Toward real-world sequencing by microdevice electrophoresis.
We report results using a microdevice for DNA sequencing using samples from chromosome 17, obtained from the Whitehead Institute Center for Genome Research (WICGR) production line. The device had an effective separation distance of 11.5 cm and a lithographically defined injection width of 150 microm. The four-color raw data were processed, base-called by the sequencing software Trout, and compared to the corresponding ABI 377 sequence from WICGR. With a criteria of 99% accuracy, we achieved average continuous reads of 505 bases in 27 min with 3% linear polyacrylamide (LPA) at 150 V/cm, and 460 bases in 22 min with 4% LPA at 200 V/cm at a temperature of 45 degrees C. In the best case, up to 565 bases could be base-called with the same accuracy in <25 min. In some instances, Trout allowed for accurate base-calling down to a resolution R as low as R = 0.35. This may be due in part to the high signal-to-noise ratio of the microdevice. Unlike many results reported on capillary machines, no additional sample cleanup other than ethanol precipitation was required. In addition, DNA fragment biasing (i.e., discrimination against larger fragments) was reduced significantly through the unique sample injection mechanism of the microfabricated device. This led to increased signal strength for long fragments, which is of great importance for the high performance of the microdevice.
Schmalzing
D
D
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Tsao
N
N
Koutny
L
L
Chisholm
D
D
Srivastava
A
A
Adourian
A
A
Linton
L
L
McEwan
P
P
Matsudaira
P
P
Ehrlich
D
D
D001483:Base Sequence; D002886:Chromosomes, Human, Pair 17; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods*; D016045:Human Genome Project; D006801:Humans; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods