I am doing RNAseq and would like to pool my library with some other lab members. The problem is we are not using the same library preparation kit (and thus the index). I would like to know whether there are any incompatible concerns?
I check the index for these kits, I would like to know what should I look for? Here is my checklist:
- check whether there are any identical indexes (which I use the index given in both user manual)
- check whether the indexes are identical after trimming off 1 base from either end
My question is do I also need to check the complement version of these indexes? And also the reverse and reverse complement?
One of my kits provides a unique Dual-indexed adapter: P7 index sequence, P5 index sequences (1 and 2) ~~ this is from the KAPA kit. On the other hand, my lab member is using the NuGEN Ovation® SoLo RNAseq kit, which only provides one index sequence (of each well) for me.
May I know which sequence I should make the comparison to?
The final question is I found that one of the "reversed" indexes from the SoLo kit is identical to one of the indexes I used after trimming one 5' base.
SoLo original index: ACCATCCT SoLo reversed index: TCCTACCA SoLo reversed and trimmed: CCTACCA #overlap found with this to next index sequence KAPA P7 Index sequence: CCTACCAT
I know that the SoLo kit claim they will have 8 random bases following the provided index. Do you think if there is a random T following the
CCTACCA will cause a problem? Or the demultiplexing algorithm can deal with the issue by knowing that the index is actually
TCCTACCA T rather than
T CCTACCAT. Of course, this is not a problem if I do not need to compare the index with its reverse sequencing in the counterpart.