Technical Support
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Good Trace data
Before we address what is constitutes to bad trace data from sequencing reactions we must first identify what is good trace data. Below is the control sequencing reaction that we perform and run concurrently with every batch of 95 reactions. This is viewed with Sequence Scanner.
The peaks are well defined plus the QV bar above shows the quality of the sequence. Using Sequence Scanner, click on Edit > Preferences Ctrl-K and change Select signal data type to Average Raw Signal to Noise Ratio. Then click OK
As you can see, the Contiguous Read Length (CRL) is well over 600 and the Trace Score is a high 51. The signal intensities from each of the base calls are high as well. Signal/Noise data is between 100 and 200. This is what constitutes to good sequencing trace data.
Early signal loss can be easily recognized by the ski slope configuration of the trace.
a) Presence of secondary structure in DNA
DNA can form complex loops and structures such as the guanine tetraplex. Use of adjuvants like DMSO can help in the reaction. If all else fails, sequence the opposite strand.
b) Repetitive regions
Repetitive regions make sequencing data look like those encountered in homopolymeric regions. It is best to sequence from the opposite direction though adjuvants can help.
c) Too much template DNA/Overloading
There is a range of DNA concentrations we can sequence reliably, too much DNA will cause premature termination of signal. If you are using FinchTV, Ctrl-I will bring up information on the trace data.
The Signal Strength clearly shows the symptoms of too much template DNA. Signal strength should be around 200-400. In this example they are well over 1000. This will ruin our expensive capillary arrays resulting in down time which is your time as well!
d) Inhibitory contaminant present in template
Perform an ethanol precipitation of the plasmid. Ensure that it is sufficiently dried from the residual ethanol before dissolving it in water. If needed, isolate another colony.
No Usable Signal
a) Primer has no annealing
Check plasmid maps and make sure you are using the correct sequencing primer for the plasmid if you are using the plasmid's flanking sequencing primers.
b) Incorrect primer sequence or poor primer design.
If you are designed an internal sequencing primer from trace data, make sure the primer sequence is designed from an area of well defined peaks.
c) Insufficient template or primer
Obtain spectrophotometric readings for your stock primers and template to determine concentration. Please check with our FAQ on how much template and primer is needed for different types of template.
d) Inhibitory contaminant present in template
Cycle sequencing reactions are very sensitive to contaminants. Ethanol, phenol and salts are the main culprits. Most contaminants originate from the template itself. Make sure your template is free from these contaminants. Performing a second acetate ethanol precipitation helps in most cases.
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Noisy data from the start
Firstly, noise in sequencing traces is defined by defined peaks with somewhat taller than usual smaller peaks that run close to the bottom of the data. These peaks often result in the basecaller assigning an N several positions. The N may signify a legitimate occurrence of two nucleotides in the case of a heterozygote. However, the case of noisy data, you have several of these N positions.
a) Multiple priming sites
Redesign the primer
b) Multiple priming sites generating PCR products
Gel purify the desired PCR product.
c) Presence of nonspecific/illegitimate PCR products
Using a separate sequencing primer or use a nested sequencing primer.
d) Too much template DNA
This is especially true when sequencing PCR products. Dilute the sample and redo the reaction.
e) PCR primers acting in both forward and reverse directions
As above, redesign the PCR primers
f) Residual primers or dNTPs from PCR
Remove the carry over reactants. If you can get a homogeneous PCR product, a spin column can do the trick. If there are mixed PCR products, gel purify the band of interest for sequencing. You can perform and Exo-SAP reaction to render the residual primers and dNTPs ineffective in the sequencing reaction.
g) Primers with high Tm or high GC content.
Redesign the primer to use one of lower Tm or GC content.
h) Primers with n-1 population
While it is not necessary to have highly purified primers for cycle sequencing, such results from poorly synthesized oligonucleotide primers rarely surface. Should your sequencing primer be longer than 30, opt to have the synthesized primers be purified.
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Noisy data further into the sequence
a) Mixed plasmid prep
This is a result from a colony pick that did not yield a pure isolate. Usually two vectors with different inserts or vector with an insert and another without. You will see a clean signal until the cloning site. There is no other way but the streak out the isolate you have and pick as many colonies as you can to obtain a pure isolate.
b) Slippage after homopolymeric region
Sequence data after homopolymeric regions are in most cases unreadable due to what is called polymerase slippage. The actual process polymerase slippage is very complicated. Sequencing through them with anchor primers may be mediocre at best. Call us if you would like to sequence through homopolymeric regions.
c) Compression
The term compression has a long history since the days when sequencing is done on large slab gels. On a modern instrument, it looks like a mixed plasmid situation except that it does not happen throughout the trace after a certain point. Only parts of the trace will produce a mixed plasmid data and then abruptly changing to a good sequence read. Temperature differences during the sequencers' run might produce compression artefacts as well. Performing the reaction with adjuvants might help. Talk to us if you encounter a sequencing compression artefact
d) Frame shift mutation
A recombination event could have occurred. Streak out the clone again and pick well defined and isolated colonies for sequencing. For PCR, this is the result of illegitimate PCR products. In this case, gel purify the PCR product.
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Noisy data with weak signal strength
Noise is always there but what concerns us is the signal-to-noise ratio. In good reactions, they will be virtually unnoticeable. This is what we call good signal-to-noise ratio. However if the signal is weak, the noise will mask out the signal and hence giving us a poor signal-to-noise ratio. If you are viewing the trace by FinchTV, Ctrl-I will bring up the chromatogram info window.
Optimally, signal strengths should be 200-400 but if they are below 100 like this example, you can be partially sure it is due to weak signal.
a) Insufficient or no template DNA or primer in the reaction.
Obtain spectrophotometric readings for your stock primers and template to determine concentration. Please check with our FAQ on how much template and primer is needed for different types of template.
b) Inhibitory contaminant present in template
Cycle sequencing reactions are very sensitive to contaminants. Ethanol, phenol and salts are the main culprits. Most contaminants originate from the template itself. Make sure your template is free from these contaminants. Performing a second acetate ethanol precipitation helps in most cases.
c) Degraded template DNA or primers
Degradation can come from various sources. Contaminating nucleases from plasmid preps are the main cause. Sources of these nucleases are from the use of bacteria without the endA1. Check your bacteria's phenotypes to be sure. Prolonged exposure to UV when purifying PCR products will degrade DNA. If possible, use non-UV alternatives for illumination like the Dark Reader. If using a UV transluminator is the only option, working quickly to reduce exposure is the only way. Avoid repeated freeze-thaws. Commonly used primers can be diluted with sterile water to working concentration and refrigerated for six months with no visible degradation.
d) Inefficient primer binding
The Tm of a primer is defined as the temperature at which 50% of the oligonucleotide and its perfect complement are in duplex. Below is the Wallace Rule for a rough estimation of the oligonucleotide primer's Tm.
Tm = 2°C(A+T) + 4°C(G+C)
This is the most commonly used formula for calculating Tm, though it is not the most accurate as it does not factor in salt or formamide concentrations. There are a lot of oligonucleotide Tm calculators in the Internet, the latest are those that use the algorithm by Allawi and SantaLucia. These are more accurate but be reminded that they are all estimates based on empirical data.
In cycle sequencing reaction, our primer/template annealing step occurs at 50°C. Therefore design oligonucleotide primers that have a Tm of between 56°C - 60°C. If you are unsure of which sequence to pick as a sequencing primer, 1st BASE offers an oligonucleotide primer design service for sequencing. |
