Bisulfite sequencing - Does strand matter?? (Sep/21/2004 )
Sorry to ask yet another potentially stupid question, but I want to ensure I have this right before I start. My CpG island I want to sequence runs 3'-->5' (i.e. the gene is on the antisense strand). When designing primers for the PCR over this region (e.g. using MethPremer) do I need to therefore use the reverse-complement of the DNA sequence obtained from the database (which runs the other direction, 5'-->3') to ensure I am sequencing the correct strand. In other words, if I sequence the opposite strand to the one containing potentially methylated sites, does this matter - or is this phenonemon symmetrical?
Thanks in advance for any help!
DNA methylation is supposed to be symmetrical, so usually just one strand (the sense strand) is mapped. However, it you map both strands at the same time, you may find they are methylated differently. Studies found that bisulfite sequencing PCR has bias toward amplifying non-methylated molecules over methylated ones, mapping both strands may give you unbiased results.
Thanks - so in theory, sequencing the antisense strand should give the same result as sequencing the sense strand? If I went on to look at quantitative levels of methylation (e.g. via SNuPe) would assays designed on either strand give identical results? This would make assay design a lot easier!
Yes, in theory, two strands should give identical result. However if PCR is involved, result from the two may not match. I am not quite familiar with SNuPe and not sure if SNuPe has the same bias.