Clinical Chemistry 56: 62-72, 2010. First published November 12, 2009; 10.1373/clinchem.2009.132639
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
clinchem.2009.132639v1
56/1/62    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chou, L.-S.
Right arrow Articles by Mao, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chou, L.-S.
Right arrow Articles by Mao, R.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2010;56:62-72.)
© 2010 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

DNA Sequence Capture and Enrichment by Microarray Followed by Next-Generation Sequencing for Targeted Resequencing: Neurofibromatosis Type 1 Gene as a Model

Lan-Szu Chou1,a, C.-S. Jonathan Liu2, Benjamin Boese3, Xinmin Zhang4 and Rong Mao1,5

1 Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT; 2 SoftGenetics, LLC, State College, PA; 3 454 Life Sciences, A Roche Company, Branford, CT; 4 Roche NimbleGen, Madison, WI; 5 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT.

aAddress correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108-1221. Fax 801-584-5114; e-mail choulb{at}aruplab.com.

Background: The introduction and use of next-generation sequencing (NGS) techniques have taken genomic research into a new era; however, implementing such powerful techniques in diagnostics laboratories for applications such as resequencing of targeted disease genes requires attention to technical issues, including sequencing template enrichment, management of massive data, and high interference by homologous sequences.

Methods: In this study, we investigated a process for enriching DNA samples that uses a customized high-density oligonucleotide microarray to enrich a targeted 280-kb region of the NF1 (neurofibromin 1) gene. The captured DNA was sequenced with the Roche/454 GS FLX system. Two NF1 samples (CN1 and CN2) with known genotypes were tested with this protocol.

Results: Targeted microarray capture may also capture sequences from nontargeted regions in the genome. The capture specificity estimated for the targeted NF1 region was approximately 60%. The de novo Alu insertion was partially detected in sample CN1 by additional de novo assembly with 50% base-match stringency; the single-base deletion in sample CN2 was successfully detected by reference mapping. Interferences by pseudogene sequences were removed by means of dual-mode reference-mapping analysis, which reduced the risk of generating false-positive data. The risk of generating false-negative data was minimized with higher sequence coverage (>30x).

Conclusions: We used a clinically relevant complex genomic target to evaluate a microarray-based sample-enrichment process and an NGS instrument for clinical resequencing purposes. The results allowed us to develop a systematic data-analysis strategy and algorithm to fit potential clinical applications.




The following articles in journals at HighWire Press have cited this article:


Home page
 Proc. Natl. Acad. Sci. USAHome page
T. Walsh, M. K. Lee, S. Casadei, A. M. Thornton, S. M. Stray, C. Pennil, A. S. Nord, J. B. Mandell, E. M. Swisher, and M.-C. King
Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing
PNAS, July 13, 2010; 107(28): 12629 - 12633.
[Abstract] [Full Text] [PDF]