Semi-automated library preparation for high-throughput DNA sequencing platforms - PubMed (original) (raw)

Semi-automated library preparation for high-throughput DNA sequencing platforms

Eveline Farias-Hesson et al. J Biomed Biotechnol. 2010.

Abstract

Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.

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Figures

Figure 1

The Parallel Semi-Automated Library Maker (PSALM) uses a NorDiag Magnatrix 8000 plus to prepare 32 samples simultaneously. DNA of 16 samples is sheared (1) and submitted to the automated steps of PSALM (Highlighted in Blue). After end-repair (2), and ligation of molecular barcoded adaptors (4), the 16 samples are pooled (6) and stored at 4°C or −20°C. A new automated cycle is started to prepare additional 16 samples. After 12.08 hours, the 2 pools containing 32 samples enter the manual steps of PSALM for size selection in 6% PAGE gel (8), nick-translation, and PCR amplification (9). A final gel purification step is done in 4% Agarose gel for removing self-ligated adaptors. Next, the library is eluted from the gel using spin columns (11). Carboxy beads substitute for columns used in the manual library preparation to clean up enzymatic reactions (3, 5, and 10), and in the steps where DNA concentration is required (7). Thirty-two libraries are prepared to enter the sequencing pipeline in 16.08 hours. In contrast, only 4 samples are processed in parallel in the manual preparation. Spin columns are used in all purification steps (3, 5, 8 and 9), and a pool with only 4 barcoded libraries is obtained after 12.49 hours (11).

Figure 2

Coverage values for each exon across all groups. Each bar has three colors. Blue shows the minimum coverage value found across all data sets. Green shows the average of the coverage values over all the data sets, and red shows the maximum coverage value from all the data sets.

Figure 3

Comparison between the purification methods used in PSALM (CarboxyBeads) and traditional manual spin column preparation (Qiagen) is presented in the histogram (a), while histogram (b) plots the average CT values obtained for quantification of barcoded libraries prepared according to PSALM. The error bars represent a 95% confidence interval. The carboxy bead method recovers an average of 93% of the initial DNA (a), while libraries prepared using the semi-automated pipeline are amplified with same number of PCR cycles (Average CT value = 15.5), which indicates similar concentration of libraries in the tested samples.

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Semi-automated library preparation for high-throughput DNA sequencing platforms - PubMed (original) (raw)