Muhammad Numan
1. For an accurate characterization/ identification of species of species more than single markers should be used...like 16s, 18s, 12s, cox1, ITs etc.
2. It depends on the size of each marker, you used.
For identification of OTUs in recent understanding ( note that adding new data could change them) - see barcoding. For identification of species - see taxonomy. OTUs are a source of additional information for the latter.
For different locus, size varies like ITS 400 and LSU 700 bases minimum required for the good identification. If you have more bases then it is good
The length to be read depends on the number of variable positions, the latter is defined by the number of OTUs. If you have N OTU to put on the tree, the theoretic minimal number of variable informative (!) sites is N-1 to have a trace of hope for obtaining a fully resoled tree. Otherwise you will depend on the (possibly wrong) coefficients of your model of molecular evolution. Safe approach to at least triple that number. But if your herd of variable sites is crowded on a short stretch of DNA, your alignment and model are doubtful. It is safe to have less than ca. 15% of variable positions.
Depends on the location (gene) of nucleotides. Some genes are conserved in even the same kingdom so we cannot use them to differentiate two species. Other regions are highly variable, so much so that they vary within a species (for example SSR markers). What you want is a locus that is variable enough to differentiate between two species. Depending on the kingdom, there are some universal markers to do that. For example, fungi are usually identified through ITS whereas animals are identified through a gene called COI. Of course, there are exceptions to these rules.
Within these regions for example, there are rules that can help differentiate between species. For example, more than 3% diversity in ITS indicates a new species.
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