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).
2
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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