Help with TruSeq gDNA library prep

[BTS]

We are currently working through the TruSeq protocol to prep gDNA for hybridization to Nimblegen probes and PE sequencing on a GAIIx... Our problem is that we attempted using Invitrogen's eGels for size selection instead of the recommended gel excision (we have the reagents in-hosue). The bands on the gel were very weak and looked to be out of the size range. I spoke with someone at Illumina about the issue and they claimed to have the same problems when using egels. Has anyone had any success/experience with TruSeq and egels?

Also, we would prefer to use Qiagen cleanup instead of bead cleanup. Has anyone attempted this with TruSeq?

Thanks for any information!

BTS

[upenn_ngs]

We have used TruSeq adapters and E-Gel size-selection, the input gDNA is 1ug (vs. 3ug for the previous protocol), so the gel band will look less intense. Additionally, the y-adapters for TruSeq are much greater in length (60bp v. 30bp), which impact the speed fragments move through the gel. The Y-adapters and concentration differences between the sample and a ladder make it very difficult to size without running a biochip.

That said, we have had success amplifying the products isolated from the E-gel, and in general less PCR cycles were needed compared with the standard adapters followed by asymmetric PCR.

[John12824]

BTS -

We orginally had the same problem with the TruSeq gDNA sample prep kit. As upenn_ngs said the large adapters significantly affect the migration of the fragments. If you follow the protocol exactly and run the library on a 2% gel prestained with SyBr gold at 120V for 2 hours the library will run near true-to-size. Not very conducive to high-throughput operations but will work. Illumina is supposedly working on changing the size-selection step.

Rather than waiting we developed our own SPRI bead protocol. Gel cut samples captured with both Nimblegen and Illumina exome probe libraries are on HiSeq now. SPRI bead samples are in the queue.

[BTS]

Thank you both for the information. Since this is a first attempt with the kit and we want to make sure that everything is done right we are going to follow the recommendations to use SyBr gold and 2% gel...

Please keep us updated on the SPRI protocol!

Thanks again.

[edawad]

upenn_ngs: can you elaborate on your experience using the E-gel with the Truseq adapters? How much was the migration affected by using truseq vs old illumina/homemade adapters? Do you do a 2nd E-gel after PCR? thanks!

[upenn_ngs]

Originally Posted by edawad

upenn_ngs: can you elaborate on your experience using the E-gel with the Truseq adapters? How much was the migration affected by using truseq vs old illumina/homemade adapters? Do you do a 2nd E-gel after PCR? thanks!

We are preparing samples for exome enrichment followed by 2x100bp reads. We collect TruSeq libraries after 18:10 on the E-gel, and amplification typically yields a mean size ~380bp (+/- 20bp, s.d. from peak ~30bp). I like the TruSeq adapters because the barcode is introduced during ligation, and the input gDNA can be lowered to 1ug. Therefore, we can load one sample/well on the E-gel collect fractions from 6 libraries in one run.

It is also important to remember that the size you select for the TruSeq libraries is that of full prepped library, whereas the y-adapter method contained only 60bp of the total 120bp adapter sequence.

[BTS]

Do you collect multiple times in a single E-gel run? We have tried collected at three different points (collect, run gel 30 seconds, collect...). Also, where does your collection point for 380 bp insert coincide with the ladder?

[upenn_ngs]

You can certainly collect multiple fractions. We did this before finding a run time that gives a reliable size range, though there is still a degree of variability between each sample and each gel. I contribute this mostly to quirks of the base module; in general, fragments on the right side of the gel (closer to the electrodes) seem to run faster. Also, running multiple gels back to back on the same base module will increase the speed fragments travel, as if the instrument is "warmed up."

We determined the run time empirically. 1ug/well is largely within the 100-500bp range it will travel differently than 1ug of discretely sized ladder, in addition, the ladder well is much smaller than the sample well. We have not found a loading concentration for the ladder that gives reliable information. That said, the larger fragment sizes (>350bp) tend to size more evenly with the ladder than smaller fragments.

[buckles]

Has anyone been using nebulized genomic DNA as input into the illumina truseq protocol? Illumina's protocol calls for 1 ug of covaris sheared DNA unfortunately we don't have a Covaris. If you have used nebulized DNA in the truseq protocol, what was your input amount after shearing? Do you cut the nebulized DNA from gel before starting the truseq protocol?

the egels have a much lower capacity than traditional gels. ~ 1ug if i recall. Definitely rinse the well once or twice. Clean up after egel selection if you are using size select. and check the expiration dates, it's pretty critical in precast gels.

[vmarshal]

Dear TruSeq'ers,

My first post.....

We are using for the first time TruSeq DNA prep. with Illlumna exome capture. Used recommended agarose gel size select with SYBR Gold, all good at this stage.
However, when we moved on to exome capture and sneaked a peak at the final product on the BioAn, we have a peak in the right size range (aroynd 370bp), but also (~ 60-70% of the total library) a large smear extended from ~ 400bp up to high MW.

Has anyone else seen this? Concerned this fraction won't generate clusters (to big) and therefore what we can get clusters/sequence from, will not be a good representation of the captured exome.

Also, John et al; can you update on how your Illumina exome capture run went?
We're about to run our products (nevertheless) on the HiSeq (next week) and will update this thread on what we find.

Many thanks for any info!

[John12824]

Hi Vmarshal,

We often, but not always, saw a high MW smear/bioanalyzer peak following the PCR enrichment step but never after capture. Tried to resolve the issue with Illumina but they were unable to help. Our only solution at the time was to run the library on a gel and cut-out the appropriately sized fragments.

As for the exome sequencing results, coverage levels were consistently below what we're accustomed to and we stopped trying to optimize the product. However, it's unclear if the decreased coverage was due to issues with library prep (likely) or the capture itself.

Worried the complexity of your sequenced library is going to be low.

FYI - I was told that Illumina has developed a gel-free, SPRI-based protocol for DNA TruSeq kits. Costs should be nearly identical to gel-cut method. You should be able to contact your Illumina rep for it.

[vmarshal]

Thanks very much John 12824 for your prompt reply. Sounds like our experience with this product has at least been similar so far.

Will give gel-cut a go in future; also shall follow up gel-free method with Illumina; surprised it isn't more "out there" already and it's what Agilent is already using; was surprised we stall had to size-select, but planning to get a "PippinPrep" for this purchase soon.

I'm also v. concerned about library complexity. We'll probably go ahead and run it on HiSeq and see what comes out (to fill a flow cell that is going on anyway); will keep this thread posted on the outcome. Hoping we don't need to drop this product and try something else (have plans to test others anyway); but shall if needs be.

Thanks again for v. helpful advice!

[Scoob]

Yes keep us posted. We are trying to decide on Truseq or Sureselect 50 for deeper exome cover and I'm worried that the reported lower coverage, combined with the greater target area with Truseq might outweigh any cost savings. Still inclined towards Sureselect until the data shows otherwise.

[csquared]

Just a quick comment on the high-molecular weight smear mentioned earlier in the thread.

The high-molecular weight smear that is often observed on the bioanalyzer after PCR amplification of libraries is often a "bubble product" that results from the complementary ends of the fragment annealing with the middle being non-complementary. Since we sequence lots of libraries prepared by other labs, we have had an opportunity to observe performance on lots of different library qualities. Our observations, while not necessarily perfectly conclusive, suggest the following:

1) If you see high-molecular weight product on the Bioanalyzer and NOT on a gel, it is the bubble product. It seems to run normally on agarose gels.

2) That product is caused by limiting primer. If you take an aliquot of the library, add PCR reagents and cycle for 2 cycles, you will see it collapse to the expected product.

3) This may be the most important: The higher molecular weight product will absolutely sequence. Therefore, if you use the bioanalyzer to quantitate libraries, you need to consider the smear in the quantitation or you may over-cluster. This is particularly concerning with the new v1.5 flowcells on the HiSeq. If you compare the bioanalysis quantitation with a real-time PCR assay such as Kappa's assay and the results show very similar molar concentrations, the library is usually good complexity and good quality. If the real-time assay shows a much larger concentration than the bioanalysis and the higher molecular weight product is there, the library may be over-amplified and complexity could be a concern. Simply looking at PCR duplicate rate may be helpful in your application to determine if this is indeed a problem.

[upenn_ngs]

it is also possible to saturate the bioanalyzer assay. if the library is highly concentrated, then 1ul might introduce too many dna fragments. consequently, these fragments may not travel evenly though the channels.

it can be useful to dilute the sample and analyze again

[EMONGS]

Originally Posted by John12824

BTS -

Rather than waiting we developed our own SPRI bead protocol. Gel cut samples captured with both Nimblegen and Illumina exome probe libraries are on HiSeq now. SPRI bead samples are in the queue.

John 1284...do you mind sharing your bead selection procedure?

Also, I have an issue with varying TruSeq DNA library sizes from 470 bp to 280 bp. I can't afford to spend time on troubleshooting right now so I was wondering if anyone has a similar problem and if so what is the remedy? Some of my initial samples were a little degraded. Will that make a difference or is it mainly the gel and excision?

[aminoda]

I have also had the problem of the presence of the broad peak that is higher than the size-selected range from the Bioanalyzer analysis after PCR. As I have seen in some other thread, when I reduced the number of cycles from 15 to 10, and only using 2 ng DNA as starting material (measured with Qubit), it completely got rid of this problem. I was using all of whatever I had at that point before.

I've heard that the TruSeq adapter ligation is so efficient now, that too much DNA is going into the PCR reaction, and so the primers run out faster, creating ssDNA, which you cannot get rid of from running the gel and size selecting, since it runs at a similar speed as dsDNA (although they seem to appear higher on the Bioanalyzer). Trust me, I did exactly this, and did not make any difference...

[pmiguel]

Reading this thread, and others on this topic, I get the impression that source of the extraneous high molecular weight products frequently visualized on bioanalyzer chips of TruSeq libraries is well understood.

However, I do not think this is the case!

Here are the hypotheses I as I understand them:

(1) "Bubble products". Until this morning I just assumed this was another way of describing "Daisy Chain" products (see below). But no. The name is the description -- two unrelated library molecules anneal to each other in the final cycle of PCR. Because their insert sequences are unrelated, only the adapter ends anneal, creating a big non-annealed "bubble" in the middle with annealed adapter "stems" on either end of the construct.

(2) "Daisy chain products". Two double stranded amplicons anneal at their adapter ends. Longer chains can form, for example a 3-mer, if both ends of an amplicon is annealed to other amplicons. Branching chains are also conceivable.

(3) "Single stranded products". This one is less clear to me. The post above, for example:

http://seqanswers.com/forums/showpos...1&postcount=18

To get single stranded products from a PCR reactions, I think you need one primer to run out while the other persists. Then only one strand is being produced in each cycle and the amplification becomes linear, rather than exponential.

But single stranded products would electrophorese faster than double stranded products. The post above claims "they seem to appear higher on the Bioanalyzer". But I am pretty sure this is not the case.

[Note add 10/12/2011: The statement above is completely wrong! ssDNA tends to run larger (for long products, much larger) than dsDNA on Agilent Bioanalyzer chips. See:

http://seqanswers.com/forums/showthread.php?t=12852

for details.]

(4) "PCR overamplification products". Run a PCR reaction for too many cycles and you will get a high molecular weight smear. One possible source of this smear is that primers have been depleted and the products themselves anneal at their ends. Once annealed the polymerase will happily extend each single-stranded product into a chimeric dimer: adapter-insert1-adapter-insert2-adapter. Longer multimers could result as well.

My guess is that all 4 of these are at work in various situations. The length of the TruSeq adapters is probably the source of these issues. What about mitigation?
[Note added 12/25/2011 -- I don't recommend the following. As I indicated down-thread, it does not work. In fact it seems to increase the amount of multimers.]
It seems like (1) and (2) can be melted out if the right kinetics can be found. How about this:

TruSeq protocol call for PCR thermal cycling:

98°C for 30 seconds
10 cycles of:
— 98°C for 10 seconds #denaturation
— 60°C for 30 seconds #annealing
— 72°C for 30 seconds #extension
72°C for 5 minutes
Hold at 4°C

How about for the last cycle doing a very slow ramp from 98 oC to 60 oC? That way the full length products will have lots of time to find each other.

Or, skipping PCR altogether is great. Except for all the issues with getting an accurate titration for clustering...

--
Phillip

[ETHANol]

The slower running fragments after amplification is from 'hybrid' fragments, i.e. fragments that are created after primer becomes limiting and the DNA fragments anneal by the adaptors sequences at the ends but not in the 'unique' sequences in the middle. Think of a bubble structure. If you add fresh primer and do one more cycle of PCR (i.e. heat to 95 1 min, 60˚ C 10 30 sec 72˚C 30 sec), your fragments will return back to the expected size.