Sanger Sequencing Troubleshooting Guide
Two options to view your results for troubleshooting:
- View the table from "Download DNA Results" within the Coreweb LIMS system to see summary data including Phred Q20 length and Phred signal intensity, or to view your raw sequence chromatogram ("View"):
- To compare your samples to others on the same run, enter your plate or order number here to compare your sequence Q20 length, signal intensity, and average Quality Value to other samples run at the same time including the DSF's internal control.
From either of these views, interpret results as follows:
- Look at the Phred Q20 length and Phred Signal Intensity:
- Plasmid sequences with good quality will have the Q20 lengths over 800 bp depending on the type of template.
- PCR product sequences will have Q20 lengths of 20 to 50 bp lower than the product length.
- Look at the Phred Signal Intensity:
- Good signal intensities will be between 500 and 4000.
- Signal intensities below 100 are background or close to background and not reliable sequence.
- Signal intensities above 4000 have too much DNA in the sequencing reaction, and will cause errors in basecalling.
- Compare your chromatograms to the examples below for further details.
Quantifying your DNA is key:
- Quantify your DNA using the UV/Vis (NanoDrop) or fluorescent quantification (PicoGreen or Sybr Green)
- UV/Vis Quantification
- The NanoDrop has a reliable lower limit of detection (LLD) of about 10ng/ul - readings below this are often NOT reliable.
- Contaminants like RNA, genomic DNA, and salts can over-estimate the concentration of DNA because they absorb at or around 260 nm.
- Good quality DNA should have A260/280 ratios around 1.8 to 2.0. Lower ratios indicate the presence of contaminants like protein and phenol that absorb at 280 nm.
- Good quality DNA should have A260/230 ratios around 1.8. Lower ratios indicate the presence of organic contaminants like salts, TRIzol, EB, EDTA, and carbohydrates.
- You may not be able to quantify PCR products due to detection limits with UV/Vis (quantities less than 10 ng/ul are not reliable on NanoDrop). If you purify your PCR products prior to Sanger sequencing: ensure that any primers leftover from the PCR reaction, unincorporated nucleotides, enzyme, buffer, and salts are completely removed. When using EtOH precipitation, make sure to remove any residual ethanol; EtOH can bind to unincorporated dyes in the sequencing reaction which can lead to dye blobs and basecalling errors. Gel-purified PCR products that produce a single band will produce the best sequencing results.
- Fluorescent Quantification - e.g. PicoGreen assays, Invitrogen Qubit assay
- Contaminants are less likely to affect quantification
- Better for PCR product quantification (lower limits in pg range)
Good UV/Vis from NanoDrop: 260/280=1.87 and 260/230=2.19
Example of PCR product on the NanoDrop
Poor UV/Vis from NanoDrop
A contaminant is present in the samples. In some cases this can cause the NanoDrop to overestimate the concentration of DNA (e.g. A260/280 = 1.53, A260/230 = 0.60, conc.= 158.5 ng/ul).
Quantification Using SYBR Green.docx
No Sequence or Not enough DNA or low DNA
- Not enough DNA
- Primer mismatch
- Priming site is absent or has a mutation
- Poor quality template, vector, or primer
- Use more DNA or re-perform the miniprep
- Increase the number of cycles for the PCR reaction
- Check and make sure the priming site is there
- Purchase new primers
- Make sure your sample submission has the correct request for primers
Too Much DNA
- Quantify samples (We recommend 15 to 30 ng/ul for a 5 kb plasmid).
- Dilute your samples
Mixed product or Double Sequence
- Multiple colonies or clones present during sample prep
- Multiple PCR products
- Multiple priming sites/Non-specific primer binding
- Primer-Template mismatch
- Contamination from water or environment
- Try a universal primer in the plasmid for verification
- re-pick colony with less dense cells
- Try another primer
- Redesign primers
Contaminant - Top Heavy or Ski Slope effect
- Most Often: Too much DNA
- High Salt concentration
- Salt or Ethanol contamination
- Excess primer
- EDTA or EB concentration too high
- Quantitate DNA samples
- If DNA is obtained by gel extraction, perform an extra wash
- Spin filter tube longer
- Dry spin column longer
- Re-precipitate DNA and perform an 80% Ethanol wash
- Elute with water
Secondary Structure - Mid Sequence Drop off or Hard Stop
- Secondary Structures (Hairpins, stem loops, palindromes)
- shRNA region, RNAi region
- GC rich region
- Sequence from the other direction or closer to the region
- Request GC Rich or HSP service types
- Linearize DNA
- Shear DNA
- Bad injection
- Inhibitory contaminant
- Dye Blobs
- Request difficult template buffer in your DSF order
- Dilute samples
- Rerun the samples
Short sequence - Primer Dimer
- Inefficient primer design
- Priming site's not there
- Primer hybridizing to itself
- Design new primers
- Double check primers for palindromes
- Ensure the last 5-6 nucleotide at the 3' end won't anneal to another region
Repeats or homo polymer
- High GC rich template
- Repeat region
- Polymerase is slipping
- Long stretches of homopolymer regions
- Request GC Rich service types
- Sequence in the opposite direction
- Use a primer that anneals at a different position
Additional Troubleshooting Resources:
3730 & 3730XL DNA Analyzers Sequencing Chemistry Guide.pdf
Automated DNA Sequencing Chemistry Guide.pdf
Automated DNA Sequencing Chemistry Guide.pdf
Here is an example of samples that had RNA contamination. It is always a good idea to run samples on a gel or BioAnalyzer for optimization.