MEGA?

I suggest you do a Bayesian inference, there are several programs such as: MrBayes and the Beast.

These are often used for this type of analysis. later also will investigate

delimiting species (consisting of discovery and validation) the ones I use are: R, TaxonDNA, R8 Geneious ...

Hello Ajijolakewu,

As Jose pointed out, using BLAST percent identity scores is not recommended since it is just a basic local alignment (thus the BLA in BLAST). This means that BLAST percent identity scores do not necessarily equal the percent sequence similarity, especially as the sequence get more divergent and become harder to align.

Fungus and bacterial assumptions are also going to be some what different given the different modes of reproduction, generation times, and if you are looking at haploid verse diploid sequences.

As far as a set percent similarity cut off for species, this is going to change drastically depending on the region of the genome you are looking at. For example, the percent dissimilarity in a mitochondrial gene like cytb is going to be much higher than that for a conservative coding nuclear exon. 

I also attached two papers on "barcoding" and species delimitation that are a good read if you are doing something like this. 

You probably still have a lot of questions, but hopefully this will help some. If you have more specific questions please post them.

Best Regards

Dear colleague, 

As Dr. Stadler already pointed out, the ITS region does not work well for identification on all groups of fungi, and often you need additional loci and morphology/physiology for satisfactory results. 

One VERY important thing to take into account is that for thousands of known fungal species there are no DNA sequences available in GenBank. So if you find a fungus that does not have any close match in GenBank, check very well the morphology and compare it with the literature before proposing a new taxon. It could be one of those species. 

Success with your research!

Hugo

 Thank you all. 

I have benefited immensely from your wealth of knowledge and expertise.

Articles attached are also of great help.

May I add that I only discovered today that I am yet to say Thank you.

Please accept my apologies!

Meanwhile, @ Dr Stadler and Dr Madrid, could it be said that morphological identifications should hence be given precedence over identifications using ITS regions?

Mr Ajijolakewu, I6S,18S BLASTs and others have limitations as noted by other contributors! Some authors opine that less than 98% in the BLAST should be re-examined for novelty. Attention is nevertheless shifting to whole genome sequencing. 

Guys, am a bit late for this answer. I just saw this post while searching for some information. I reckon sometimes some common sense is important. With too much reliance on DNA sequence data, we can be neglecting important characters (morph). For instance, even if one of my species is somewhat similar in terms of DNA seq to others in GB, i can still go ahead with giving a taxon novel species status if the morphs and other aspects are remarkably different. I attached a paper here which discusses some of these aspects:

@all Is any work on vegetative compatibility of fungi? i want to discuss them?

Hi Ajijolakewu

You can read this reference = Similarity Searches on Sequence Databases

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=16&cad=rja&uact=8&ved=2ahUKEwjZgP-iwezgAhUMKewKHRQkC08QFjAPegQIAxAC&url=https%3A%2F%2Fembnet.vital-it.ch%2FCoursEMBnet%2FPages06%2Fslides%2FBLAST_FASTA.pdf&usg=AOvVaw0Q9ShyWBYi9OVbKoFdiL4D

Ref.2 = BLAST Glossary

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=2ahUKEwjZgP-iwezgAhUMKewKHRQkC08QFjAOegQIBxAB&url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fbooks%2FNBK62051%2F&usg=AOvVaw2SPjGRz5ZPcZR9n1QVc_Rv

Ref.3 = An Introduction to Sequence Similarity

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=14&cad=rja&uact=8&ved=2ahUKEwjZgP-iwezgAhUMKewKHRQkC08QFjANegQIARAB&url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC3820096%2F&usg=AOvVaw2EL9QkgdTAfmuTnC0H_RCB

= BLAST: Identity % and Similarity %

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=13&cad=rja&uact=8&ved=2ahUKEwjZgP-iwezgAhUMKewKHRQkC08QFjAMegQICBAB&url=https%3A%2F%2Fwww.biostars.org%2Fp%2F195017%2F&usg=AOvVaw1r5uMvpPKvSxZyEt9eALll

Also = Protein Similarity Score

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=17&cad=rja&uact=8&ved=2ahUKEwjZgP-iwezgAhUMKewKHRQkC08QFjAQegQIAhAC&url=http%3A%2F%2Fdigitalcommons.law.scu.edu%2Fcgi%2Fviewcontent.cgi%3Farticle%3D1371%26context%3Dchtlj&usg=AOvVaw3TlqgX0IcPSbBAEaoGcyGe

Ref.4 = A brief tutorial on BLAST

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=18&cad=rja&uact=8&ved=2ahUKEwjZgP-iwezgAhUMKewKHRQkC08QFjARegQICRAC&url=https%3A%2F%2Fnature.berkeley.edu%2Fgarbelotto%2Fdownloads%2FBLASTtutorial.doc&usg=AOvVaw1jx77M04BaZq99ypR6dUvy

See this = BIOINFORMATICS TUTORIAL

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=19&ved=2ahUKEwjZgP-iwezgAhUMKewKHRQkC08QFjASegQIBBAC&url=http%3A%2F%2Fabacus.bates.edu%2F~ganderso%2Fbiology%2Fbio242%2FBioinformatics_Tutorial_2016.pdf&usg=AOvVaw0BdKhUvzqGXjghggz3Cnd3

See this = Sequence comparison (of DNA or Protein data)

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=20&cad=rja&uact=8&ved=2ahUKEwjZgP-iwezgAhUMKewKHRQkC08QFjATegQIABAC&url=http%3A%2F%2Fhpc.ilri.cgiar.org%2Fbeca%2Ftraining%2Filri_addis%2FBlast.pdf&usg=AOvVaw0eH35HyPnv0KQ6t-cQk-r1

Check = similarities between biological sequences

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=11&ved=2ahUKEwjVlYG6w-zgAhUJyaQKHUPFBdc4ChAWMAB6BAgKEAE&url=https%3A%2F%2Fgenomebiology.biomedcentral.com%2Farticles%2F10.1186%2Fgb-2001-2-10-reviews2002&usg=AOvVaw2Y8U9B6m9n8EaxJBeoHz6r

Read = statistical significance in biological sequence comparison

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=13&cad=rja&uact=8&ved=2ahUKEwjVlYG6w-zgAhUJyaQKHUPFBdc4ChAWMAJ6BAgIEAI&url=http%3A%2F%2Ffaculty.virginia.edu%2Fwrpearson%2Fpapers%2Fstat_gen_00.pdf&usg=AOvVaw1qCGHNbI1XlfrKe7I-XgG4

Used ITS and LSU (LROR & LR5) region for DNA sequence..

Then performed combine genes phylogenetic analysis using PAUP or Raxml software to confirm new species or not.

BLAST percentage similarity is depend on your sequence quality, so on the basis of BLAST result you can not conclude new species.

There is no set percentage of genetic difference to say when something becomes a different species. It could be as little as one base pair, if that is something that's not polymorphic and is absolutely consistent between populations. But, typically, it's going to be defined in terms of convergent lines of evidence, like consistent morphogological, ecological, or biogeographical differences that correspond with a consistent sequence difference.

I once asked my advisor the same question, and he simply answered "One must make a taxonomic descision". (That seemed to be his favorite answer in a lot of contexts :-)

Keep in mind as well, the percent difference between species in fungi is much greater than it is with animals. Take Saccharomyces cerevisiae vs. Saccharomyces eubayanus two similar species that are capable of hybridization, and in fact, it's the hybrid of these that's used to make lager beer. The percent base-pair difference between these two would be as great as that between mammals and birds!

I also have similiar problem with my BLAST results. I used CO1 gene from Glossoscolecidae. The BLAST result is, my query has a high query cover (93-95%) and 0.0 E-value, but it has low percent identity (82-87%). Could it be a novel species ? Fyi, the morphological identification also said that it is a novel species...

please help me, for the following sequence : GCAAAAGCTCAAATTTGAAATCCCCCGGGAATTGTAATTTGAAGAGATTTGGGTCCGGCCGGCRGGGGTTAAGTCCACTGGAAAGTGGCGCCACAGAGGGTGACAGCCCCGTGAACCCCYTYAAMGCMCTCATCCCAGATCTCCAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATACCGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGCAAGCAGACACTTAACTGGGCCAGCATCGGGGCGGCGGGRARCAAAACCACCGGGGAATGTACCTTTCGAGGATTATAACCCCGGYCYYWAYTYCCWYRYYRCCCCGAGGCCTGCAATCTAAGGATGCTGGCGTAATGGTKGCARGTCGCCCGTCTTS

I'm using BLAST to find the identity, the results come from lower identity , why?

actual problem is sorting of the BLAST sequence according to percentage identity but automatically it goes by E value. Please help me !

I suggest to read this link:

https://openwetware.org/wiki/Wikiomics:Percentage_identity

The subject is broad to address because it depends on several factors to determine an acceptable or ideal percentage or percentage range, I list the reasons:

It depends on the marker used. It can be a highly conserved gene or a gene with a medium/high rate of evolution. In some organisms some genes are so conserved that 99% identity may suggest that it is another species or even a genus; other genes are not so conserved that 95% identity suggests that it is the same species and that 5% difference is only NIS that do not affect the activity of the enzyme that is encoded or the non-coding region.

The size of the marker that was used is of great importance. For example, let's assume that they used the gene encoding Beta-galactosidase, which is about 2000 nt in size, however, the oligos they used only amplify a 500 base pair fragment, which means that only 1/4 of the entire gene is known and analyzed. The questions that would arise at this point are: How variable or conserved is this amplified region? Does it contain SNPs, with respect to the reported sequences? It would not be the same 99% identity in a 500 nt sequence as in a 2000 nt sequence.

The amount of sequences that are deposited in the databases should be considered, since the more sequences there are, the more robust and reliable the study is with bioinformatics analysis.

It should be remembered that in order to compare one organism with another, alignment only provides 50% of the information; the other 50% is supported by phylogeny. Phylogeny considers the size of the marker, the type of marker, the SNPs and the species phylogenetically most related to the study sample.

To be able to say that it is the organism in question, it is not enough to use a single marker, the ideal is to do massive sequencing or at least a multigenic study of at least the use of three or four markers and perform concatenated and non-concatenated phylogeny.

On the other hand, I can tell you that the organism studied also has a great influence. For example, in viruses and in certain genera, it is necessary to have more than 99% of identity to be the virus in question. In fungi and considering the marker, region and size, 94 % suggests that it is the fungus in question.