Troubleshooting
DNA SEQUENCING TROUBLESHOOTING
Purity and concentration of DNA template are crucial factors to obtain good sequencing results.
- Plasmid must have the right concentration and purified from E.Coli, genomic DNA, RNA, endotoxin and salt.
- PCR product must be single band, clean and nice, and should not have any kind of fluorescent label (primers or dNTPs)
Good sequence generally begin around base 20 and is represented by tall and distinct peaks with a little overlap.
Below an example of an excellent sequence:
The following examples can help you to examine the different structure of all type of sequencing data:
Short DNA Trace Read Lengths
Identification of short DNA read
Peaks appear broad, bad resolved and progressively deteriorated through out the run. Loss of resolution may start from somewhere in the middle of your sequence.
Causes of short DNA sequencing read lengths
- Too much DNA template
- Too much primer
- "Dirty" DNA template
Solving short trace reads
- Too much DNA template: Check your samples concentration loading them onto an agarose gel. Do not rely on simply spectrophotometer readings. Spec reading are often inaccurate, in particular for plasmid templates.
- Too much primer: Check that the oligonucleotide primer concentration is correct.
- "Dirty" DNA template: Check that the template is clean.
Failure of DNA sequencing reaction
Identification of failed reactions
Failed reactions are indicated by N’s or by a noisy baseline
Causes of failed DNA sequencing reactions
- Poor quality DNA
- Too much DNA template
- Wrong choice of primer
- Degraded primer
- Failed oligonucleotide synthesis
Solving DNA sequencing reaction failures
- Poor quality DNA: Prepare your template using plasmid miniprep kit suitable for sequencing reaction.
- Too much DNA template: Excess of template can kill the sequencing reaction. Check template concentration onto an agarose gel before sequencing.
- Wrong choice of primer. Be sure that the primer binding site is present. Sometimes the “ universal” primers (fw & rev) could be the wrong choice, be aware to select the right sequence that works with the plasmid you choose.
- Degraded primer. Avoid the use of old diluted primer stocks. Store your primers in 10mM Tris/0.1mM EDTA (pH 8.5) rather than water.
- Failed oligonucleotide synthesis. The bad quality of your primer, could be invalidate the successful of your sequencing reaction.
Contaminated DNA Preparation
Identification of contaminated DNA preparation
The peaks are not spaced and they overlap making the entire sequence unreadable. Sometimes could happen that sequence data is clear ‘till the end of the insert cloning site, then become unreadable.
Causes of mixed template sequencing traces
- Two or more templates are present in the sequencing reaction
- Presence of leftover primers
- Two priming sites are present in DNA template
- Poor quality PCR template containing multiple DNA fragments
- Aspecific primer annealing in the sequencing reaction.
Solving mixed template sequencing problems
- Two or more templates present in the reaction. Make sure that only one DNA template is present. Prepare a new plasmid prep making sure that only one colony is selected.
- Presence of leftover PCR primers: Even low levels of the PCR primers can cause mixed signal problems, especially if they have a high annealing temperature. Remove PCR primers using suitable clean-up protocols.
- Two priming sites are present in DNA template. Use a different primer if you suspect the presence of two annealing sites.
- Poor quality PCR template containing multiple DNA fragments: Clone the PCR product or purify it cutting out agarose gel before sequencing.
- Aspecific primer annealing in the sequencing reaction: Check the predicted melting temperature of the sequencing primer.
Presence of complex structure
Identification of complex structure
A good quality trace stops abruptly. It is caused by the inability of the dNTPs and ddNTPs molecules to be incorporated into the secondary structure during the sequencing cycle.
Causes of complex structures
- Presence of hairpins. Regions of DNA template can fold back on themselves and form hairpin structures that polymerase cannot pass through.
Solving complex structures
- We strongly suggest to sequence the DNA template in both directions.
Homopolymeric regions
Identification of homopolymeric regions
Long repetition of the same base can cause problems due to polymerase “slippage” in the repeating region. The sequence data appear perfect and clean through the homopolymer region, after, the data is noisy because of the presence of multiple sequences.
Causes of sequencing trace signal sudden stops
- Long repeated nucleotides in the template. These repetition can form hairpins or single stranded conformational structures that sequencing polymerase has difficulty passing through. Long regions composed by any type of bases are problematic, in particular the “Guanidine stretch”.
Solving overcoming DNA sequencing hard stops
- Use a custom primer annealing just outside the mononucleotide or dinucleotide run region.
Identification of heterozygous peaks
A single peak position may have two peaks of different colors instead of just one. This is common when sequencing a PCR product derived from diploid genomic DNA, where polymorphic positions will show both nucleotides simultaneously. Note that
the basecaller may list that base position as an 'N', or it may simply call the larger of the two peaks.
Causes of double nucleotide peaks
- Presence of point mutation in your DNA template
- PCR product derived from diploid genomic DNA with polymorphic position
Solving heterozygous SNPs
- Clone your PCR product before sequencing
Multiple peaks due to primers with “n-1” population
Identification of “n-1” presence
The data is visible but the use of “n-1” primer generates a second sequence lagging one base to the right or the left of the main peak.
Causes of “n-1” in the primer preparation
- Poor quality synthesis of sequencing primers.
- Primer degradation from the 3’ end.
Solving “n-1” due problems
- Poor quality primer. Synthesize a new oligonucleotide to obtain a primer suitable for sequencing. HPLC purification of the primers is not necessary for typical oligos (<30bp) but sometimes additional purification can be beneficial.
- Primer degradation from the 3’ end. Synthesize a new oligonucleotide.
Identification of Primer Dimer Problems in Traces
The trace signal is mixed in the early regions, while the late regions are not mixed.
The signal drops from very high early in the trace (usually in the first 100 bases) to much lower levels.
Causes of Primer Dimers in Sequencing Reactions
- Contamination of the template with smaller PCR products
- Too low annealing temperature
- Primers forming self-binding structure
Solving Primer Dimer Problems
- Contamination with small PCR products: Cut your PCR product out of agarose gel to isolate a single band
- Low annealing temperature: Raise the annealing temperature and/or use a shorter annealing time.
- Self-annealing primers: Check the sequence of your primer and be especially careful of partial matches (8-9 base pair match can cause problems if at the 3' end of the primer).
Dye Terminator Peaks
Identification of Dye Terminator Peaks
Blobs of dye terminators are characterized by large peaks, usually early in the sequence (between 70 to 90bp and approximately at 100bp). Unincorporated BigDye terminator can partially obscure the real peaks.
Cause of Dye Terminator Peaks
- Reaction ratio not balanced
Solving Dye Terminator Peaks
- Use more DNA template
- Decrease the amount of BigDye terminator in the sequencing reaction.
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