Toward Real-World Sequencing by Microdevice Electrophoresis

Abstract

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 µm. 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°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.Significant advancement in the technology of DNA analysis is expected from the use of microfabricated electrophoretic devices for sequencing and genotyping. In this approach photolithography, combined with wet-etching and thermal wafer bonding, is used to construct enclosed intricate microchannel structures in glass and fused-silica substrates; these structures are then utilized for electrophoresis (Harrison et al. 1993). It has been speculated that these devices will allow DNA separations approaching the theoretical limits of electrophoresis and in a format that will reduce analysis time and extend parallelism and automation (Freemantle 1999), which might hence increase throughput well beyond current capillary array machines. For example, in recent experiments we have demonstrated genotyping at 10-to 100-fold reduced analysis times on microdevices when compared to capillaries and slab gels, respectively (Schmalzing et al. 1997. DNA sequencing of single-color pGEM and four-color M13 DNA standard sequencing samples has been demonstrated on 3.5-, 11.5-, and 7-cm-long microdevices (Woolley et al. 1995;Schmalzing et al. 1998;Liu et al. 1999). The feasibility of ultra-high sample throughput has been proven through still modest multiplexing up to 96 microchannels (Simpson et al. 1998;Koutny et al. 1999). However, to the best of our knowledge, all published studies on DNA sequencing by microdevices have been performed using DNA standard samples such as M13 or pGEM. Practical sequencing must deal with additional factors such as variable salt and template concentrations Salas-Solano et al. 1998), highly samplespecific compression regions, and the interplay between electr...

Peer Reviewed: Plastic Advances Microfluidic Devices

“…There are several examples of DNA sequencing, including four-color analysis, in glass-etched microchannels (13)(14)(15). Five hundred bases can be sequenced in 20 min at 99.4% accuracy if all four base types (A, C, G, and T) are simultaneously analyzed using a corresponding set of four spectrally distinguishable dyes to label each DNA fragment according to its terminal base (16).…”

DNA sequencing and multiplex STR analysis on plastic microfluidic devices

“…As the use of the GeneScan 700 sizing standard precluded evaluation of the resolving capability of these three devices for single-base separation of short fragments (i.e., less than 75 bases), the results suggest a minimum separation of 75-400 bases for the 6-cm device, 75-500 bases for the 10-cm device, and 75-600 bases for the 18-cm device. With the aid of sophisticated software, data processing and analysis, quality-sequencing read lengths offered by these devices may be extended to fragments that have wider size ranges in which the achieved R values could be smaller than 0.5 [10]. Recently, R values more than 0.5 have been reported for glass devices using 2-4% LPA as sieving matrices for single-base separations of ,70-250 bases on 6-cm [5] and ,50-500 bases on 11.5-cm [5,10] channels.…”

“…With the aid of sophisticated software, data processing and analysis, quality-sequencing read lengths offered by these devices may be extended to fragments that have wider size ranges in which the achieved R values could be smaller than 0.5 [10]. Recently, R values more than 0.5 have been reported for glass devices using 2-4% LPA as sieving matrices for single-base separations of ,70-250 bases on 6-cm [5] and ,50-500 bases on 11.5-cm [5,10] channels.…”

Emerging technologies in DNA sequencing

“…Woolley and Mathies (1995) demonstrated the first application of DNA sequencing using a microfabricated glass device in 1995, reporting single-base resolution using their four-color scanner technology. Data quality and readlengths have improved significantly since then, because of an increase in the effective separation lengths with run times of 30 minutes or less (Table 2) (Woolley and Mathies 1995;Liu et al 1999;Schmalzing et al 1999;Backhouse et al 2000;Koutny et al 2000;Liu et al 2000;Salas-Solano et al 2000;Simpson et al 2000;Boone et al 2002;Paegel et al 2002;Shi and Anderson 2003). For example, Liu et al (1999) reported 99.4% accuracy over 500 bases in 20 minutes, with an increase in separation length from 3.5 cm to 6.5 cm.…”