In a typical ddRad protocol we bring samples to a similar concentration before multiplexing and sequencing. However, I was wondering if two different genomes from 2 species (e.g. c-value of 1.8 and 6.1) even at same concentration (e.g.10ng/ul), will result in more base pairs from one species than the other at a 1ul ? I will have less copies from the bigger genome and more copies of the smaller? Could I expect that the bigger genome will have a larger coverage than the smaller one on a next-gen plataform?
I guess the opposite. The larger genome (with fever copies per ng) additionally should have more restriction sites than the smaller one and for this two reasons a lover coverage per site/fragment. You will recieve a marked ammount of fragments from the larger one that are not comparable because they are simply not present in the smaller one.
Hi
This may also be affected by the sequence composition of the genomes to a certain extent
Hello Amanda,
If you assume the same percentage of restriction sites in each genome (fair assumption if the gc content is about the same) then you should generate more loci in the larger genome species with a concomitant decrease in reads per loci (low coverage) and no difference in the number of raw reads between the species. To adjust for this I would suggest reducing the size selection for the larger genome species.
In the lab I was in, which did ddRAD for a range of species (c-value range from ~ 1 to 4) we would first start with a test digest, either in silco (if we had a genome) or in vitro (i.e. run a digest on a TapeStation and estimate from the distribution of the fragments) to estimate the number of loci captured in a given size selection. These estimates can be crude and sometimes off but gives us a starting point. We would then do a test library for each species or genus and multiplex multiple species on a single lane of sequencing. We would then adjust our size selection and number of individual per lane to get the desired number of loci and coverage we wanted.
Hope this helps,
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