Hello all.
From a previous project, we have a 'NimbleGen SeqCap EZ Choice Library'-design that we would now like to use for a new project on our Ion Torrent PGM.
I am very new to next-gen sequencing, but it looks like I should be able to prepare a library (fragmented to appropriate PGM-size, PGM-adapter ligated, nick translated, size selected, amplified, and purified) according to the PGM protocols and then do the capture following NimbleGens protocol, followed by template preparation and sequencing according to the PGM protocols. The main problem seems to be the differences in the oligos used, i.e. during the NimbleGen capture 'hybridization enhancing'-oligos are added to the mixture. Can anyone please explain to me in detail what these are, how they are designed and how I might create a set corresponding to the PGM-adapters used?
Also, according to the NimbleGen protocol a ligation-mediated PCR is performed after the capture. Can't I just do a PGM-protocol amplification here and achieve the same or am I missing a point here?
Any advice would be greatly appreciated. Thanks in advance.
Best regards
Christian
From a previous project, we have a 'NimbleGen SeqCap EZ Choice Library'-design that we would now like to use for a new project on our Ion Torrent PGM.
I am very new to next-gen sequencing, but it looks like I should be able to prepare a library (fragmented to appropriate PGM-size, PGM-adapter ligated, nick translated, size selected, amplified, and purified) according to the PGM protocols and then do the capture following NimbleGens protocol, followed by template preparation and sequencing according to the PGM protocols. The main problem seems to be the differences in the oligos used, i.e. during the NimbleGen capture 'hybridization enhancing'-oligos are added to the mixture. Can anyone please explain to me in detail what these are, how they are designed and how I might create a set corresponding to the PGM-adapters used?
Also, according to the NimbleGen protocol a ligation-mediated PCR is performed after the capture. Can't I just do a PGM-protocol amplification here and achieve the same or am I missing a point here?
Any advice would be greatly appreciated. Thanks in advance.
Best regards
Christian
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