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Old 02-03-2014, 05:08 PM   #1
mbk0asis
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Default TruSeq DNA PCR-free vs NANO DNA kit

Hi.
I am planning to purchase one of TruSeq DNA kits, 'PCR-free' or 'Nano'.
On their manual, there seemed to be no differences regarding on the contents except PCR related reagents. What I'm curious about is whether the 'PCR-free' kit has more reagents (i.e. higher concentrations or volumes) than 'Nano' since 'PCR-free' kit requires more starting materials.
If someone out there has tried both kit, please let me know about it!

Thanks!
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Old 02-24-2014, 03:55 PM   #2
zaakwalton
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Quote:
Originally Posted by mbk0asis View Post
Hi.
I am planning to purchase one of TruSeq DNA kits, 'PCR-free' or 'Nano'.
On their manual, there seemed to be no differences regarding on the contents except PCR related reagents. What I'm curious about is whether the 'PCR-free' kit has more reagents (i.e. higher concentrations or volumes) than 'Nano' since 'PCR-free' kit requires more starting materials.
If someone out there has tried both kit, please let me know about it!

Thanks!
Hi,

I am not sure with the nano kits, as they are new to me as well, but the adapters are different in the PCR-free kit compared to the standard TruSeq kit.

The standard TruSeq kit uses Y-shaped adapters that require PCR to create a library that will anneal to the flow cell--or something like that. Otherwise, the buffers should be identical, though it is possible that concentrations will differ. For your purposes, they are not interchangeable because the adapters will not work between kits. At least, I know they will not work from TruSeq without running PCR.

I have learned this the hard way.
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Old 02-25-2014, 04:23 AM   #3
SS00
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Quote:
Originally Posted by zaakwalton View Post
Hi,

I am not sure with the nano kits, as they are new to me as well, but the adapters are different in the PCR-free kit compared to the standard TruSeq kit.

The standard TruSeq kit uses Y-shaped adapters that require PCR to create a library that will anneal to the flow cell--or something like that. Otherwise, the buffers should be identical, though it is possible that concentrations will differ. For your purposes, they are not interchangeable because the adapters will not work between kits. At least, I know they will not work from TruSeq without running PCR.

I have learned this the hard way.
Zaakwalton, has this change from Y-shaped adapters been confirmed?

I myself have used the old kit & adapters PCR Free (without one cycle of PCR at the end) with no problems with clustering.

Looking at the kits, I would say the only difference between them is that one contains additional PCR reagents and one does not.
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Old 02-25-2014, 09:15 AM   #4
zaakwalton
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Originally Posted by SS00 View Post
Zaakwalton, has this change from Y-shaped adapters been confirmed?

I myself have used the old kit & adapters PCR Free (without one cycle of PCR at the end) with no problems with clustering.

Looking at the kits, I would say the only difference between them is that one contains additional PCR reagents and one does not.
I did fail with QPCR when making libraries with the TruSeq kit, and not doing PCR enrichment.

When you say "old kit," is this a kit before TruSeq, or are you referring to the TruSeq kits? the older Illumina kits and adapters should work without PCR.

But, I do know that I made the same assumption, comparing TruSeq kit with PCR vs the kit without PCR, thinking that only the PCR reagents were not included. The adapters are different; the product numbers are not identical between the two (unlike the difference between the DNA TruSeq/RNA/ChIP seq kits, where the only difference is in concentration).

At least, this is what seems to have happened to me after submitting about 4 dozen non-PCR'd libraries using the standard TruSeq kit with y-shaped adapters. After PCR, everything was fine.
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Old 03-04-2014, 12:44 AM   #5
SS00
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That's interesting. I used the old Truseq Kits, including v2, (not old PE kits) without any cycles of PCR using 500ng DNA input and regularly got qPCR values of over 2nM and regular cluster numbers. That was some time ago though, I recently did the same, but with cDNA and will sequence them soon and let you know as I am a bit worried now!

I am quite interested as to the difference of the adapters as I thought the Y shaped adapters were specifically to allow different sequences at the 5' and 3' end of the libraries, thereby allow Paired End Sequencing with P5/P7 annealing to the flowcell. I wonder how Illumina would achieve this without Y shaped adapters, or did they just eliminate the need for unique ends?

Quote:
Originally Posted by zaakwalton View Post
I did fail with QPCR when making libraries with the TruSeq kit, and not doing PCR enrichment.

When you say "old kit," is this a kit before TruSeq, or are you referring to the TruSeq kits? the older Illumina kits and adapters should work without PCR.

But, I do know that I made the same assumption, comparing TruSeq kit with PCR vs the kit without PCR, thinking that only the PCR reagents were not included. The adapters are different; the product numbers are not identical between the two (unlike the difference between the DNA TruSeq/RNA/ChIP seq kits, where the only difference is in concentration).

At least, this is what seems to have happened to me after submitting about 4 dozen non-PCR'd libraries using the standard TruSeq kit with y-shaped adapters. After PCR, everything was fine.
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Old 03-05-2014, 02:26 AM   #6
lorendarith
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My experiences are the same as SS00 describes.

The adapters from the Illumina TruSeq DNA (FC-121-2001, FC-121-2002; discontinued now and replaced with TruSeq Nano DNA FC-121-4001, FC-121-4002) and TruSeq DNA PCR-Free kit (FC-121-3003) are the same or at least similar.

Before they discontinued the kit I regularly used the TruSeq DNA adapters in conjunction with other library prep kits to make Illumina PCR-free libraries and it always worked. I initially synthesized my own Illumina-like adapters (also Y-shaped), but the originals from the kit performed simply better because who knows what all sorts of modifications it has that we don't know about.

The only difference the adapters from TruSeq DNA (discontinued) and TruSeq DNA PCR-Free might have, could be that the PCR-free ones might have some additional modifications for even better adapter ligation.
To test if one yields better ligation than the other I tested both of the adapters from the two different kits and found no difference in ligation efficiency whatsoever.

I think that you could even use the adapters from the new TruSeq Nano DNA kit, but the obvious difference might be that these adapter stocks have a lower concentration than the older kit the since the input requirements are changed - from 1µg to 100ng (or 200ng), and the adapter:fragment ratio has remain the same.

Y-shaped adapters work fine without PCR, because before clustering on the flow cell the strands get denatured anyhow. Illumina recommends that if you have problems with clustering you could try doing one PCR-cycle, so that the strands are denatured and renatured once properly.

Hope this helps!

Last edited by lorendarith; 03-05-2014 at 02:29 AM.
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Old 05-15-2014, 09:59 PM   #7
Jeremy
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The TruSeq DNA PCR-Free kit claims to have "Superior genomic coverage with radically reduced library bias and gaps" compared to the original TruSeq DNA kits. I have been recommended to do multiple library preps (5 to be specific) for sequencing a whole genome (2.3 Gb), but this is most likely based on results of the original TruSeq DNA kits. Do the new TruSeq DNA PCR-Free kits remove the need to sequence multiple libraries to get good coverage or is it still recommended to do more than one?
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Old 05-16-2014, 01:40 AM   #8
nucacidhunter
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Discontinued TruSeq DNA sample prep kit was shown to have GC bias and it was tracked to polymerase used in amplification step. PCR free kit alleviates any bias caused by PCR and also PCR duplicates, so results in more even coverage across the genome. Nano kit also claims has better coverage than old DNA kit. I have not seen any comparison of coverage or other metrics between Nano and PCR-free libraries either in publication or Illumina application notes. Maybe someone can update this thread if there is data.

To obtain 50x coverage of a species with 2.3 Gb genome by 2x100 cycle sequencing, 575,000,000 fragments need to be sequenced. Average output of a lane of HiSeq is 180 M reads (fragments). If my math is right 3.2 lanes (4 to be sure) of sequencing would be required. So the number of library preps should be calculated based on the yield of PCR-free kit. If the library yield is sufficient for QC and input into 4 lanes, one library should be suffice.

Last edited by nucacidhunter; 05-16-2014 at 02:00 AM.
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Old 05-17-2014, 08:15 AM   #9
FWOS
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Hi,

Just wanted to note that the Illumina nano and per free adapters are both Y adapters. The Y is created by improper base pairing at the 3' and 5' ends of the adapter, but that goes away once the sample is amplified. Might seem obvious retrospectively, but worth noting.

I have run both kits many times and would only recommend the Nano kit if you have low input. The kits are almost identical except for PCR obviously. The main reason for better coverage and less bias in the PCR-free kit is simply the lack of PCR bias. So you won't have any PCR duplicates or PCR errors. It's not clear how much bridge amplification can muddy the waters, but we have found the PCR Free kit to be fantastic. If dealing with lower input I would recommend trying the Kapa Hyper kit. The Kapa Hyper kit can be stopped post ligation and qPCR'd to determine whether or not PCR is necessary (same can be done if using a nano kit).

Hope this helps!
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