Sequencing FAQ

Frequently Asked Questions

A: Samples should be submitted in a total volume of 12 µl of DNA and primer mix. Sequencing Handout
Plasmid DNA 500 ng
PCR 10 ng/100 bp* (500bp needs 50 ng)
SsDNA 100 - 200 ng
BACs and Cosmids 1 - 2 µg
Bacterial Genomic DNA 2 - 3 µg

A: Yes we have the following primers we offer with no charge:
M13 Universal Primer(-20) Forward
5'-GTAAAACGACGGCCAGT-3'

M13 Universal Primer (-40) Forward
5'-GTTTTCCCAGTCACGAC-3'

M13 Reverse Primer (-24)
5'-GGAAACAGCTATGACCATG-3'

M13 Reverse Primer (-48)
5'-AGCGGATAACAATTTCACACAGGA-3'

SP6 Sequencing Primer
5'-GATTTAGGTGACACTATAG-3'

T3 Sequencing Primer
5'-AAATTAACCCTCACTAAAGG-3'

T7 Sequencing Primer
5'-TAATACGACTCACTATAGGG-3'

T7 Terminator Primer
5'-GCTAGTTATTGCTCAGCGGT-3'

A: A Core Prepared sample is where the cycle sequencing reaction and clean up is performed by the Core Facility and loaded on the sequencer.
A: User Prepared sample is where the customer does the cycle sequencing reaction and clean up and the Core Facility resuspends the sample in liquid and runs the sample on the sequencer.

A: All Core Prepared samples that are in the Core Facility at 10 am will be run that day.
All User Prepared samples that are in the Core Facility at 1 pm will be run that day.
Whole plate samples can be run when they are brought in or as scheduling permits.

.. Run you sample out on a gel to make sure that there is only one template present.
.. Check your primer sequence and make sure there aren’t two primer binding sites on your template.
.. Clean up your sample again to try and get rid of any contamination.

A: Phred Quality Scores

Phred is a base-calling program for DNA sequence traces. The program was developed by Drs. Phil Green and Brent Ewing, and is copyrighted by the University of Washington.

Phred's base-specific quality scores are one of the most innovative features in Phred. After calling bases, Phred examines the peaks around each base call to assign a quality score to each base call. Quality scores 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:

Phred quality score	Probability that the base is called wrong	Accuracy of the base call
10	1 in 10	90%
20	1 in 100	99%
30	1 in 1,000	99.9%
40	1 in 10,000	99.99%
50	1 in 100,000	99.999%

It has been shown that Phred's error probabilities are very accurate - if Phred assigns a quality score of 40 to a base, the chances that this base is called incorrectly are indeed just 1 in 10,000. This high accuracy has been observed for sequences generated at different laboratories, each using a different combination of sequencing enzymes, fluorescent dyes, and gel run conditions.

The high accuracy of Phred quality scores make them an ideal tool to assess the quality of sequences. The most commonly used method is to count the bases with a quality score of 20 and above (sometimes called "high quality bases") or 30 and above ("very high quality bases"). By looking at individual sequences, failed reactions or low-quality reads can easily be identified. When looking at collections of sequences, the effect of different sequencing methods on sequence quality can be directly measured. This allows straightforward quality control in sequencing projects, and can give easily available measures to optimize sequencing operations.