DNA Sequencing Technical Support

1. Good Sequence Trace

 

2.  Early signal loss

 

3. Abrupt signal loss

 

4. Noisy data with weak signal

 

5. No usable signal

 

6. Noisy data with strong signal / high level of background noise

 

7. Difficult template (homopolymer, repetitive sequence)

 

8. Noise up to a specific point

 

9. Noise after a specific point

 

10. N-1 pattern

 

11. Dye blob

 

 

 

 

1. Good Sequence Trace

It is important to identify what calls as a good sequencing trace. Below is the control of sequencing reaction that we perform and run concurrently within each of our processing batch. 

This is viewed with Sequence Scanner.


A good sequencing trace data will contain: -

  1. Continous long stretch or un-interupted good quality value (QV) of the basecall. The QV will be displayed as rectangle bars at the upper part of the electropherograme.

    They are 3 categories of QV bars: -
    1. Blue: high quality with >= QV 20
    2. Yellow: medium quality with QV 15 to 19
    3. Red: low quality with QV < 15
  2. The peaks are well defined with almost no or very minimum of background signal.

    For sequencing trace of plasmid DNA, we will not able to see the end of the trace (or we called as stop point) as the template size is larger than what the electrophoresis can separate. After 850 bases, the resolution of the electrophoresis will gradually lost and there will be more low quality QVs.

    For sequencing trace of PCR product which shorter than 1000bp, you will find a clear stop point at the very end as below:

 

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2. Early signal loss

Pattern: Ski slope configuration of the trace. 

The sequence starts with high quality peaks but become messy in the downstream. The raw data will shows a good signal intensity at the beginning of the sequence and decreases when the sequence continues. 

This is viewed with Sequence Scanner.

Cause:

  1. Having too much starting template DNA. 
    Too much starting temple DNA will cause the used up of the chemistry at the beginning stage and thus little was left over for longer extension.
    When the actual DNA template size given is too much shorter (e.g. 750bp) than what indicate in the order form (e.g. 1500bp), we may overload template DNA during the cycle sequencing.
  2. Not enough of template DNA. 
    When there is not enough of template DNA, there will be less extending fragments generated in the sequencing reaction and thus the signal decrease when the sequencing continue.
    When the actual DNA template size given is too much longer (e.g. 1500bp) than what indicate in the order form (e.g. 750bp), we may put too little template DNA during the cycle sequencing.
  3. Present of salt contaminant in the sample. 
    Excessive salts in the sample will give rise to premature termination because it affects the activity of the polymerase which leads to overabundance of short fragments generated.
  4. Difficult region within the template DNA.
    DNA polymerase had difficulty to process through some particular sequence context, for example homopolymer & repetitive sequences.
     

Solution:

  1. We 100% quantify each template DNA before cycle sequencing. We will thus able to calculate the optimum volume required for each template DNA. Thus, providing us the correct or approximate size (with less than 100 bases different from the actual size) of DNA template will prevent too much/ too little template DNA used in the cycle sequencing. Please click here for our samples and primers requirement.
  2. Any commercial column purification kit will able to remove the salts effectively. A good advice for researcher that purifying bulk samples, we highly recommend processing the purification in several batches carefully.  For purification kit using high salt as binding buffer, a skillful laboratory practitioner will prevent salts from contaminating the final elution. Alternatively, we also provide purification service before cycle sequencing. Please enquire.

 

 

 

 

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3. Abrupt signal loss

Pattern: The signal is good before and it suddenly terminates or drops without continued basecalling.
This is viewed with Sequence Scanner.

Cause:

  1. Difficult region found in the sequence which stops the polymerase reaction.
    DNA polymerase had difficulty to process through some particular sequence context, here is the homopolymer, repetitive sequence and high GC or AT content.
  2. Formation of DNA secondary structure.
    The complementary regions fold up on themselves forming hairpin structures that the polymerase cannot pass through.

Solution:

  1. Sequence from the opposite strand.
  2. Order our Difficult Template Sequencing Service, which uses an alternative cycle protocol to sequence the difficult regions.
  3. Difficult region and DNA secondary structure from PCR product template tend to fail to be sequenced; cloning will be an option to get stable and thus longer read. 
    Alternatively, we provide cloning under our Molecular Biology Services before cycle sequencing. Please enquire.
     

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4. Noisy data with weak signal

Pattern: Undefined peaks, no continuous read length (CRL), and majority having low quality QV bars.
This is viewed with Sequence Scanner.


 


 

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5. No usable signal

Pattern: Data showed only 5Ns. 
This is viewed with Sequence Scanner.

Cause:

  1. Too little DNA template or primer in the reaction.
  2. Inhibitory contaminant present in template.
    Present of contaminant (e.g salt, ethanol, phenol, RNA, etc.) will inhibit the binding of primer or polymerase to the DNA template in order to start the reaction.
  3. Degraded template DNA or primers.
  4. No priming site.


Solution:

  1. Quantify your template DNA through gel electrophoresis; and if possible together with spectrophotometer e.g Nanodrop. As long as there is a visible DNA band when 1uL of your purified template DNA loaded in agarose gel, it will be sufficient to be sequenced. Larger template DNA will require more DNA amount to get the optimum reads. Please click here for our Samples and primers requirement ).
  2. Someone may say making sure of the ODA260/280 ratio is between 1.8 to 2.0. However, there are not many customers are willing to perform this extra checking unless something seriously went wrong. In our opinion, as long as you are using a commercial kit, and they are not expired, the quality of the purified sample should be working well for sequencing typically. The contamination that leads to no usable signal usually is the serious case, until the template is being degraded.
  3. Bad quality of RNase premix that used during the plasmid DNA extraction & purification process will degrade the DNA easily (especially during the transportation). Typically, Rnase doesn’t degrade DNA, but contamination with Dnase is the main reason. It happens when you see good DNA bands in your lab; but we see nothing when receive your samples. Change a new batch of Rnase will solve the problem.
    If you need our help to run agarose gel quantification before cycle sequencing, it is available as 1 of our sample-add-on service.
    Make sure the template DNA and primer are freshly prepared before send for sequencing service. Plasmid DNA that extracted more than 4 months ago tends to give shorter reads.
  4. If you are sequencing a purified plasmid DNA, we highly recommend you to use the vector’s sequencing primer for your first reaction to avoid disappointment.
    For purified PCR products, most of the researcher will use one of their PCR primer as sequencing primer. As long as the design of PCR primers also fulfill the requirements as a sequencing primer, it will be fine.

 

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6. Noisy data with strong signal / high level of background noise

Pattern: Multiple overlapping peaks with strong signal from beginning. The secondary peak from the same position may be the same, lower or higher than the major peak. 
This is viewed with Sequence Scanner.



Cause:

  1. Unspecific priming site. 
    Present of more than 1 region within the template DNA, which complement to the primer sequence. This cause the priming site of the sequencing primer is not specific, and thus they will be more than 1 type of extension products produced from a single reaction tube. During the capillary electrophoresis, they only do the job of size separation. If the extension product is not unique at each size, the signal will be mixed.
  2. Trace of excess PCR primers present in the sample.
    If the PCR products are not purified before sequencing, the excess PCR primers will act as sequencing primer in the sequencing reaction.
    Each primer will give different cluster of extension products, which lead to mixed signals.
  3. Present of more than one template DNA.
    1. Occasionally, we will found some purified PCR products show more than 1 DNA band in the gel photo. Most of the commercial PCR Clean-up kits will not able to remove the contaminated double stranded DNA or primer-dimmers. If the sequencing primer can binds on these unspecific DNA bands or primer-dimmers, it will generate different cluster of extension products and being analysed together to the major product, which leads mixed signal. It is very easy to identify the mixed signal under this category. For example: 
      -you have an unspecific DNA band at the size of 250bp, and your major product is at the size of 750bp; you will found the mixed signal will stop after the region of 250-base, followed up with the clean signal until the end of the trace.
    2. There is a frame shift deletion from your target band. 1 frame shift = 1 amino acid = 3 basepairs. If your actual target PCR product size is 750bp, a frame shift deletion sample will have both 750bp and 747bp of PCR products. You will not able to distinguish such a small different in size using any type of agarose gel. But the sequencing result will show different end point with 3 bases of size in different. The mixed signal will start immediately after the mutation site, due to different signal of extension products from the same reaction tube. This observation is however limited to the product size. When the size of PCR products is greater than 850bp, it is very difficult to judge the different of the end point since the resolution of CE is getting worse beyond 850 bases.
  4. Primer with too low annealing temperature.
    When the Ta is lower than 48oC, we will follow your recommended Ta on the order form. The same Ta may works for your PCR, but not necessary works for sequencing. PCR is an exponential reaction that involves 2 primers, but sequencing is a linear reaction that involves 1 primer.



Solution:

  1. Redesign primer specifically for sequencing. If you are sequencing a plasmid DNA, the vector primer usually works very well.
    Alternatively, we help our customers to design and synthesis sequencing primer. Please enquire.
  2. Someone can optimize their PCR protocol to have depleted primers during the end of PCR, so they can send their unpurified PCR products directly for sequencing. However, sometime it will fail when it lost control. The best safe guard is to purify your PCR products before sequencing. Alternatively, please order our purification service before sequencing. Please enquire.
  3. Examine your purified DNA sample on agarose gel before sending the sample for sequencing. If there is more than 1 DNA band, purify them with gel extraction purification rather than PCR clean-up. If you are bothering with the samples that showing mutation, the best solution is to clone the fragment and pick many colonies for sequencing. You should justify the cost of cloning only if you are really interested with the mutated fragment. Otherwise, put it aside and sequence the next sample.
  4. If you are using 1 of your PCR primer to be your sequencing primer, you may simply compliance the rules of sequencing primer when select the best primer that designed by the primer software. 

 

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7. Difficult template (homopolymer, repetitive sequence)

Pattern:

  1. Noisy data after a long stretch of mono- (homopolymer) or di-nucleotide repeat.
  2. Noisy data after a region of the template DNA that is especially high in one or two of the nucleotides.
    This is viewed with Sequence Scanner.



Cause:

  1. Polymerase did not pair correctly with the template.
    The polymerase dissociates and re-hybridized in a different location when meet with a long stretch of mono- or di-nucleotide repeat generating various sized fragments and creating mixed signal after the region. This normally will generate a slippage pattern in the results. 
    This condition tends to be more problematic in PCR products. 
     

Solution:

  1. If the sample is PCR products, clone the fragment into a holding vector will help to stabilize the sequencing through the same homopolymer region. It is advisable to test on a few samples firstly before use on large number of samples.
    We provide cloning services too. Please enquire.
  2. Sequence from the opposite strand.
    Although the sequencing will still fail during passing through the same homopolymer region, but the alignment will be successful. E.g. AAAAAAAAAA… will align with TTTTTTTTTT… from the opposite strand. You probably will lose the information of how many repeats of A. But if this is not what you are looking for, it is still worth for trying to save the cost from cloning.
  3. Use anchored sequencing primer
    • The most common anchored primer is 21TV, where T is repeated for 21 units, followed by a degenerated primer of V (mixed of G, C and A). It looks like this: TTT TTT TTT TTT TTT TTT TTT V.
    • The anchored primer will bind specifically on the homopolymer region, and allows the polymerase to work normally right after them. It works very well when there is only 1 homopolymer region found within the template DNA. This however will lose some information (~ 20 to 30 bases) after the priming site, which is 1 of the disadvantage for BigDye Terminator v3.1. If you need to recover this lose region, you need to have the other sequencing primer that either sequence from the opposite strand until reaching the homopolymer region, or the original primer that binds upstream from the anchored primer.

 

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8. Noise up to a specific point

Pattern: Data showing noisy peaks from the start until a specific point. The continue data showing good QVs. 
This is viewed with Sequence Scanner.

Cause:

  1. Contamination of primer-dimmers.
    The contamination of primer-dimmer will generate noise up to 150 bases from the beginning of the sequencing trace. Normally primer-dimmer can be seen as a cloudy band/ spot in the bottom of agarose gel quantification.
  2. Contamination of smaller fragment/ illegitimate products.
    Instead of seen as a cloudy band/ spot in the bottom of agarose gel quantification, smaller fragment/ illegitimate products will be seen as DNA band. In some cases, they are not easily detected using agarose gel when the loading volume is smaller than 5uL. They are not necessary generate high level of noise, especially when there is a high similarity of their sequence with the target fragment. But you can trace them easily by detecting the end point of extension products that produced by these smaller fragment/ illegitimate products in the beginning of the sequence. Their end point is just like the other PCR products that usually end by a high A peak by the PCR Taq polymerase.



Solution:

  1. The quick way is to reduce the amount of PCR primers during conduct the PCR. It works fine only if your PCR primers do not have any degenerate base(s).
    When you design your PCR primers, use the software to choose the best primer sequence that does not perform self-dimmers.
  2. If you able to recover this region from your opposite sequencing result, what you need to do is probably just cut off the noise region before perform the pairwise alignment. If your PCR products is very short (<250bp), the only way to resolve this is to gel purify the template DNA before sequencing.


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9. Noise after a specific point

Pattern: Data is good at the beginning turns to double or more peaks after a specific position. 
This is viewed with Sequence Scanner.



Cause:

  1. Mixed preparation of a plasmid DNA sample.
    There is more than 1 colony is picked and extracted for sequencing. The noise is found right after the multiple cloning site (MCS).
  2. Frameshift mutation.
    Insertion or deletion of nucleotide may occur in plasmid or PCR product. The noise will starts right after the mutation site.



Solution:

  1. Re-extract the plasmid DNA from the single colony to re-sequencing.
  2. The best solution to sequence the mutated samples is still to clone the fragment and pick many colonies for sequencing. You should justify the cost of cloning only if you are really interested with the mutated fragment. Otherwise, put it aside and sequence the next sample.

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10. N-1 pattern

Pattern: Overlapped peaks throughout with the second peaks generally 1 base smaller and being the same base as that of the true base immediately to the right. It looks like a small “tail” before the peak of the correspondent nucleotide. This is viewed with Sequence Scanner.



Cause:

  1. Poor synthesis quality of the sequencing primer because of low coupling efficiency.
    If the contamination of N-1 primers is higher than 10%, it will be detected from the sequencing reads and affects the QVs.
    The bad sequencing primer will affect each reaction without a miss.
  2. Mis-priming
    The same sequencing primer works very well for the other template DNA in the same submission, but only shows this pattern on some selected template DNA.


Solution:

  1. Ask replacement from your primer synthesis Company. A new synthesis usually will resolve this problem immediately.
  2. Re-design the sequencing primer by extend the primer length will help to increase the priming specificity. We can certainly help you in the sequencing primer design and synthesis. Please enquire.

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11. Dye blob

Pattern: The sequence contain dye blob may or may not affect the basecalling.  
This is viewed with Sequence Scanner.



Cause:

  1. Unincorporated dye terminator molecules did not remove efficiently during the step of post sequencing clean-up.
  2. Degraded template DNA
     

Solution:

  1. When we see dye blobs, we will automatically re-run the reaction. If you notice this in your results and you are very sure that the template DNA is freshly prepared, kindly contact us to request for the re-run.
  2. Re-prepare the fresh template DNA for re-sequencing.

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