Can I do multiple rounds of SWGA and should I alternate primer sets?
Multiple rounds of SWGA will further increase the enrichment of the target genome. However they might also increase the unevenness of coverage. So you may need to test this on your samples and determine if the additional rounds of SWGA produce the desired result.
Alternating primer sets does not consistently to produce better coverage or enrichment than using the same primer set for both rounds.
Alternating primer sets does not consistently to produce better coverage or enrichment than using the same primer set for both rounds.
Should I use methylation dependent restriction enzymes to digest my host DNA prior to SWGA?
Only if necessary. We have found the digest step to be inconsistent when used prior to SWGA and currently only use methylation digest for samples with exceeding small proportions of target DNA (<0.01%).
Methylation dependent restriction enzymes have been used to digest primate host DNA prior to Plasmodium SWGA (Sundararaman, 2016). These enzymes likely fragment the methylated primate DNA into small enough pieces that much of the host DNA does not amplify at all, yielding more selectivity. However, methylation dependent digest can be problematic. First, it requires that the background DNA contain the appropriate methylation motifs. Second, methylation dependent enzymes likely digest the target DNA to some degree and prevent SWGA from working.
References
Should I digest contaminating mitochondrial DNA prior to SWGA?
Only if necessary. Mitochondrial DNA digestion was used by Leichty and Brisson prior to SWGA amplification of Wolbachia from D. melanogaster. This was done because they observed that D. melanogaster was over-amplified by the SWGA reaction. The drawback to any digestion is the fragmentation of target DNA. This will lead to a loss of amplification and subsequent sequencing coverage at and around digested positions.
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