Illumina library quantification

[deepakpatilp]

Can you please share your experiences about quantification of final illumina library? Also which is best of the following library quantification?
1. Bioanalyzer
2. Qubit
3. qPCR

I am planning to send 200 bp to 500 bp for sequencing. How should I go about quantification of such a library?

Deepak

[aplany]

Qbit seems to be the most reliable method for quantification for us.

[HESmith]

A search of the forum will find several threads that address this topic.

[pmiguel]

Originally Posted by HESmith

A search of the forum will find several threads that address this topic.

That said, I don't think there is good answer to this oft repeated question yet.

The problem is not so much with perfect libraries -- where
(1) nearly every molecule is an amplicon (has adapters on both ends)
(2) the size distribution of the amplicons is even
(3) there are aren't small amplicons hidden amongst the large ones (by adapter-adapter annealing?)
(4) the solution is free of substances that confound quantitation.

I think most bench ninjas get to the "perfect library" point quickly and thus can't understand the travails the rest of us mere mortals have to deal with. Sad, because they are the ones who know the right path to traverse to get a good outcome almost every time. But they tend to be completely incapable of describing how they do it in a way someone without their intuition/outlook can absorb.

Anyway, I would say, read those earlier posts to get a sense of the issues you may face as a whole. Don't let the issues paralyze you, just let you knowledge of them guide you.

Each of the techniques you mention does have weaknesses:
(1) The bioanalyzer is not designed to give accurate estimations of the amount of a sample -- rather it would be better at telling you the length distribution. Also, the bioanalyzer lull you into thinking you know more than you do -- specifically it doesn't distinguish between single stranded and double stranded molecules. Further migration of single stranded molecules is slower than I would naively expect, such that ssDNA can run slower than dsDNA of the same length. Nevertheless a very useful tool.
(2) Double stranded fluorescence assays can tell you the amount of dsDNA in a solution. Some fluors, like pico green, barely fluoresce at all in the presence of ssDNA, nucleotides, oligos. Also this assay tends to be insensitive to compounds that light up other assays like a Christmas tree. (eg, phenol with a UV spec). But it still is not perfect. Ethidium bromide, for example, in a library shifted the emission spectrum of a fluor we were using (either ribo green or pico green) so dramatically, we had to purify the sample before it could be quantitated.
(3) qPCR is also impacted by EtBr. At least the type of qPCR that uses a SYBR green assay. Further, the presence of small amplicons mixed in your library (possibly, as mentioned above, complexed with larger molecules in such a way that they do not show up on gels or ds bioanalyzer assays) can cause inaccuracies in this method as well.

--
Phillip

[protist]

We use Qubit to quantify ng/uL and Bioanalyser to get correct peak size for 10 nM dilution calulations.

[biochembug]

Hi Friends

Thanks all for posting replies. Some people whom I talked here at my place suggested qPCR can be best. Anyone knows whether some company provides any standard for it? Of course I would be trying qubit and bioanalyzer.

Biochembug

[Heisman]

Originally Posted by biochembug

Hi Friends

Thanks all for posting replies. Some people whom I talked here at my place suggested qPCR can be best. Anyone knows whether some company provides any standard for it? Of course I would be trying qubit and bioanalyzer.

Biochembug

For a standard it would be best to use a library (prepared similarly) that you have already sequenced.

[ETHANol]

The biggest problem here (for me at least) is that I prepare and quantitate the library and then I send it out to a core facility to sequence. It makes it difficult to figure out what goes wrong, why, how it can be fixed/optimized. When I get poor cluster generation I don't even know if it is from too much or too little DNA. I think a lot of people are in this same situation. So we just sort of muddle along looking for something that works and nothing ever really does. Bottom line is the quantification step is a problem. All the methods work and don't work. I use qPCR but as said that has problems too.

[BIG_SNP]

We use the KAPA Biosciences kit. It comes with standards and everything you need to quant your adapted libs. We use this routinely with the bioanalyzer to QC our libraries before sequencing.

[Smriti]

I am looking to purchase one of these- Qubit or NanoDrop, which one is better in terms of accuracy and long run? Please provide your input.

[protist]

Originally Posted by Smriti

I am looking to purchase one of these- Qubit or NanoDrop, which one is better in terms of accuracy and long run? Please provide your input.

Personally I would choose the Qubit, while the Qubit will not give you a 260/280 ratio it is far more accurate for low ng quantities. Added to that it is far cheaper. We have been using ours for library and RNA input quantification for over 3 years and have had no problems.

[lterhune]

Originally Posted by Smriti

I am looking to purchase one of these- Qubit or NanoDrop, which one is better in terms of accuracy and long run? Please provide your input.

We have used both of these in our lab but almost always go with the Qubit. The Nanodrop seems to be less accurate with smaller concentrations, and can skew dramatically high or low if any contaminants are in the sample. The Qubit is also selective for dsDNA. We have had some minor problems with the Qubit (can be 10-20% off), however, it is much better than any alternative we have. Just make sure to spec your Qubit standards ahead of time, as we received a bad lot once which made our readings up to 50% high (Invitrogen replaced them for free).

[NextGenSeq]

We never use our Qubit. All our libraries are QCed by Bioanalyzer to check for adapter artifacts and quantified by QPCR.

If you have a good library and your NanoDrop is calibrated properly the QPCR and OD260 concentration should agree within 20%

[GW_OK]

Originally Posted by BIG_SNP

We use the KAPA Biosciences kit. It comes with standards and everything you need to quant your adapted libs. We use this routinely with the bioanalyzer to QC our libraries before sequencing.

Same thing in my lab. It's the best way to go, I've found.

[ETHANol]

With regards to Qubit you have to be careful as a significant portion of your library my not be double stranded DNA. As primers become limiting in the PCR enrichment step bubbles and daisy chains of molecules form as the adapters anneal on fragments with non-complementary inserts.

So if you use Qubit it is extremely important to not do too many cycles of PCR enrichment. Given the exponential nature of PCR this is easier said then done.

[Smriti]

Thanks everyone for your input!
Right now I am looking for a preliminary estimation on % ds DNA recovery from my experiments. So, Qubit looks best to me after reading posts on this thread.

[pmiguel]

A word of caution about qPCR -- don't forget there are two types. Most commonly one uses the SYBR green, a dye the fluoresces in the presence of dsDNA. But there is also "Taqman" style qPCR, that is probe based. If you know the average size of your amplicons, the former should work fine modulo minor (hopefully) differences in your amplification yield due to sequence composition biases. TaqMan should be insensitive to the size of amplicon.

The specific issue that throws off SYBR green style qPCR is the presence of small amplicons (usually primer dimer or adapter dimers) in a library. If you have done a size selection on your library, you might be fooled into thinking you have no small amplicon contamination. However if you size-selected on dsDNA, this may not be the case. Small amplicons can anneal to the ends of larger ones, effectively "hiding" from size selection. You need to QC you library as ssDNA to detect this. Fortunately this issue is rare for libraries constructed with the TruSeq kits.

If you do get the right titer, and the small amplicons do not predominate, then your results will be okay. Some of your sequence reads will be wasted on primer-dimers. But not that big a deal. If you undershoot on your titer, then you are potentially in big trouble. First, this can result in overclustering. Second, under conditions of over clustering Illumina's infamous susceptibility to low-diversity sequence in the first 4 bases of amplicons can cause major issues. This is especially confusing because it can result in low cluster densities--even though the lane is packed with clusters.

--
Phillip

[FredericRaymond]

Hello,

I have a related question. In the illumina Nextera protocols, they suggest to use the HS DNA bionalyzer chips. Would it be ok to use the 7500 DNA chips for library validation? I will also quantify by picogreen and, most probably, validate libraries by PCR.

Thank you,

FR

[NextGenSeq]

The Bioanalyzer chips you use depend on the yield of your library. We find if our libraries are 10 ng/ul or less in concentration we have to use the HS chips. If they are more you can use regular DNA chips.

However, with DNA 7500 you may have a hard time resolving any adapter artifacts at 120bp. We usually use the DNA 1000 chips.

[Genquest]

Hello everyone,
I am pretty new to this forum and stumbled on this post as even I am facing problem with qPCR quantification method
1.Has anyone encountered with, a higher qPCR concentration than the estimated concentration by pico green??
2. Also if assumed that qPCR is more accurate(w.r.t pico green) as it would give conc. of ssDNA ligated with adapters,then the cluster generation should be accurate with qPCR conc. but it doesn't happen so...the flow cell (V3,Hiseq 2000) remains under clustered.Can any one think of possible reasons??
3. This trend seems to change with every library.Is absolute quantification the right approach for Sybr green qPCR method?Also I am using the PhiX control provided by Illumina as my standard.
4.Do people take the "exact concentration of libraries" as determined by qPCR for cluster generation or they compare it with pico green to see the relative trend of concentration?

Would appreciate if any of the queries are resolved!!
Thanks in advance