The right answers to frequently asked questions
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What are IUPAC bases? Which results will I receive?
DNA base call identification is based on the nomenclature system
issued by the International Union of Pure and Applied Chemistry
(IUPAC). At positions of ambiguity, the following IUPAC codes are
used:
| G/T = K |
C/T = Y |
A/C/G = V |
| A/G = R |
A/C = M |
C/T/G/ = B |
| G/C = S |
A/G/T = D |
A/C/G/T = N |
| A/T = W |
A/C/T = H |
What are Q20 (Q30 / Q40) bases?
Eurofins Genomics is using a quality base calling software which examines the peaks around each base call to assign a quality score (Q) to each base call. Quality scores (Q) range from 4 to about 60, with higher values corresponding to higher quality. The quality scores are logarithmically linked to error probabilities, as shown in the following table:
| Quality Score | Probability of a wrong base call | Accuracy of a base call |
|---|
| Q 10 |
1 in 10 |
90 % |
| Q 20 |
1 in 100 |
99 % |
| Q 30 |
1 in 1.000 |
99.9 % |
| Q 40 |
1 in 10.000 |
99.99 % |
| Q 50 |
1 in 100.000 |
99.999 % |
Which sequencing method do you use?
For all our Sanger Sequencing Services we are using the cycle sequencing technology (dideoxy chain termination / cycle sequencing) on ABI 3730XL sequencing machines.
Could you explain the cycle sequencing technique?
Cycle sequencing is a modification of the traditional Sanger sequencing method. The components are DNA, primer, heat resistant DNA polymerase, 4 dNTPs, 4 ddNTPs (dideoxy terminator nucleotides) fluorescently labelled with four different dyes and buffer containing Mg++ and K+. The single primer binds to the complementary DNA strand and is extended in a linear mode.
This extension continues until by chance and depending on the complementary base a particular ddNTP is incorporated. Because of the latter's dideoxy-configuration the polymerase cannot add any other base to this fragment and the extension is terminated. Thus at the end of the selected number of cycles, numerous fragments with different lengths and one labelled nucleotide at the end are generated.
Stoichiometric manipulation of the reaction components ensure that the fragments of every possible length starting from n+1 say 2000 bases are generated with n being the number of bases in the primer. The key difference between the traditional Sanger method and cycle sequencing is the employment of a thermo stable DNA polymerase. The advantage of using such a polymerase, is that the sequencing reaction can be repeated over and over again in the same tube by heating the mixture to denature the DNA and then allowing it to cool down to anneal the primers and polymerise new strands. Therefore less template DNA is needed than for conventional sequencing reactions.
After a post sequencing reaction cleanup, the samples are electro kinetically injected into the array of 96-capillary sequencers. The negatively charged fragments migrate toward the anode by size, the smallest ones move fastest. Their tagged ddNTP terminators can be read as the fragment's base sequence. A laser beam excites these dye molecules as the fragments reach a detection window, producing fluorescent signals that are collected from all 96-capillaries at once, spectrally separated and focused onto a CCD (charge coupled device) camera. Sophisticated optical and electronic devices produce a colour readout that is translated with the help of sequence analysis software into a sequence.
What is the quality report?
The quality report is the trace file in *.pdf format in which the quality value of each single base is shown in colour code below each single peak. Different colours represent the four specified quality ranges.
Are there special features regarding the standard DIN EN ISO 17025?
Under the conditions listed below, only simplified test reports may be provided.
Subject: Simplified test reports according to DIN EN ISO 170252018, (Section 7.8)
Testing Laboratory (Accreditation): Eurofins Genomics Europe Applied Genomics GmbH (D-PL-13372, ) Eurofins Genomics Europe Sequencing GmbH (D-PL-17038)
For accredited examinations at these two companies, test results may only be reported in simplified form, i. Requirements of the standard DIN EN ISO 17025: 2018 (section 7.8) are not fully applicable to this type of transfer of results.
These results can therefore also be reported as short test reports, electronically generated result reports, Excel spreadsheets or as result files.
If you have any questions, please contact the Customer Care Team.
How can I view the .ab1 trace files
There are many softwares on the market which you can use for testing or buying.
For your convenience you can download and install our GATCViewer software for free.
What does "DONE_QV_NOT_MET" mean?
In order to ensure that the sequencing quality is not affected by a technical failure, an internal quality control is performed with each sequencing run. In addition to that, a sophisticated software is analysing each plate considering parameters like quality values (QV) and reading lengths.
If you see "DONE_QV_NOT_MET" as a result of your sequencing reaction, it means that the internal quality control has passed, but some sample quality values have not been met. (e.g. sample or primer concentration or volume, sequence design or structure etc.).