Sanger sequencing troubleshooting guide. GATC Biotech AG

Size: px

Start display at page:

Download "Sanger sequencing troubleshooting guide. GATC Biotech AG"

Eustace Skinner
6 years ago
Views:

1 Sanger sequencing troubleshooting guide GATC Biotech AG April, 2017

2 Introduction All sequencing data generated at GATC Biotech is carefully analysed before it is delivered to the customer. In cases where issues arise with the sequencing data, we recommend using the following guide to commonly encountered problems and possible solutions. 1 Signal drops or stops abruptly Problem: A sudden drop or loss in signal is usually caused by secondary structures in the DNA. These secondary structures tend to form in GC-rich or AT-rich genomic regions. Often, the polymerase stops reading fluently in these areas, causing the appearance of short reads or no reads at all on the chromatogram. Solution: Use different primers or apply optimised sequencing protocols for secondary structures (available with SUPREMERUN) 2 Unreadable sequence after a mononucleotide stretch Problem: Sequences that contain large mononucleotide stretches could show overlapping peaks after the stretch. Whether or not the problem occurs depends on the length of the stretch and how well the primer binding works. Solution: Try sequencing with both a forward and reverse primer to cover the stretch. GATC Biotech

3 3 Presence of large peak at beginning of sequence Problem: The sequence starts with a large, unreadable signal. The following peaks appear in low intensity and the read length is often too short. This could be caused by primer dimers leading to weak primer binding or by presence of super concentrated DNA. If the DNA is in super concentrated, the template and primers could be depleted in the early cycles of the sequencing reaction, resulting in an excess of short DNA fragments. These fragments could block the capillary and inhibit the reaction flow. Solution: Reduce concentration of DNA or primer 4 Presence of dye blob on chromatogram Problem: Unincorporated dye terminators appear between positions 50 to 80 bp and are superimposed over the real peaks. The dye blobs often result from failed or impure sequencing reactions and can be affected by template concentration and quality, as well as by impurities of EDTA, salt, protein or solvents Solution: Use a primer which binds upstream of the region or check quality of template by gel electrophoresis and spectrophotometer GATC Biotech

4 5 Repetitive region in sequence Problem: Sequences with repetitive regions often show a signal decrease. Solution: Apply optimised protocol for sequencing of repetitive regions (available with SUPREMERUN) 6 High initial peak and no readable sequence Problem: Super concentrated DNA often causes high initial peaks in the chromatogram and no readable sequence afterwards. Solution: Make sure your template and primer concentrations and volumes correspond to our sample requirements. GATC Biotech

5 7 Presence of flat peaks Problem: Peaks appear flat or could blast out of range whenever the DNA concentration is too high. Solution: Make sure your template and primer concentrations and volumes correspond to our sample requirements. 8 Empty or noisy chromatogram Problem: The sequencing reaction has failed. This could be due to improper primer or template concentrations or due to primer binding issues. Solution: Perform a quality control check of your DNA by gel electrophoresis prior to sending us the samples GATC Biotech

10 Base line is highly elevated in raw data Problem: Raw data of chromatogram shows highly elevated base line.

6 9 Presence of additional small peaks / N-1 primer binding Problem: The primer is binding n-1, giving rise to small peaks underlaying the correct peaks shifted n-1. This scenario is often caused by primer degradation. Solution: Repeat sequencing with resynthesized primer. 10 Base line is highly elevated in raw data Problem: Raw data of chromatogram shows highly elevated base line. This problem could arise due to high content of salt in the sample. Solution: Use a desalting kit to further purify the DNA template before repeating the sequencing. GATC Biotech

11 Double sequence with two or multiple peaks in the same location Problem: The sequence starts off with good quality and then turns into a mixed sequence.

7 11 Double sequence with two or multiple peaks in the same location Problem: The sequence starts off with good quality and then turns into a mixed sequence. This could be caused by the accidental picking of two colonies during DNA preparation, resulting in sequencing of more than one insert. Solution: Prepare a new DNA sample. 12 Mixed Template Problem: The sequence has multiple peaks in the same location from the beginning onwards. This could arise due to multiple PCR fragments being present in the reaction or if more than one primer was added per reaction. The problem could occur in cases of multiple primer binding sites on the template strand of if the PCR reaction was not cleaned well following DNA preparation. Solution: Make sure you provide only one template with one primer per sequencing run. Ensure the template you are sequencing has only one binding site for your primer of choice. Perform careful PCR purification to avoid residual salts in the reaction. GATC Biotech

8 Copyright GATC Biotech K-1478 v GATC Biotech AG European Genome and Diagnostics Centre Jakob-Stadler-Platz Konstanz Germany Tel. +49 (0) Fax +49 (0) GATC Biotech AG European Custom Sequencing Centre Gottfried-Hagen-Straße Köln Germany

Sanger Sequencing: Troubleshooting Guide

Sanger Sequencing: Troubleshooting Guide If you need help analysing your Sanger sequencing output, this guide can help. CONTENTS 1 Introduction... 2 2 Sequence Data Evaluation... 2 3 Troubleshooting... 4 3.1 Reviewing the Sequence... 4 3.1.1