Results From the DNA Sequencing Research Group’s General Survey of Core Facilities, 2000

Dina Leviten, George Grills, Susan Hardin, Margaret Robertson, Theodore Thannhauser and James VanEe.

DNA Sequencing Research Group

Survey Goals

• The goal of this survey was to come up with a detailed summary of the composition and configuration of the average DNA sequencing core laboratory.

• For this survey, we had 37 labs submit information.

DNA Sequencing Research Group

Year the Core Was Established

• Mean = 2.3 years • Median = 6 years • Range = 1-15 years

Year Seq. Lab Established

7 6 5 4 3 2 1 0 1984 1985 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999

Year

DNA Sequencing Research Group

Number of Reactions Run in 1999

Number of Reactions in Last 12 Months 20 15 10 5 0 11 16 4 4 2 <10K 10-20K 21K-30K 31K-40K

Number of Reactions

>40K • Mean = 18,813 • Median = 13,000 • Range = 268 – 80,000 DNA Sequencing Research Group

Principal Investigators/Lab Groups Supported

• Mean = 87 • Median = 80 • Range = 3 - 225 PI's/Lab Groups Supported 15 10 5 0 10 11 10 4 2 >50 51-99 100-150 151-200 Number PI's/Labs Supported >200 DNA Sequencing Research Group

Turnaround Time

• Five of the respondents have a set cut-off time to help define the turnaround time for samples.

• Mean = 53 hours • Median = 48 hours • Range = 20 - 96 hours DNA Sequencing Research Group

Services Provided Besides DNA Sequencing

Services Other than DNA Sequencing 60% 50% 40% 30% 20% 10% 0% Pr im er d O es lig ig Te n o s m yn pl th ate .

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rtP CR O th er Services DNA Sequencing Research Group

Physical Resources

Square feet of lab space: • Mean = 545 • Median = 368 • Range = 28 - 5,000 Square feet of office space: • Mean = 127 • Median = 90 • Range = 0 - 1,200 DNA Sequencing Research Group

Subsidized vs. Self-Supporting

• Subsidized = 62% Percent subsidized Mean = 26% Median = 27.5% Range = 4 - 100% • Self-supporting = 38% DNA Sequencing Research Group

Service Fees

• Most facilities do not charge for editing or expedited service.

– In-house=customers at your institution/company – Outside=customers not at your institution/company – reaction provided=customer performed reaction – reaction not provided=seq. reactions performed by lab • Other charges: – Printing – Primers – Fragment assembly – Reactions that don't work Service Fees $40.00

$35.00

$30.00

$25.00

$20.00

$15.00

$10.00

$5.00

$0.00

In house/rxn.

provided Outside/rxn.

provided In house/rxn.

not provided Outside/rxn.

not provided Service Provided Minimum Maximum Median Mean DNA Sequencing Research Group

Facility Director Education

BA/BS 14% MS 8% phD/MD 78%

Director of Facility

• Mean = 6 years • Median = 5 years • Range = 1 - 12 years Scotch: 31 responses • single malt = 52% • blend = 16% • Mother Theresa = 10% • no pref = 23% Only brand that had more than one vote (2) • Glenfiddich DNA Sequencing Research Group

Staff

• FTE’s working in the core: Mean = 2 Median = 2 Range = 0.1 - 8 • Experience of staff: Mean = 2 years Median = 3 years Range = 0 - 10 years DNA Sequencing Research Group

Instrumentation

Instrumentation/Manufacturers Number of sequencers in the core facility: – Mean = 3 – Median = 2 – Range = 1 - 15 373A_24wtr 6% 377_36 2% 373S 6% 377_96 17% 373S_XL 21% 310 1% 3700 5% ALFexp 377_18 1% 1% CEQ2000 1% Other 5% 377_48/64X L 37% licor4200LD 2% DNA Sequencing Research Group

Templates

Type of Templates DS Plasmid 61% PCR Products 25% PAC 2% YAC Other 8% SS Plasmid 5% BAC 4% Cosmid 2% Genomic 1% other DNA Sequencing Research Group

Robotics

• 3 Respondents use robotics – 2 Beckman BioMek 2000’s – 1 Qiagen BioRobot 9500 • Determination of Quality/Quantity – Agarose gel – OD 260 – Both Agarose gel and OD 260 DNA Sequencing Research Group

Template Preparation

Recommended Template Preparation Alkaline Lysis Cesium Chloride 8% 15% Commercial Kit 77% Determination quantity/quality: •OD260 = 47% •Agarose gel = 42% •Flourescent assay = 8% •Other = 3% Most labs don’t require documentation of template purity Recommended Commercial Kits Qiagen 73% Promega 15% BioRad 2% MOBIO 4% Centricon Concert 2% 2% PE/ABI 2% DNA Sequencing Research Group

Sequencing Chemistry - Dyes

Dye Types – Dye Terminator = 99.6% – Dye Primer = 0.4% Flourescent Dyes Used PE BigDye 77% PE Big Dye dGTP 1% Other 4% Other 4% PE dRhod.

10% PE old Rhod.

8% Amersham ET dyes DNA Sequencing Research Group

Sequencing Chemistry - Enzyme

Sequenase V2 3% Amersham TSII 1% Enzyme Used Taq Gold (Beckman) 3% PE TaqFS 93% DNA Sequencing Research Group

Reaction Cleanup

Format of post-PCR cleanup: •Single tube = 64% •96-well plate = 25% •Both = 11% Spin Column Manufacturers Type of Post-Reaction Cleanup NH4OAC/E tOH ppt 3% Other 8% NaOAC/Et OH ppt 31% Spin column 58% Other 26% Edge Biosystems 29% Millipore 15% Bio-Rad 12% Princeton Separations Pharmacia 10% 8% DNA Sequencing Research Group

Gel Mixes/Capillary Matrices

Gel/Capillary Matrices Sources FMC LongRanger - Singel 33% Other 24% FMC Long Ranger - 50% 44% PE POP6 3% Bio-Rad 6% Amresco Gene PLUS6% 3% Homemade 2% Other 6% Amresco Gene-PAGE PLUS CONC.

3% DNA Sequencing Research Group

60 40 20 0

Gel Information

Length of Gel/Capillary 24cm 36cm 48cm Length (cm) 50cm • Routine standards are run: Yes = 89% No = 11% Plate/Capillary Manufacturers PE Biosystems array 3% Sigma 5% The Gel Company CellarSTO 4% P 2% Sooner Scientific 6% Other 6% PE Biosystems 74% DNA Sequencing Research Group

Data Analysis/Basecaller

•By far the most prevalent data analysis software used is the PE Biosystems Version 3.3 (69%).

•The most used basecallers were ABI 100 (59%) and ABI 50 (19%).

DNA Sequencing Research Group

Additional Software/Tools Used

26 Responses (multiple answers per response): • 19 use Sequencher (GeneCodes) • 8 use The Wisconsin Package (Genetics Computer Group) • 3 use Lasergene (DNASTAR) • 2 use DNAsis (Hitachi Genetic Systems) • 2 use AutoAssembler (PE Biosystems) DNA Sequencing Research Group

Data Management/Distribution

Judging Quality of Data Confidence values 6% Visual inspection 52% Accuracy of standards 42% • Most labs check the quality of the data by two of the above methods (73%) and 11% check all three.

Results Distribution • 21/37 Store results forever • 16/37 Store results for 0.25-6 months. (Avg. 2 mo.) • Hard Copy (32/37) – Range 1-100% – Median 100% • Floppy/Zip disk (20/37) – Range 1-100% – Median 42.5% • Electronic (35/37) – Range 5-100% – Median 99% DNA Sequencing Research Group

Data Storage/Archiving

Media Storage Types Hard drive/RAID 12% Other 6% CD's 24% Opt. Disk 12% Mag. Tape 10% Jaz disks 14% Zip disks 22% • Data Storage – 30/37 Store data forever – 7/37 Store data for 1 36 months • Mean = 11 months • Median = 6 months DNA Sequencing Research Group

Information Management Systems

• 28/33 use in-house designed/modified program or programs like Excel and Paradox for tracking/billing.

• 5/33 are moving or have recently moved to a commercial product for tracking/billing – All 5 commercial LIMS systems are from different vendors.

DNA Sequencing Research Group

Future Directions of Core Facility

• Longer reads, better tracking, less expensive.

6 months 6-12 months 12-18 months 18+months no plans Plans to add new instrumentation

4 PE Biosystems 3700 2 PE Biosystems 377's 3 PE Biosystems 377's 1 3700 or M egaBACE 1 PE Biosytesms 3700 1 377 or 3700 2 PE Biosytesms 3700 1 PE Biosystems 377 1 3700 or M egaBACE 1 waiting for midsize capillary 1 PE Biosystems 3700 19 DNA Sequencing Research Group

Suggestions for Future Studies

• Manual editing differences from lab to lab • Stress levels of different labs (customer pressures) • List of tips for different procedures in sequencing (PCR, P1 seq., repeats in sequence...) • More 3700 information • Length of Reads required by customer DNA Sequencing Research Group

Average Core Facility Profile

• The average core facility is 7 years old and runs ~40,000 sequencing reactions per year.

• They support 111 PI’s/lab groups with a turnaround time of 38 hours.

• This lab offers at least 2 other services and has on average 2,500 sq. ft. of lab space and 600 sq. ft. of office space.

DNA Sequencing Research Group

Average Facility Profile Continued...

• The lab is subsidized 48% and usually does not charge extra for services such as editing and expedited samples.

• The director of the facility has 6 years of experience and likes single malt Glenfiddich scotch, the rest of the staff has 5 years of experience.

DNA Sequencing Research Group

Instrumentation/Reactions

• Average of 7 sequencers (95% from PE Biosystems).

• Sequencing DS-DNA templates (61%), PCR products (21%) and SS-DNA (5%).

• Qiagen Template preparation, with quality checked by both agarose gel and OD 260 .

• Dye Terminator using TaqFS, cleaned up with spin columns (58%, Edge) or EtOH pptn. (31%).

DNA Sequencing Research Group

Gel Information

• FMC is the gel matrix of choice (83%), where read length is usually 36cm (51%) or 48cm (43%).

• PE Biosystem plates/arrays are used (77%) and standards are usually run (89%).

• The analysis software is PE Biosystems Version 3.3, where basecaller ABI100 is used.

DNA Sequencing Research Group

Data Management/Distribution

• Visual inspection and quality of the reads of the standards are the way quality of a sequencing run is checked.

• Customers get hard copy and electronic copies of their data.

• Data is kept on disks (Jaz, Zip and Optical, 48%) forever (81%).

DNA Sequencing Research Group

Other Tools

• Sequencher (GeneCodes) is the most prevalent tool used. (73%) • An in-house designed or modified program is used to track samples and do billing.

• Another sequencer will be done in 6-12 months from PE Biosystems.

• Future directions are to do more for less.

DNA Sequencing Research Group

Survey Differences 1998 vs. 2000

• Average age of lab was up 3 years.

• Number of reactions run, up by ~30,000.

• Number of PI’s/lab groups supported doubled.

• Turnaround time was reduced by 10 hours.

• Outside service charges were 1/2 the price.

• Trend towards dye terminators. (88% vs. 99.6%) DNA Sequencing Research Group

1998 vs. 2000 Continued

• BigDyes replaced most Rhodamine use.

• For data analysis software, labs have moved from PE Biosystems Ver. 3.0 to Ver 3.3.

• Number of labs participating in survey was down from 59 to 37 which may be due to a shorter time available for entering the survey.

DNA Sequencing Research Group

Acknowledgements

• All the labs who participated in the survey.

• Web site for this poster is : – http://abrf.org/ABRF/ResearchCommittees/DS RG/DSRG2000/ Note: This is case sensitive!

DNA Sequencing Research Group