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   & Fragment Analysis
 

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  Genomic Services
• Oligo Synthesis
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• Real-Time PCR
• Pyrosequencing
• Next Gen Sequencing
• DNA Sequencing
   & Fragment Analysis
• Single Cell Genomics
• RNA/DNA QC
‡ Bioanalyzer QC
‡ Fragment Analyzer QC
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• Peptide Synthesis
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‡ Mass Mapping
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DNA Sequencing/Fragment Analysis
 

DATA ANALYSIS

Current instrumentation, software, and PAN personal are reliable, but errors can occur.  It is the responsibility of customers to evaluate their data, even when it’s good.  Please download your chromatogram (.ab1 files) and examine your sequences!!!


 

Good Data
Fair or Poor Data
Bad Data

Below are graphical examples of common DNA sequencing issues. Most fair/poor sequencing data require simple adjustments to fix. The PAN Facility offers FREE reruns and/or resequencing of most fair and poor data. Bad data usually requires a little more troubleshooting, luckily the PAN Facility offers free troubleshooting assistance. If you have any questions or concerns about your data please contact us at dnaseq@stanford.edu or 650-723-3189.

**Due to changes in ABI polymer formulations, please be aware the first ~20 bases of a sequence maybe unreadable due to poor resolution.

 


 


Good Data (top)

Description

How to get it

-Data should have sharp, evenly spaced peaks with a clear baseline.
- Read length should be between 500-600 bases.
-Signal strength should be between ~100-700.

-Good quality template.
-Good primer design.
-Correct concentration of both template and primer.
-Fresh reagents.

 


 

Poor Resolution (top)

Description

Cause

Solution

-Broad unresolved peaks throughout read.

-Degraded reagents on capillary instrument.
-Excess salts.

-Request sample be re-run.
-Request dilution of sample and rerun.

 


 

Dye Blobs (top)

Description

Cause

Solution

-Broad florescent signal spanning multiple sequencing peaks

-Sephadex clean-up failed to remove unincorporated dye terminators.
-Fluorescent contaminant in sample.

-Request sample be cleaned-up and re-run.

 


 

Dye Spikes (top)

Description

Cause

Solution

-Intense fluorescent signal obscuring  few sequencing peaks

-Air bubble or contaminant.

-Request sample be re-run.

 


 

Top Heavy (top)

Description

Cause

Solution

-Excessive signal intensity followed by an early drop-off of sequence read.

-Too much template or primer resulting excess of short fragments.

-Request sample be diluted and resequenced or rerun.

 


 

Weak and Noisy Signal (top)

Description

Cause

Solution

-Weak peak height and signal strength.  High background noise.

-Template and/or primer concentration too low.
-Poor quality of template and/or primer.
-Excess salts or contaminant present.
-Capillary failed top pickup sample.

-Double check template and primer concentration.
-Clean-up template.
-Double check primer design.
-Request samples be rerun.

 


 

Missed Calls (top)

Description

Cause

Solution

-Peak annotation is missing, or incorrect, or represented with an "N"

-Limitation of software algorithm to recognize base correctly. Sometimes due to overlapping peaks, compressed peaks - poor peak spacing, weak signal, poor resolution of peak.

-Edit sequence manually.
-Disregard or trim degraded sequence region.
-Sequence region to verify correct sequence.

 


 

No Data (top)

Description

Cause

Solution

-No peaks detected.  Signal strength <25 for all bases.

-Template and/or primer concentration too low or absent.
-Priming site not present or wrong primer used.
-Excess salts or contaminants present.
-Capillary failed to pickup sample.

-Double check template and primer concentration.
-Double check primer site is present and correct primer was added.
-Clean-up template.
-Request sample be rerun.

 


 

Homopolymer Region (poly A, T, C, G) (top)



Description

Cause

Solution

-Sequence drop-off  or double peaks after homopolymer region

-Polymerase slippage

-Sequence in opposite direction.
-Redesign primers closer to homopolymer region.
-Redesign polyN primer with wobble base.
-Request sequencing with alternative chemistry.

 


 

Repeat Region (top)

Description

Cause

Solution

-Sequence drop-off after repeat region

-Depletion of dNTPs or presence of secondary structure.

-Request sequencing with dGTP chemistry
-Sequence from opposite direction.
-Redesign primers closer to repeat region.

 


 

Secondary Structures / Hairpins (top)

Description

Cause

Solution

-Abrupt decrease or loss of sequencing

-Secondary structure/hairpin restricting the polymerase from move through the region. 

-Request sequencing with dGTP chemistry.
-Sequence from opposite direction.

 


 

Contaminated Sequence (top)

Description

Cause

Solution

-Overlapping peaks after stretch of good sequence

-Plasmid prep contaminated – clean sequence typically vector region, overlapping sequence typically from two different inserts
-Human error – two templates mixed together.

-Pick new single clone and reprep.

-Request sample be resequenced. Possibly provide fresh aliquot of sample.

-Overlapping peaks from the very beginning

-Human error – two different templates mixed together.
-Two primers added to sample.
-Two priming sites on template.

-Request sample be resequenced.
-Submit new sample with one primer
-Redesign primer with specific binding site.

 


Helpful links (top)

Applied Biosystems - DNA Sequencing Chemistry Guide
http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_041003.pdf

Qiagen - Guide to Template Purification and DNA Sequencing
http://www.ibt.lt/sc/files/QIAGEN%20Guide%20to%20Template%20Purification%20and%20DNA%20Sequencing.pdf

Roswell Park Cancer Institute DNA Sequencing
http://www.roswellpark.edu/shared-resources/biomolecular-resource-facilities/dna-sequencing/troubleshooting-your-data

Oregon State University – Center for Genome Research & Biocomputing
http://corelabs.cgrb.oregonstate.edu/sequence/troubleshoot

University of Michigan - DNA Sequencing Core
http://seqcore.brcf.med.umich.edu/