ABRF, DNA Sequence Committee Report 1996

submitted: 6/3/96, published: 6/18/96, last updated: 12/3/96, © 1996 ABRF

DNA SEQUENCING OF A MODERATELY DIFFICULT TEMPLATE: EVALUATION OF RESULTS FROM A THERMUS THERMOPHILUS UNKNOWN TEST SAMPLE.

Association of Biomolecular Resource Facilities (ABRF) 
DNA Sequence Committee
9650 Rockville Pike, Bethesda, MD 20814

Pamela Scott Adams  W. Alton Jones Cell Science Center, Lake Placid, NY
Mary Kay Dolejsi    Fred Hutchinson Cancer Research Center, Seattle, WA.
Susan Hardin        University of Houston, Houston, TX .
Sheena Mische       Rockefeller University, New York, NY.
Betsy Nanthakumar   Johns Hopkins University, Baltimore,
Harold Riethman     Wistar Institute, Philadelphia, PA.
John Rush           Howard Hughes Medical Institute, Harvard Medical School Boston, MA 
Paul Morrison       Dana-Farber Cancer Institute, Boston, MA.

Acknowledgements

ABSTRACT

The Association of Biomolecular Research Facilities (ABRF) DNA Sequence Committee (DSC) completed a study of 95 ABRF member facilities that offer DNA sequencing as a core service. A Thermus thermophilus DNA template and a survey questionnaire was sent to these facilities. The objective was to evaluate the sequencing data obtained from core laboratories when analyzing a moderately difficult template. Because of recent changes in enzymes used for DNA sequencing the results will be useful to determine what improvement these new enzymes have made to DNA sequencing data in a core facility setting.
The questionnaire was used to survey the DNA sequencing services offered by member laboratories.

METHOD

A library of Thermus thermophilus DNA was prepared by inserting random partial Sau3A fragments of genomic DNA into BamHI-cleaved pUC18 DNA. The library was transformed into strain XL1-Blue (Stratagene), selecting ampicillin resistance and screening for white colonies on Xgal-IPTG plates. This particular clone was chosen at random from the library and designated ABRF-DNA96, the first test sample distributed by this committee. The DNA was prepared using a Qiagen "Mega-prep" kit according to the manufacturer. The sample was diluted to 1 microgram/microliter , dispensed in 20 microliter aliquots and dried down in a SpeedVac. Samples, along with a sample specific questionnaire, a general survey questionnaire, a floppy disk and a return envelope addressed to a third party, were mailed to 95 ABRF facilities that offer DNA sequencing as a service. Samples were sent on January 16, 1996. The deadline for return of of data was set for February 10, 1996. This deadline was later extended to February 27, 1996.

RESULTS

The sequence of ABRF-DNA96

Some of the CONDITIONS used by study participants to sequence this sample in table form arranged by length of read.

TROUBLE SPOTS

% ACCURACY of all submissions sorted by machine type and whether they were edited or not.

Results from the GENERAL SURVEY about DNA Sequencing Core Facility conditions.

Summary

In its first study, the DNA Sequence Committee sent a test sample to 95 facilities that perform automated DNA sequencing to evaluate how well they could sequence a moderately difficult template. The test sample chosen by the Committee was a double-stranded plasmid containing a GC-rich Thermus thermophilus DNA insert (69% GC over 926 bp). A survey questionnaire was included with this test sample to determine how DNA sequencing services have changed since the last survey was conducted two years ago. The Committee received 73 sequencing datasets from 50 facilities, and 53 completed surveys were returned.

Sequencing ABRF-DNA96. Sequences submitted by study participants were ranked according to length of entirely correct sequence, i.e., the longest segment of sequence without ambiguous assignments, miscalls, deletions, or insertions. We chose this criterion for ranking because we felt it most closely matches the expectations of collaborating researchers submitting samples, who rely on facilities to provide entirely correct data. The average length of entirely correct sequence was 284 bases, and the longest was 615 bases.

We noticed that gel length, manual data review by facility personnel and sequencing chemistry influenced sequence accuracy and length. Although only 17% of the datasets were obtained with 48 cm or longer plates, these tended to cluster near the top of the ranking: four of the top ten responses were obtained on ABI 373 Stretch instruments with 48 cm plates. Even though 70% of the datasets were not edited, six of the seven top-ranked responses were. Several facilities submitted both edited and non-edited datasets: comparing these for each facility showed that, on average, editing increased the length of entirely correct sequence by 157 bases. Nearly all study respondents submitted data obtained using dye-terminator sequencing chemistry and the enzyme TaqFS. Some facilities submitted data they obtained with and without DMSO in the sequencing reactions. In most cases DMSO increased the length of entirely correct sequence; however many of the top-ranked responses did not use DMSO.

DNA Sequencing Facility Survey. The survey showed the number of facilities performing automated DNA sequencing and the number of samples sequenced per year continues to grow: 56% of survey respondents sequenced 1000-5000 samples last year, and the total number of samples sequenced by the respondents was 223,548 corresponding to almost 100 million bases. Based on the survey, a typical DNA sequencing facility offered primer design and synthesis, DNA analysis and database searches, but not template preparation services. Of the templates sequenced, 76% were double-stranded plasmids, and 21% were PCR products. Fluorescent techniques were used by all contributing facilities, dye-terminator chemistry was used by 82%, and two-thirds used ABI's TaqFS enzyme.

The DNA Sequence Committee linked the contents of this report to the ABRF web page and will modify the report as it refines its analysis of the data. Several datasets that did not make the submission deadline will be added to the analysis as time allows.

The committee would like to thank all participants in this study for making it possible. Each participant is a collaborator on a report like this and your responses are appreciated.

Other web sites are encouraged to make links to this report. Please send any comments or suggestions about this report to the committee by sending email to Paul Morrison, paul_morrison@dfci.harvard.edu