When I contacted Sage they replied, "Blue Pippin users who use TruSeq kits, perform size selections at 500-600bp to achieve the desired 450 peak." Yet Sage later made this sound like an approx. range (hence my post).
The Blue Pippin gels we're using (1.5% cassettes) have internal standards, and do Not have ethidium bromide. A marker is loaded with ea. sample.
Another user just tried the 500-600 bp setting (different DNA source, but again Blue Pippin / TruSeq DNA), and that shifted the peak from the prior median of ~300 to ~350 bp. It's close to good enough, but a 30 bp insert (100 bp, PE, + ~120 bp for the adapters) seems a tad short. He has a lot of undesirable products at higher sizes as well -- presumably PCR artifacts with smaller/lower quantity peaks around 1.2-1.4 kb. (We have less input DNA available and cut the reagent volumes in 1/2 , so last time I didn't have the odd bubble peaks; this time we increased from 150 ng to 300 ng and I've been wondering about cutting the # of PCR cycles...).
In talking about this the lab is concerned with potential issues/biases with the PCR (eg amplifying adapters pre-PCR) so we may see if we can just offset the Blue Pippin settings by trial and error. Given we have low input DNA to start I hesitate to go to the manual gels per Illumina's protocol, as that can (I gather) give lower yield, and do want to avoid adapters dimerizing or annealing to the product etc. I'm also not sure if we'd have to use 2X the PCR reagents if we do some cycles before and others after the size selection step; at present we're working with the TruSeq kits and have 28+ libraries so that could get expensive. Trial & error optimization to guess the apparent size of Y-ligated adapter+library DNA seems rather inefficient ... but we might be able to make it work with the 2 extra libraries I made this time for troubleshooting.
Thank you again for all of your insight! This is my first time with the DNA TruSeq kits (and the cantankerous Blue Pippin; I love the precision of the instrument ... but adapting it for use with TruSeq is frustrating).
The Blue Pippin gels we're using (1.5% cassettes) have internal standards, and do Not have ethidium bromide. A marker is loaded with ea. sample.
Another user just tried the 500-600 bp setting (different DNA source, but again Blue Pippin / TruSeq DNA), and that shifted the peak from the prior median of ~300 to ~350 bp. It's close to good enough, but a 30 bp insert (100 bp, PE, + ~120 bp for the adapters) seems a tad short. He has a lot of undesirable products at higher sizes as well -- presumably PCR artifacts with smaller/lower quantity peaks around 1.2-1.4 kb. (We have less input DNA available and cut the reagent volumes in 1/2 , so last time I didn't have the odd bubble peaks; this time we increased from 150 ng to 300 ng and I've been wondering about cutting the # of PCR cycles...).
In talking about this the lab is concerned with potential issues/biases with the PCR (eg amplifying adapters pre-PCR) so we may see if we can just offset the Blue Pippin settings by trial and error. Given we have low input DNA to start I hesitate to go to the manual gels per Illumina's protocol, as that can (I gather) give lower yield, and do want to avoid adapters dimerizing or annealing to the product etc. I'm also not sure if we'd have to use 2X the PCR reagents if we do some cycles before and others after the size selection step; at present we're working with the TruSeq kits and have 28+ libraries so that could get expensive. Trial & error optimization to guess the apparent size of Y-ligated adapter+library DNA seems rather inefficient ... but we might be able to make it work with the 2 extra libraries I made this time for troubleshooting.
Thank you again for all of your insight! This is my first time with the DNA TruSeq kits (and the cantankerous Blue Pippin; I love the precision of the instrument ... but adapting it for use with TruSeq is frustrating).
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