Cost-Effective Paramagnetic Bead Technique for Purification of Cycle Sequencing Products

Table 1

Summary of results of cycle sequencing purification methods examined in this study.

Technique	Sequence length/quality^a	Cost, US $^b; (per sample)	Time needed^c; (per 96 samples)	Advantages	Disadvantages
BioMag Carboxyl beads	815±1/97±3	0.006–0.015	25 min	Low cost; Quick; Good results	Moderate potential for user error; Occasional blobs
Proprietary size exclusion columns	816±1/99±1	2.2–2.6	3-4 h	Best results	Costly; Time consuming; Moderate potential for user error
Proprietary; in-well; technique	816 ± 1/99 ± 1*	1.94	37 min	Quick; Good results; Low potential for user error	Costly; Short shelf life; Occasional blobs
Ethanol precipitation	744 ± 35/87 ± 11**	~0.001	1 h 45 min	Lowest cost	Loss of amplicon; Low quality results; High potential for user error

aSequence length and quality scores for overlapping fragments were obtained directly from Sequencher 4.8 software (Gene Codes Corporation, Inc., Ann Arbor, MI). Prior to analyzing the data, raw sequences were trimmed by aligning all of the sequences of one reaction and determining the unusable part of the longest sequence and cutting all sequences at that base. Measurements and standard deviation are calculated using triplicate results of 4 different reactions (primers used were con2, con3, VP7R and 9con1L). Measurement based on *1 and **2 fewer sequences due to low quality of final sequence.
bCost per reaction was calculated by totaling the cost of consumables needed to clean one 10 𝜇L cycle sequencing reaction and does not account for the cost of equipment.
cTime needed to purify 96 samples was measured from the moment the plate containing the cycle sequencing reactions was removed from the thermocycler after cycle sequencing to the time purification was completed and the 96-well septum was placed on the plate.