Open Access Highly Accessed Method

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

Sheila Fisher1, Andrew Barry1, Justin Abreu1, Brian Minie1, Jillian Nolan1, Toni M Delorey1, Geneva Young1, Timothy J Fennell1, Alexander Allen1, Lauren Ambrogio1, Aaron M Berlin2, Brendan Blumenstiel3, Kristian Cibulskis3, Dennis Friedrich1, Ryan Johnson1, Frank Juhn4, Brian Reilly1, Ramy Shammas1, John Stalker1, Sean M Sykes2, Jon Thompson1, John Walsh1, Andrew Zimmer1, Zac Zwirko14, Stacey Gabriel2, Robert Nicol1 and Chad Nusbaum2*

Author Affiliations

1 Genome Sequencing Platform, Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02141, USA

2 Genome Sequencing and Analysis Program, Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02141, USA

3 Genetic Analysis Platform, Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02141, USA

4 Foundation Medicine, One Kendall Square, Suite B6501, Cambridge, MA 02139, USA

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Genome Biology 2011, 12:R1  doi:10.1186/gb-2011-12-1-r1

Published: 4 January 2011

Additional files

Additional file 1:

Table S1a and S1b - cost comparison. (a) Cost model comparison of whole genome shotgun to whole exome sequencing. (b) Performance metrics of whole genome shotgun compared to whole exome sequencing with a control sample.

Format: DOCX Size: 39KB Download file

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Additional file 2:

Comparison of targeted capture methods. Table comparing scaled solution hybrid selection to other approaches.

Format: DOCX Size: 39KB Download file

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Additional file 3:

Automated SHS process map. A powerpoint file showing a process map for the solution hybrid selection method.

Format: PPTX Size: 70KB Download file

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Additional file 4:

Manual SHS protocol. A word document outlining the manual protocol.

Format: DOCX Size: 147KB Download file

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Additional file 5:

DNA shearing optimization. Profiles of sheared genomic DNA from unoptimized (blue) and optimized (red) conditions are shown. The size distribution from optimized conditions has a larger fraction of product DNA in the desired size range of 120 to 150 bases. The sharp peaks at approximately 20 and approximately 1,500 bases represent size standards.

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Additional file 6:

Shearing rack CAD drawing. A PDF showing the CAD drawing and dimensions for the shearing rack adapter for the Covaris unit.

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Additional file 7:

Optimization of pond PCR cycle number. For each number of PCR cycles tested, red bars (left-hand y-axis) show number of unique molecules per library, in millions; green bars (right-hand y-axis) show percent duplicated sequences. Data were generated in a controlled experiment using high quality human female DNA purchased from Promega (Madison WI, USA, catalogue number G1521). Patient samples typically demonstrate lower performance likely due to lower sample quality.

Format: PDF Size: 104KB Download file

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Additional file 8:

Reagent reservoir CAD drawing. A PDF showing the CAD drawing and dimensions for the low volume custom reservoir used for reagent dispensing.

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Additional file 9:

Optimization of hybrid selection wash conditions. Results for three sets of conditions are shown: manual protocol from Gnirke et al. [14], with three 500-μl washes; unoptimized automated protocol, with three 150-μl washes; optimized automated protocol, with six 150-μl washes. Shown are percent sequenced bases on target for a controlled bait set.

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Additional file 10:

Improved process control with transition from manual to automated capture. Implementation of the automated capture protocol greatly reduced sample to sample variability as measured by the percent of bases on or near the target. Data from 550 samples from the production process are shown. Samples in the gray box (the first 110) were performed manually, and samples on the white background represent the first group run with the automated protocol.

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Additional file 11:

Comparison of DNA recovery between manual NaOH denaturation and automated 'off-bead' enrichment. Total yield of DNA in nanograms is shown.

Format: PDF Size: 95KB Download file

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Additional file 12:

Sequencing metrics definitions. A Word document that defines the sequencing metrics used to measure process performance.

Format: DOCX Size: 26KB Download file

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Additional file 13:

Fingerprint bait sequences. A Word document listing the sequences of baits used in the fingerprint panel.

Format: DOCX Size: 13KB Download file

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Additional file 14:

Automated SHS library construction protocol. A Word document detailing the automated SHS library construction protocol.

Format: DOCX Size: 583KB Download file

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Additional file 15:

Automated SHS hybridization and capture protocol. A Word document detailing the automated hybridization and capture protocols.

Format: DOCX Size: 249KB Download file

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