DNA SEQUENCING

RESEARCH GROUP

Annual Report 2000

The 2000 DNA Sequencing Research Group (DSRG)members include Laurence Hall, John Hawes, TimHunter, Emily Jackson-Machelski, Kevin Knudtson,Dina Leviten, Margaret Robertson, and TedThannhauser. Ad hoc members include Duane Bart-ley, George Grills, Kathryn Lilley, and James VanEe,and the Executive Board Liaison is Mark Lively.

We believe our mission is to

• Promote and encourage communication andcooperation among member laboratories thatcarry out DNA sequencing

• Conduct studies to assess the capabilities of DNAsequencing laboratories and to disseminate thefindings of these while they are still accurate andrelevant

• Provide materials that help member laboratoriesevaluate their performance and that promoteexcellence in DNA sequencing

• Provide a mechanism for sharing of informationrelevant to the organization, administration andfinancial management of DNA sequencing labo-ratories.

Besides being an informational resource, thegroup conducts studies to assess the capabilities ofDNA sequencing core laboratories. Two studies wereconducted and analyzed this year. The bacterial arti-ficial chromosome (BAC) study had two parts; thefirst was to determine a robust protocol for BACsequencing, and the second was to find a rigorousprotocol/kit to purify BAC DNA that, whensequenced, gives clean and accurate data. This studywas conducted in three steps:

1. An initial external study in which BAC DNA wassent and sequenced using the participants’ protocol.Data and protocols were submitted for analysis.

2. An internal study, coming up with a “standard”protocol within the DSRG.

3. A tertiary study in which the initial external studywas repeated using a standard protocol.

The results from part 1 of this study suggest thatthere are two critical determinants of sequence qual-ity when dealing with BAC templates. These are theamount of template used and the amount of cyclesused in the PCR reaction.

An online poster of the results can be viewed athttp://brcweb.biotech.cornell.edu/abrf01_bac_poster.pdf. However, because of a statistically insignificantnumber of participants, part 2 of this study is stillopen for submissions, and information on the studyresults will be disseminated later.

The group conducted a general survey to comeup with a detailed summary of the composition andconfiguration of an average DNA sequencing corefacility. Compared with the 1998 study, we found sev-eral interesting changes:

• The average laboratory age was 3 years.

• The number of reactions run was up by about30,000.

• The number of PIs or laboratory groups sup-ported doubled.

• The turnaround time was reduced by 10 hours.• Outside service charges were one-half the price.

• There was trend toward dye terminators (88%versus 99.6%).

• BigDyes replaced most rhodamine use.

• For data analysis software, laboratories havemoved from PE Biosystems Ver. 3.0 to Ver 3.3.

• The number of laboratories participating in thesurvey was down from 59 to 37, which may be aresult of the shorter time available for entering thesurvey.

Journal of Biomolecular Techniques12:27–29 © 2001 ABRF

FRAB

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The results were presented at ABRF 2000 and can befound at http://www.abrf.org/ABRF/ResearchCom-mittees/DSRG/general2k/.

Two studies are being prepared for launch thiscoming year. The Never Ending Study (NES) will bereopened to include new technologies. This studyanalyzes the effect of different DNA sequencing meth-ods on the quality of resulting data using a standardtemplate. A website has been developed for the NESas a database and resource (http://nes.biotech.cornell.edu/nes).

The second study will determine whether singlenucleotide polymorphisms (SNPs) can be sequencedaccurately using the equipment and chemistries cur-rently used in participating laboratories. Our groupcame up with a list of questions and launched aninternal study (DSRG only) to determine whetherthese questions could be answered when we launchthe study externally. Note that we are screening forSNPs, not discovering them. The questions we askedand the information we gained from our internalstudy follow:

• What software is available for analyses? Can thissoftware handle high throughput? We initiallytried Polyphred and Sequencher. We opted forSequencher because of its accuracy and ease ofuse. We determined that Polyphred needs moreexploring and that other software, unknown tous, may also work well for the analysis of SNPscreening.

• Can mutations or mixes be detected with oneread (forward or reverse)? We found that in mostcases you need more than one read to correctlyidentify the SNP.

• Does slab gel versus capillary electrophoresisshow a difference? No.

• Dye primer versus dye terminator chemistry? No.

• Can laboratories detect these mutations or mixes?Yes, but determining the exact percentage of themixture was more difficult to predict than from a50:50 mixture.

• What is the lowest percent mixture accuratelydetectable? We could detect a 70:30 mixture withconfidence, but lower percentage mixtures werenot identified as confidently.

These are preliminary results from our group,and our conclusions may alter after a larger exter-nal population of data is analyzed. The study willbe launched in the near future, and the group isparticularly interested in assessing mutations andmixed base detection and may include insertionsand deletions.

In 2001, look for the following events:

• Launching the SNP study, possibly adding dele-tions and insertions

• Reopening the NES to include new technologies • Continuing to collect samples from the BAC

study, part 2• Getting ready to launch the 2001–2002 General

Survey.

Thank you to all our participating laboratories!Thanks also to the BAC study members: ProfessorJune Nasralla, Tom Stelick, Bill Enslow, Tatyana Pyn-tikova, and Farhad Dhabhar (Cornell University), aswell as to the SNP study participants: The Institute forGenomic Diversity, Professor Steve Kresovich (CornellUniversity), Heather Lin (University of Utah), andChristine Hansen (ICOS Corp.). For more informa-tion, see http://www.abrf.org.

FRAGMENT ANALYSIS

RESEARCH GROUP

The Fragment Analysis Research Group (FARG) recentlycompleted an educational study focusing on a commonproblem associated with dinucleotide markers used infragment analysis and its possible solution. Typically,dinucleotide markers amplify the correct-size productsbut often amplify a secondary product caused by anontemplated nucleotide addition. This can hinderidentification of the correct peak during analysis. Thesolution presented in the study involved the use oftailed reverse primers and demonstrated the efficacy ofthis method for improving accuracy of allele calling. Theresults were presented in a poster and during theResearch Group presentation at the ABRF 2001 meetingin San Diego. The poster and the Research Groupreport are available by accessing the ABRF web site:http://www.abrf.org/Research Groups/FARG.

The members of FARG would like to express theirappreciation to all researchers who participated inthe study. As a token of our appreciation, we haveprepared certificates of appreciation for all partici-pants. However, because the study was anonymous,any participant interested in receiving a certificateshould contact the FARG chairperson through theABRF web site.

The FARG report during the conference was fol-lowed by a 30-minute panel discussion about real-time quantitative PCR in a core facility setting. The dis-cussion briefly touched areas such as service setup,pricing, and instrumentation to help those interestedin starting a real-time quantitative PCR service in theirfacilities.

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FARG also sponsored a tutorial session duringthe ABRF conference. Tutorial speakers LynnPetukhova and Caprice Rosato discussed basicmethodology and problem-solving methods for slabgel and capillary electrophoretic fragment analysis.

The topics included ways to improve speed and accu-racy while reducing the cost by multiplexing. Bothtutorial presentations are available by accessing theABRF web site/Research Groups/FARG.

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