APPLICATION INFORMATIONTECHNICAL� INFORMATION

IntroductionAutomated DNA sequence analyzers have increasedthe throughput and ease of use of an application thathas become a core technique for many areas ofgenetic analysis. For large de novo sequencing proj-ects, automated DNA sequence analyzers have obvi-ously shown their advantage and strength. Whilemost DNA sequencing is confirmatory in nature,with required read lengths of 700 bases or less,some researchers need to maximize base readlengths to economize on laboratory resources.

Although the CEQ™ Genetic Analysis Systemproduces sequence read lengths greater than 700bases using the standard sequencing methods provid-ed with the instrument, longer sequencing readlengths are possible by modifying the separationparameters. This bulletin presents a strategy forincreasing base read lengths by 20% or more by mod-ifying the separation voltage, duration, capillary tem-perature, and injection time parameters as comparedto the existing CEQ LFR1 sequencing separationmethod. Applying this new separation method canachieve average 98% accuracy base read length cut-offs greater than 900 bases with some individual sam-ples obtaining 98% accuracy cutoffs of 1000+ bases.

MethodDNA PurificationOvernight cultures of DH5α cells containingpUC18 as a control plasmid and pUC19 vector con-taining a 1.2 kB Glucouronidase gene insert (pGus)were grown overnight in rich bacterial media with100 µg/mL ampicillin to an optical density of 4-5.The cultures were transferred to deep square-well

plates at 1 mL per well. Plasmid DNA preparationwas performed on a Biomek® FX LaboratoryAutomation Workstation from Beckman Coulterusing both the Promega Wizard* SV 96 PlasmidDNA Purification System and the QiagenQIAprep* 96 Turbo Miniprep plasmid DNApurification kit. The Biomek FX program forthe Promega Wizard has been described previ-ously in Beckman Coulter Application BulletinA-1907A.(1) This Biomek FX program wasmodified for use with the Qiagen QIAprepDNA purification kit.

DNA Sequence ReactionSince many DNA prep technologies yield alarge quantity of supercoiled plasmid whichcan affect the performance of sequencefragment separations, appropriate preheattreatment of plasmid DNA for any particular celltype and purification method combination should bedetermined by performing a temperature-versus-timematrix.(2) In addition, for obtaining longer read lengthseparations, the preheat treatment should be as gentleas possible in order to relax the supercoiled plasmidswhile not nicking them so much that producinglonger fragments during the cycle sequencingreactions becomes problematic. Both of theDH5α-grown plasmids in this study were preheattreatment optimized using a matrix of threetemperatures (65°C, 76°C, and 86°C) and four time

T-1975A

METHOD FOR LONG SEQUENCING READ LENGTHS ON THECEQ GENETIC ANALYSIS SYSTEM

G e n e t i c A n a l y s i s

Doni Clark, Jim Thorn, and Keith RobyBeckman Coulter, Inc.

lengths (1 to 4 minutes). The final preheat treatmentcondition for both of these purification methods thatmaintained appropriate signal strength for the longersequencing fragments was 3 minutes at 86°C.

Sequencing reactions were performed with boththe pGus plasmid and the pUC18 control plasmidusing the CEQ™ Dye Terminator Cycle SequencingKit chemistry (CEQ DTCS Kit, Beckman Coulter,Inc., PN 608000). All reactions used 70 fmol of plas-mid DNA template which was thermal cycled as sug-gested in the CEQ DTCS kit protocol. While thisquantity of DNA template worked well in our stud-ies, many researchers may need to adjust this tem-plate quantity for their specific needs based on tem-plate type and size. Another means of increasing theoverall signal and hence the amount of longer frag-ment signal is to increase the number of cycles in thethermal cycling parameters. This can be combinedwith resuspension of the final sequencing products inless Sample Loading Solution (30 µL instead of40 µL). It should be noted that incrementally increas-ing signal strength by increasing the effective con-centration of the sequence fragments in the samplecauses more sample to be injected onto the capillary

which negatively affects the resolution of the longerfragments. Long fragment signal strength and resolu-tion are both critical factors for achieving long baseread lengths; therefore, a balance must be achievedthat provides adequate signal strength for detectionbut not so much as to negatively impact resolution.Purification of the sequencing reaction products wasperformed by the plate precipitation techniquedetailed in Application Bulletin A-1903A.(3) Afterdrying the plates, the sequencing products wereresuspended in 40 µL Sample Loading Solution,overlaid with mineral oil, and loaded onto the CEQ8000 Genetic Analysis System. Note: This methodwas not tested on the dual-plated CEQ 8800 System.

Results and DiscussionTo obtain longer sequencing read lengths, the sepa-ration method parameters were modified to reducethe voltage and increase the data collection durationover a range of capillary temperatures. After per-forming a series of voltage versus temperatureexperiments, it was found that the following separa-tion method yielded the most consistent results(Figure 1).

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Figure 1. Sample Setup module Method tab.

Using the new separation method parameters, acomparison was performed against the defaultLFR-1 separation method supplied with the CEQ8000 Genetic Analysis System. For the pUC18DNA prepared with the Promega kit, 24 individualsamples were run. All other sample sets for both thecontrol pUC18 and pGUS DNAs represent a total of48 individual samples per DNA purification chem-istry and separation method. Comparison of peakresolution is performed in Figure 2. Visually, peakresolution is similar between the standard LFR1separation method and the new longer read methoduntil approximately base 400. Beyond that point,the new longer read separation method clearlydemonstrates improved peak resolution.

Note: Capillary lifetime studies were not testedwith the new, longer-read separation method.

Table 1A shows representative base read length98% accuracy cutoffs for each plasmid, separationmethod, and purification chemistry combination.The sample names are coded as such: QpUC18 andQpGUS represent the template DNAs purified bythe Qiagen chemistry, while pUC18 and pGUS fromTable 1B are the DNA templates purified by thePromega chemistry. 86c 3min indicates the preheattreatment used for all samples, and LFR1 or 3kV 55cindicate, respectively, the standard CEQ Genetic

Analysis System sequencing method or an abbrevia-tion of the new optimized separation method.

The 98% accuracy cutoff results were averagedand compared for each set of templates (Table 2). Ineach combination of DNA purification chemistryand plasmid template, the new separation methoddemonstrated an increase of greater than 20% forthe base read length 98% accuracy cutoff values.

Use of this separation method increases theoverall base sequence yield from a single reactionby approximately 20% and thereby maximizes theamount of sequence information obtained from asingle reaction. In some instances where DNA tem-plates may not yield sufficient fluorescent signal, itmay be helpful to increase either the number ofcycles during the sequencing reaction thermalcycling, or to increase the injection time duration inthe separation method. Be aware, though thatadding too much DNA template to the sequencingreaction to boost signal strength by making moreproduct or increasing the injection time too muchwill greatly impact the resolution of the longersequencing fragments and should be avoided. Thisis an important point to consider since, in order toachieve the longest possible sequencing readlengths, it is absolutely critical to maintain resolu-tion quality.

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Figure 2. Comparison of separation methods for pGUS demonstrating improvement in resolution of longer readseparation method at bases 510 to 600.

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Table 1A. Qiagen QIAprep Base Read Length 98% Accuracy Cutoff

Qiagen QIAprep 96Turbo MiniprepResults Name

TotalRead

Length

98.00%CutoffBase #

Qiagen QIAprep 96Turbo MiniprepResults Name

TotalRead

Length

98.00%CutoffBase #

QpUC18 86c 3min 3kV 55c.A07_03110103KY

1042 972QpGUS 86c 3min 3kV 55c.A09_03110608V9

1037 906

QpUC18 86c 3min 3kV 55c.B07_03110103KY

1083 996QpGUS 86c 3min 3kV 55c.B09_03110608V9

905 905

QpUC18 86c 3min 3kV 55c.C07_03110103KY

1055 974QpGUS 86c 3min 3kV 55c.C09_03110608V9

900 900

QpUC18 86c 3min 3kV 55c.D07_03110103KY

1061 1005QpGUS 86c 3min 3kV 55c.D09_03110608V9

1063 961

QpUC18 86c 3min 3kV 55c.E07_03110103KY

1271 1032QpGUS 86c 3min 3kV 55c.E09_03110608V9

1012 911

QpUC18 86c 3min 3kV 55c.F07_03110103KY

1050 1020QpGUS 86c 3min 3kV 55c.F09_03110608V9

1033 906

QpUC18 86c 3min 3kV 55c.G07_03110103KY

1115 1026QpGUS 86c 3min 3kV 55c.G09_03110608V9

969 950

QpUC18 86c 3min 3kV 55c.H07_03110103KY

997 936QpGUS 86c 3min 3kV 55c.H09_03110608V9

1022 912

QpUC18 86c 3minLFR1.A10_03111209JW

788 788QpGUS 86c 3minLFR1.A12_03110720XN

770 768

QpUC18 86c 3minLFR1.B10_03111209JY

803 803QpGUS 86c 3minLFR1.B12_03110720XN

790 761

QpUC18 86c 3minLFR1.C10_03111209K0

798 796QpGUS 86c 3minLFR1.C12_03110720XN

788 767

QpUC18 86c 3minLFR1.D10_03111209K1

807 807QpGUS 86c 3minLFR1.D12_03110720XN

778 778

QpUC18 86c 3minLFR1.E10_03111209K3

803 796QpGUS 86c 3minLFR1.E12_03110720XN

744 744

QpUC18 86c 3minLFR1.F10_03111209K5

804 801QpGUS 86c 3minLFR1.F12_03110720XN

791 766

QpUC18 86c 3minLFR1.G10_03111209K7

806 795QpGUS 86c 3minLFR1.G12_03110720XN

798 761

QpUC18 86c 3minLFR1.H10_03111209K9

810 810QpGUS 86c 3minLFR1.H12_03110720XN

711 711

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Table 1B. Promega SV96 Base Read Length 98% Accuracy Cutoff

Promega Wizard SV 96Plasmid DNA Results Name

TotalRead

Length

98.00%CutoffBase #

Promega Wizard SV 96Plasmid DNA Results Name

TotalRead

Length

98.00%CutoffBase #

pUC18 86c 3min 3kV 55c.H05_03103001P3

1059 1025pUC19gus 3kV 55c.A04_03102121MM

1169 959

pUC18 86c 3min 3kV 55c.B04_03102922FY

1075 961pUC19gus 3kV 55c.B04_03102121MM

1029 943

pUC18 86c 3min 3kV 55c.C04_03102922FY

1025 1004pUC19gus 3kV 55c.C04_03102121MM

1060 977

pUC18 86c 3min 3kV 55c.D04_03102922FY

1029 1023pUC19gus 3kV 55c.D04_03102121MM

1100 985

pUC18 86c 3min 3kV 55c.E04_03102922FY

1054 996pUC19gus 3kV 55c.E04_03102121MM

1015 971

pUC18 86c 3min 3kV 55c.F04_03102922FY

1049 1022pUC19gus 3kV 55c.F04_03102121MM

1077 981

pUC18 86c 3min 3kV 55c.G04_03102922FY

1124 982pUC19gus 3kV 55c.G04_03102121MM

1104 1017

pUC18 86c 3min 3kV 55c.H04_03102922FY

1108 1030pUC19gus 3kV 55c.H04_03102121MM

1071 1044

pUC18 86c 3minLFR1.A03_03102920TH

742 730Gus 8643 40 µLLFR1.A05_031018054A

769 769

pUC18 86c 3minLFR1.B03_03102920TH

823 769Gus 8643 40 µLLFR1.B05_031018054A

833 812

pUC18 86c 3minLFR1.C03_03102920TH

830 801Gus 8643 40 µLLFR1.C05_031018054A

811 811

pUC18 86c 3minLFR1.D03_03102920TH

826 800Gus 8643 40 µLLFR1.D05_031018054A

847 837

pUC18 86c 3minLFR1.E03_03102920TH

802 789Gus 8643 40 µLLFR1.E05_031018054A

833 809

pUC18 86c 3minLFR1.F03_03102920TH

818 801Gus 8643 40 µLLFR1.F05_031018054A

821 821

pUC18 86c 3minLFR1.G03_03102920TH

807 786Gus 8643 40 µLLFR1.G05_031018054A

799 789

pUC18 86c 3minLFR1.H03_03102920TH

803 783Gus 8643 40 µLLFR1.H05_031018054A

796 796

Table 2. Increase in Base Read Length 98% Accuracy Cutoff

Average98% Cutoff

% Increase of 98%Cutoff

DNA prep/Plasmid LFR1 3kV 55CPromega pUC18 767 958 25

Promega pUC19gus 798 964 21

Qiagen pUC18 786 977 24

Qiagen pUC19gus 759 930 23

References1. Quick Start Guide to Purifying Plasmids on the

Biomek® FX Using Beckman Coulter WizardSV 96 Reagents. Beckman Coulter ApplicationInformation Bulletin Quick Start Guide A-1907-QS (2001).

2. Improved Sequencing of Plasmids on the CEQ2000 by a Simple Template Pre-HeatingProcedure. Beckman Coulter ApplicationInformation Bulletin A-1872A (1999).

3. Roby, K., Gull, H. A Rapid and EfficientMethod for the Post-Reaction Clean Up ofLabeled Dye Terminator Sequencing Products.Beckman Coulter Application InformationBulletin A-1903A (2001).

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