I am a student currently working on a project that requires the sequencing of DNA from two species of California sunflowers. For the record I am using the ddRADseq method. Although I am familiar with size selection methods, I am not familiar with how to choose the size. The general range my mentor gave me is around 300-600 base pairs. What are the advantages and disadvantages for smaller sizes and bigger sizes. Thank you!
Often if the size of the larger piece of DNA gives a wider area of study
regards
Thank you Hassan Nima . The species I am researching has a genome size of 3061 Mbp. Do you know if there are any methods for size selecting based on genome size?
Something to keep in mind is that the likelihood of having problems with paralogs during your bioinformatic pipeline increases for species that underwent genome duplication. Hence I would choose the largest fragment possible.
Matthijs P. van den Burg that is very useful information. Thank you!
Olivia Grace Durant
Reviews that would be interesting to read for your question are these, although more recent reviews might be out there as well:
https://www.nature.com/articles/nrg2626
https://www.nature.com/articles/nrg3012
Because at the first instance you do not how often the enzymes cut the genomic DNA, I would recommend that after the dd you run a gel to see the size distribution of the digested DNA. That can depend very much on the used restriction enzymes. It might be in the 300-600 bp range but might be outside. If so you select other enzymes. But when you do the gel purification, the isolated fragments are never of a discrete size, they are a population of different size fragments. Therefore you should go for the most narrow area as possible to cut out from the gel, but still you will get a range of fragments. Do not worry about that. The size selection also depends on the length of sequenced reads and calculate the size of the adapters and choose a sequencing with longer reads just to sequence your inserts too, not just the adapters. But usually the 300-600 bp is fine, we normally go for 300.
Check this out too: http://www.bit.ly/ddRAD
Good luck.
It is better to do primer walking first, an initial confirmation of sequence, then proceed for your desired length
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