Sequencing troubleshooting

Roswell Park Cancer Institute DNA Sequencing

A very good site with information on interpreting your chromatograms. Includes chromatographic illustrations of common sequencing problems along with suggested solutions.

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University of Michigan DNA Sequencing Core

A good source of information on all aspects of sequencing.

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Troubleshooting table

The following information is from the Applied Biosystems 3730/3730xl DNA Analysers Sequencing Chemistry Guide, Appendix B and the BigDye Terminator v 3.1 Cycle Sequencing Kit Protocol.

Sequencing troubleshooting table

Observation Possible Cause Recommended Action
Weak signal
  • Quantity of template or primer in the sequencing reaction is too low.
  • Excess salt present in the sample.
  • Bad post-reaction clean-up
  • Refer to the Sequencing Protocol for recommended quantity of template.
  • Clean up the sample using a spin column or a 70% ethanol wash.
  • Repeat sample preparation.
High background
  • Dirty template, bad primers, bad post-reaction clean-up.
  • Refer to the Automated Sequencing Guide page 3-16 for information on how to clean up dirty templates.
Top-heavy data
  • The amount of template in the sequencing reaction is too high, creating an excess of short fragments that are preferentially injected into the capillary array.
  • Diluted reactions.
  • Refer to the Sequencing Protocol for the recommended amounts of template to use in the sequencing reaction.
Blank lanes or no signal
  • Cycle sequencing reaction has failed.
  • Bad post-reaction clean-up.
  • Repeat the cycle sequencing reaction, adjust primer and template concentration.
  • Repeat the sample preparation.
Multiple, overlapping sequences in the data ( PCR templates)
  • More than one template present in the reaction (i.e. secondary PCR products) due to lack of specificity.
  • The majority of clean-up procedures for PCR products are designed to remove unincorporated nucleotides and residual PCR primers, not secondary PCR products. Use agarose gel electrophoresis to detect the presence of secondary PCR products.
  • Optimise the PCR conditions and/or use a Hot Start method.
  • Purify the PCR products using a gel before sequencing.
Multiple, overlapping sequences in the data (cloned DNA templates)
  • More than one sequence present in the reaction due to mixed plaques or colonies.
  • Re-isolate the DNA from a pure colony and re-sequence.
  • When picking bacterial colonies for growth and DNA isolation, choose a colony that is well isolated.
  • With M13 plaques, use fresh plates for plaque picking.
  • Check the DNA purity by running it on an agarose gel.
Multiple peaks in the same position at some points (pull-up peaks or bleedthrough)
  • Very strong signals saturates the instrument's detector, causing the signals to be truncated.
  • The Sequencing Analysis software underestimates the amount of signal at these positions, therefore underestimating the amount of spectral overlap to correct.
  • Very strong signals are common when sequencing short PCR fragments, because the sequencing reaction is often very efficient. Use the recommended amount of template for PCR products.
  • Overloaded sample. Decrease the amount of template used in the reaction. Check the recommendation for template amount.
Excess dye peaks at beginning of sequence
  • Incomplete removal of unincorporated, fluorescently labelled ddNTPs
  • Use only room-temperature alcohol. Cold alcohol will also precipitate unincorporated dye terminators.
  • Do not use denatured alcohol. Denatured alcohol has inconsistent quality. The concentration of the alcohol and purity of the additives can vary.
  • Spin samples for the recommended times (spinning too long precipitates more dye with the sample).
  • With microfuge tubes, aspirate the supernatant rather than decanting (decanting leaves excess ethanol on the sides of the tube).
  • Use ethanol/ EDTA precipitation protocol to remove unincorporated dye terminators.
  • With spin columns, take care to load the sample on the centre of the gel surface. Follow the instructions for use. Note: Do not touch the gel surface with the pippette tip.
Difficulty sequencing GC -rich templates, resulting in weak signal
  • The DNA is melting at a higher temperature due to the high proportion of GC base pairs.
  • Note: Even a template that has a fairly average base composition overall can have a very GC -rich region that affects its ability to be sequenced.
  • See the 3730/3730xl Sequencing Chemistry Guide, Appendix B for suggestions.
Secondary structure in the template, making it difficult to obtain good sequencing data beyond the region of secondary structure.
  • Self-annealing DNA
  • Refer to the 3730/3730xl Sequencing Chemistry Guide, Appendix B for suggestions.
Noisy data throughout the sequence with low signal strength
  • Insufficient DNA in the sequencing reactions
  • Degraded template
  • Old or mishandled reagents
  • Thermal cycling conditions incorrect
  • Use more DNA in the sequencing reactions. Load or inject more of the resuspended sequencing reactions.
  • Prepare fresh DNA and repeat the reactions.
  • Use fresh reagents.
  • Calibrate the thermal cycler regularly
  • Use correct thermal cycling parameters
  • Use correct tube for your thermal cycler
  • Set ramp rate to 1° C /sec
Noisy data throughout the sequence with good signal strength
  • Inhibitory contaminant in the template
  • Multiple templates in the sequencing reaction
  • Multiple priming sites
  • Multiple primers when sequencing PCR products
  • Primer with N-1 contamination
  • High signal saturating the detector
  • Clean up the template.
  • Examine the template on an agarose gel to be sure only one template is present.
  • Make sure the primer has only one priming site. If necessary, redesign the primer.
  • Purify your PCR template to remove excess primers.
  • Use HPLC -purified primer
  • Use less DNA in the sequencing reactions.
Noise up to or after a specific point in the sequence
  • Mixed plasmid separation
  • Multiple PCR products
  • Primer-dimer contamination in PCR sequencing
  • Slippage after repeat region in template
  • Make sure you have only one template.
  • Optimise your PCR amplification.
  • Make sure there is no sequence complimentarity between the two PCR primers.
  • Make sure your sequencing primer does not overlap the sequences of the PCR primers.
  • Use a Hot Start technique such as with Amplitaq Gold polymerase.
  • Try an alternate sequencing chemistry.
  • Use an anchored primer.

Fragment analysis troubleshooting

The most common problems encountered with fragment analysis are:

Off-scale data

Trial dilutions of your original PCR reaction in order to determine the optimal loading concentration for the 3130xl. The optimal signal strength is 150 - 4000 RFUs . GUDSF staff will be able to advise you on the optimal concentration.

Poor or non-specific amplification

For advice please refer to Troubleshooting PCR Amplification, page 11-1 and Optimising PCR, page 6-1 of the GeneScan Reference Guide.

Incomplete 3' A nucleotide addition

This is the DNA polymerase catalysed addition of a single nucleotide (usually adenosine) to the 3' end of the two strands of a double-stranded DNA fragment. This results in a denatured PCR product that is one nucleotide longer than the target sequence. The longer product is referred to as the "plus A" form. This results in a "split peak" electrophoretic pattern. For advice on how to avoid problems created by the incomplete 3' A nucleotide addition please refer to Optimising PCR, page 6-18 of the GeneScan Reference Guide.

Stutter

Stutter occurs during PCR amplification of di-, tri- and tetranucleotide microsatellite loci, producing minor products that are 1-4 repeat units shorter than the main allele. Stutter may be caused by polymerase slippage during elongation. For advice regarding this phenomenon please refer to Optimising PCR, page 6-21 and Troubleshooting Microsatellite Analysis, page 8-25 of the GeneScan Reference Guide.

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