Troubleshooting Sequencing Data
Observation Possible Causes Recommended Actions
No recognizable sequence (see page 7-10)
Insufficient template Quantitate the DNA template. Increase the amount of DNA in the sequencing reactions. See
page 3-17.
Inhibitory contaminant in template Clean up the template. See page 3-16.
Insufficient primer Quantitate the primer. Increase the amount of primer in the sequencing reactions. See page 3-19.
Primer has no annealing site Use a primer that is complementary to the template.
Poor primer design or incorrect primer sequence
Redesign the primer. See page 3-18.
Missing reagent Repeat reactions following the protocol carefully.
See page 3-21.
Old or mishandled reagents Use fresh reagents. See page 3-20.
Thermal cycler power failure Repeat reactions.
Thermal cycling conditions Calibrate the thermal cycler regularly.
Use the correct thermal cycling parameters.
Use the correct tube for your thermal cycler.
Set ramp rates to 1 °C/second.
Extension products lost during reaction cleanup
Ensure that correct centrifugation speeds and times are used for precipitation and spin column procedures. See page 3-33
Extension products not resuspended Resuspend sample pellet in loading buffer or TSR carefully.
Lane tracking failure (ABI 373 or ABI PRISM 377 DNA Sequencer)
Check lane tracking. Retrack and reextract lanes if necessary.
Electrokinetic injection failure (ABI PRISM 310 Genetic Analyzer)
Repeat injections.
Noisy data throughout sequence, with low signal strength (see page 7-11)
Not enough DNA in the sequencing reactions
Use more DNA in the sequencing reactions.
Load or inject more of the resuspended sequencing reactions. See “Preparing and Loading Samples for Gel Electrophoresis” on page 3-50 or “Preparing and Loading Samples for Capillary Electrophoresis”
on page 3-53.
GC-rich template or GC-rich region in template
Increase the denaturation temperature to 98 °C.
Add DMSO to a final concentration (v/v) of 5%.
Incubate the reaction at 96 °C for 10 minutes before cycling.
Double all reaction components and incubate at 98 °C for 10 minutes before cycling.
Add 5–10% glycerol or 5–10% formamide to the reactions.
Linearize the DNA with a restriction enzyme.
Shear the insert into smaller fragments (<200 bp) and subclone.
Amplify the DNA using 7-deaza-dGTP in the PCR, then sequence the PCR product.
Expired or mishandled reagents Use fresh reagents. See page 3-20.
Thermal cycling conditions Calibrate the thermal cycler regularly.
Use the correct thermal cycling parameters.
Use the correct tube for your thermal cycler.
Set ramp rates to 1 °C/second.
Lane tracking failure Check lane tracking. Retrack and reextract lanes if necessary.
Noisy data throughout sequence, with good signal strength (see page 7-11
Contaminated template Clean up the template. See page 3-16.
Multiple templates in sequencing reaction
Examine your template on an agarose gel to see that only one template is present. See page 3-16.
Multiple priming sites Ensure that your primer has only one priming site.
Redesign the primer if necessary. See page 3-18.
Multiple primers when sequencing PCR products
Purify your PCR template to remove excess primers. See page 3-12.
Primer with N–1 contamination Use HPLC-purified primers.
High signal saturating detector Use less DNA in the sequencing reactions or load less on the gel or into the capillary.
Incorrect run module Use the correct run module. See page 6-2.
Incorrect instrument (matrix) file Use the correct instrument file for your sequencing chemistry. See page 6-7 for information on creating instrument files.
Noise up to or after a specific point in the sequence (see page 7-12)
Mixed plasmid preparation Ensure that you have only one template. See
“Plasmid DNA Templates” on page 3-6 and
“Determining DNA Quality” on page 3-16.
Multiple PCR products Ensure that you have only one template. See
“Preparing PCR Products for Sequencing” on page 3-12 and “Determining DNA Quality” on page 3-16.
Primer-dimer contamination in PCR sequencing
Optimize your PCR amplification. See page 3-10.
Make sure there is no sequence complementarity between the two PCR primers.
Use a sequencing primer that is different from either of the PCR primers.
Ensure that your sequencing primer does not overlap the sequence of the PCR primers.
Use a Hot Start technique, e.g., AmpliTaq Gold DNA Polymerase.
Slippage after repeat region in template
Try an alternate sequencing chemistry. See page 2-15.
Use an anchored primer. See page 7-36.
Poor mobility correction (see page 7-13)
Incorrect dye set/primer (mobility) file Use the correct mobility file. See page 6-5.
Incorrect Peak 1 Location for data analysis
Choose a new Peak 1 Location. See page 6-15.
Gel with very different separation properties from the gel matrices that were used to construct the dye set/primer (mobility) files
Use the correct dye set/primer file for your gel type.
Refer to the ABI PRISM DNA Sequencing Analysis Software User’s Manual.
Early signal loss (see page 7-14)
Region of secondary structure in the template
Sequence the opposite strand.
Use a sequencing primer that anneals at a different position.
Try an alternate sequencing chemistry. See page 2-15.
Incubate the reaction at 96 °C for 10 minutes before cycling.
Increase the denaturation temperature to 98 °C.
Increase the extension temperature by 2–3 °C.
GT-rich regions with BigDye terminators (see page 7-34)
Decrease the extension temperature in cycle sequencing to 55 °C or 50 °C.
Increase the magnesium ion concentration by 1 mM.
Sequence the opposite strand.
Try an alternate sequencing chemistry. See page 2-15.
Early signal loss (see page 7-14)
GC-rich region in template Increase the denaturation temperature to 98 °C.
Add DMSO to a final concentration (v/v) of 5%.
Incubate the reaction at 96 °C for 10 minutes before cycling.
Double all reaction components and incubate at 98 °C for 10 minutes before cycling.
Add 5–10% glycerol or 5–10% formamide to the reactions.
Linearize the DNA with a restriction enzyme.
Shear the insert into smaller fragments (<200 bp) and subclone.
Amplify the DNA using 7-deaza-dGTP in the PCR, then sequence the PCR product.
Poor lane tracking, such that tracker line diverges from the data
Check lane tracking. Retrack and reextract lanes if necessary.
Poor quantitation of primer Quantitate the primer. See page 3-19.
Poor quantitation of template Quantitate the DNA template, especially with PCR products. See page 3-17.
Excess dye peaks at the beginning of the sequence in dye terminator chemistries (see page 7-27)
Poor removal of unincorporated dye terminators
Choose the Start Point for data analysis to exclude the excess dye peaks. See page 6-18.
Follow the protocols for excess dye terminator removal carefully. See page 3-33.
Refer also to the Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions User Bulletin (P/N 4304655). This document can be obtained from the PE Applied Biosystems WWW site
(http://www2.perkin-elmer.com/ab/techsupp/pdf/ga/
ub/Precipitation_UB.pdf).
When using Centri-Sep spin columns, be careful to load the sample onto the center of the gel surface.
Do not touch the gel surface with the pipet tip. See page 3-34.
IMPORTANT When using BigDye terminators, be sure to hydrate the column for at least 2 hours.
Spin samples in the centrifuge for the
recommended times. Spinning too long precipitates more dyes with the sample.
When working with microcentrifuge tubes, aspirate the supernatant rather than decant it. Decanting leaves excess ethanol on the sides of the tube.
Broad, red peak between base 200 and 350 (see page 7-28)
Poor removal of unincorporated dye terminators
Follow the protocols for excess dye terminator removal carefully. See page 3-33.
Refer also to the Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions User Bulletin (P/N 4304655). It can be obtained from the PE Applied Biosystems WWW site (http://www2.perkin-elmer.com/ab/techsupp/
pdf/ga/ub/Precipitation_UB.pdf).
When using Centri-Sep spin columns, be careful to load the sample onto the center of the gel surface.
Do not touch the gel surface with the pipet tip. See page 3-34.
IMPORTANT When using BigDye terminators, be sure to hydrate the column for at least 2 hours.
Pull-up peaks/
bleedthrough (see page 7-22)
Total signal strength above 4000 Quantitate the DNA template (see page 3-17). Use less template.
Load or inject less of the resuspended sequencing reactions. See “Preparing and Loading Samples for Gel Electrophoresis” on page 3-50 or “Preparing and Loading Samples for Capillary Electrophoresis”
on page 3-53.
Stop peaks in dye primer chemistry
Primer-dimer contamination in PCR sequencing (see page 7-24)
Optimize your PCR amplification. See page 3-10.
Make sure there is no sequence complementarity between the two PCR primers, especially at the 3´
end.
Use a Hot Start technique for the PCR amplification used to generate the sequencing template, e.g., AmpliTaq Gold DNA Polymerase.
Use a dye terminator sequencing chemistry.
Default fragments in PCR sequencing of plasmid inserts (see page 7-24)
Ensure that you have only one template. See
“Plasmid DNA Templates” on page 3-6 and
“Determining DNA Quality” on page 3-16.
DNA sequence composition (see page 7-30)
Use a dye terminator sequencing chemistry. See page 2-2.
Sequence the opposite strand.
Compressions (see page 7-31)
Sequence-dependent region of anomalous mobility, particularly with dye primer chemistries
If using dye primer chemistry, try a dye terminator sequencing chemistry. See page 2-2.
Sequence the opposite strand.
Increase the denaturing ability of the gel or polymer by using higher run temperatures or denaturing agents such as formamide.
Note This can decrease the resolution of the gel or polymer and give shorter read lengths.
