Update on the Continuation of the DNA Sequencing Research Group 2000 Study:

An evaluation of the methods used to sequence and isolate bacterial artificial chromosomes.
T. Thannhauser1, L.S. Hall2, J. Hawes3, T. Hunter4, D. Leviten7, K. Knudtson6, E. Jackson-Machelski5, M.A. Robertson8 and J. VanEe1.
1
Cornell University, Ithaca NY; 2Albert Einstein College of Medicine, Bronx NY; 3Indiana Univiserity School of Medicine, Indianapolis, IN; 4University of Vermont, Burlington VT;
5
Washington University School of Medicine, Saint Louis MO; 6Univ. of Iowa, IA; 7ICOS Corp., Bothell WA; 8Cimarron Inc., Houston TX.

Introduction: Table 1. Results of Quality Analysis

At the 2000 meeting of the Association of Biomolecular Resource Phred Score RunTime
Facilities (ABRF) the DNA Sequencing Research Group (DSRG) presented the Figure 1. Effect of # of Cycles on Sequence Quality I n s t r u m e n t Filename Q20 Q30 Q40 WTR (hr) Chemistry
results of a study in which the protocols used to sequence bacterial artificial
chromosomes (BACs) in member laboratories were surveyed in an attempt to 700
correlate methodology with sequencing outcome. The results of this study suggest 4200L-2 5949A.scf 8 0 5 6 6 5 4 6 0 66 cm 12 IRDye800 Acyclo-Terminators
that there are two critical determinants of sequence quality when dealing with BAC 600
templates:
3100 9696a.abi 5 3 7 4 7 1 3 9 1 50 cm 2.5 BigDye Terminators

Avg. Phred q20

500
amount of template used 9696B.abi 5 0 3 3 9 9 2 1 7 50 cm 2.5 BigDye Terminators
400 200ng
number of cycles in the thermocycling program
500ng
300 1000ng 3700 7553a.abi 6 2 7 5 5 3 4 0 7 50 cm 2.8 BigDye Terminators
The correlation between these factors and sequencing quality were not
7553b.abi 6 7 3 5 2 8 3 3 3 50 cm 2.8 BigDye Terminators
as strong as expected. It was concluded that the weak correlation was due to the 200
accumulation of uncertainty arising from systematic differences in the experimen-
tal parameters used in the participating laboratories. To eliminate this uncertainty 100 373S-36 3303C.abi 21 5 4 36 cm 14 Old rhod terminators
the DSRG conducted an internal study on the effects of these factors under 9701A.abi 4 7 2 2 8 9 58 36 cm 12 BigDye Terminators v2
0
rigorously controlled conditions. The results of this internal study are presented
25 50 75 100
below and were used to develop an optimized protocol for sequencing BAC
templates. # Cycles 0255A.abi 6 3 9 4 6 7 2 5 0 48 cm 15.5 BigDye Terminators
Member laboratories were asked to sequence a sample of the standard 0255B.abi 4 5 1 2 9 2 1 1 5 48 cm 15.5 BigDye Terminators
BAC template using the optimized procedure to test its efficacy. These results are 373S-48 0583A.abi 5 9 7 4 9 1 4 2 1 48 cm 17 BigDye Terminators
also presented and clearly demonstrate the effectiveness of this protocol.
Findings: 0583B.abi 6 5 1 5 7 8 4 7 4 48 cm 17 BigDye Terminators
For samples containing >1 g of template a standard 25 cycle thermocycler program 4666.abi 5 0 0 48 cm 18 BigDye Terminators
About the Template: is sufficient to produce high quality sequence.
The standard BAC used for this study was selected at random from a For samples containing <1 g of template increasing the number of cycles to 50 will
library of Brassica oleracea. A stock of this clone was used to inoculate and grow improve sequence quality. 3358A.abi 5 5 3 4 9 1 3 6 3 36 cm 8 BigDye Terminators
cultures from which the BAC DNA was purified using the Midi Prep Kit from Increasing the number of cycles in the thermocycling program beyond 50 has little 3358B.abi 4 6 0 3 3 3 1 2 7 36 cm 8 BigDye Terminators
Qiagen. effect on sequencing quality even for very low amounts of template (0.2 g). 8488A.abi 5 0 3 4 0 7 2 1 7 36 cm 8 BigDye Terminators
This study was intended to explore the effects of template size on
sequencing success. This template does not represent a significant challenge 377-36-2X 8488B.abi 5 3 6 4 3 6 2 6 4 36 cm 8 BigDye Terminators
Based on these findings the following protocol is recommended for sequencing BAC templates:
from the point of view of GC content (Avg. about 45%). 8980c.abi 4 5 1 3 7 6 2 7 4 36 cm 9 BigDye Terminators
Recommended BAC Sequencing Procedure: 8980d.abi 4 6 1 2 6 9 1 2 6 36 cm 9 BigDye Terminators v2
Methods: Template quantity: 1000 ng 8980e.abi 3 5 1 2 7 0 1 7 1 36 cm 9 BigDye Terminators
To determine the correlation between amount of template used and length Number of cycles: 50
8980f.abi 4 6 5 3 1 8 1 8 7 36 cm 9 BigDye Terminators v2
of thermocycling program to sequence quality an experimental program was carried Reaction volume: 20 l
out using the following standard operating procedures: Primer: 10 pmoles total
Chemistry: Big Dye Terminator 2088A.abi 8 0 4 6 9 9 5 6 0 48 cm 16.5 BigDye Terminators
Experimental Program Sequencing Chemistry Volume Big Dyes: 8 l 2088B.abi 7 4 2 6 3 1 5 2 0 48 cm 16.5 BigDye Terminators v2
(2 X 200 ng) X 25, 50,75, 100 cycles Reaction Volume: 20 l Cycling conditions: Hot start 95 C for 5 min, 96 C for 30 sec, 50 C for 20 sec, 60
2454A.abi 6 5 2 5 5 8 3 9 1 48 cm 14 BigDye Terminators
(2 X 500 ng) X 25, 50,75, 100 cycles T7 Primer: 10 pmol C for 4 min x 50 cycles, hold 4 degrees
3380A.abi 6 1 3 4 3 9 2 6 3 48 cm 18 BigDye Terminators v2
(2 X 1000 ng) X 25, 50,75, 100 cycles Chemistry: Big Dye Term.
Samples of the standard BAC template and T7 primer were then sent to participating 377-48-2x 4503.abi 6 9 0 5 9 2 4 6 3 48 cm 16 BigDye Terminators
Total of 96 samples Premix Volume: 8 l
ABRF laboratories who were asked to sequence them using the recommended procedure. The 5677A.abi 7 7 2 6 8 6 5 6 4 48 cm 18 BigDye Terminators v2
sequencing results were returned to the DSRG electronically and analyzed for sequence quality
Thermocycling Conditions Electrophoretic Conditions 5677B.abi 7 6 2 6 5 0 5 2 5 48 cm 18 BigDye Terminators v2
as described above. The results of this analysis are shown in Table 1.
Hot Start 95C 5 min. 377 Sequencer 5677C.abi 6 0 2 3 9 5 1 7 0 48 cm 18 BigDye Terminators v2
96C 30 sec. 36 cm WTR Conclusion/ Future Directions: 5677D.abi 6 3 3 4 0 1 1 4 1 48 cm 18 BigDye Terminators v2
50C 20 sec. 0.2mm combs The recommended procedure for sequencing BAC templates (see above) is bac_study 3 5 1 1 9 5 69 48 cm 10 BigDye Terminators
60C 64 lanes capable of generating high quality sequencing results using the ABRF's standard BAC
4 min. Acknowledgements:
repeat for N cycles Module: 36E-1200
template.
However this procedure needs to be tested by sequencing a variety of different The authors would like to thank the following people for their many contributions to this study: Professor
4C Hold 10 hours June Nasrallah (Cornell University) for providing the BAC clone used in this study; Tom Stelick, Bill
BAC templates before it can be considered fully validated.
Temperature 48C Preliminary reports from laboratories that have adopted this procedure suggest that Enslow, Tatyana Pyntikova and Farhad Dhabhar (Cornell University) for their excellent technical
it will prove generally effective. assistance.
The performance level of each submission was judged by sequence References:
quality. To determine sequence quality the unedited sequences were analyzed by 1) Ewing, B.,Hillier, L..,Wendl, M.C.and Green,P. (1998) Base-calling of automated sequencer traces using
phred (1,2).The total number of base calls with a quality value >20 (phred score ) phred: I. Accuracy assessment. Genome Research 8:175-185.
for each sequence was determined with the program quat (written by J.V.), 2) Ewing, B. and Green, P.(1998)Base-calling of automated sequencer traces using phred: II. Error probabili-
The results of these experiments are summarized in Figure 1: ties. Genome Research 8:186-194.