Evaluation of oligonucleotide sequence capture arrays and comparison of next-generation sequencing platforms for use in molecular diagnostics.
BACKGROUND
Next-generation DNA sequencing (NGS) techniques have the potential to revolutionize molecular diagnostics; however, a thorough evaluation of these technologies is necessary to ensure their performance meets or exceeds that of current clinical sequencing methods.


METHODS
We evaluated the NimbleGen Sequence Capture 385K Human Custom Arrays for enrichment of 22 genes. We sequenced each sample on both the Roche 454 Genome Sequencer FLX (GS-FLX) and the Illumina Genome Analyzer II (GAII) to compare platform performance.


RESULTS
Although the sequence capture method allowed us to rapidly develop a large number of sequencing assays, we encountered difficulty enriching G+C-rich regions. Although a high proportion of reads consistently mapped outside of the targeted regions, >80% of targeted bases for the GAII and >30% of bases for the GS-FLX were covered by a read depth of > or =20, and > 90% of bases for the GAII and > 80% of bases for the GS-FLX were covered by a read depth of > or =5. We observed discrepancies among sequence variants identified by the different platforms.


CONCLUSIONS
Although oligonucleotide arrays are quick and easy to develop, some problematic regions may evade capture, necessitating sequential redesigning for complete optimization. Neither sequencing technology was able to detect every variant identified by Sanger sequencing because of well-known drawbacks of the NGS technologies. The rapidly decreasing error rates and costs of these technologies, however, coupled with advancing bioinformatic capabilities, make them an attractive option for molecular diagnostics in the very near future.
Hoppman-Chaney
Nicole
N
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA. hoppmanchaney.nicole@mayo.edu
Peterson
Lisa M
LM
Klee
Eric W
EW
Middha
Sumit
S
Courteau
Laura K
LK
Ferber
Matthew J
MJ
D011125:Adenomatous Polyposis Coli / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics; D005091:Exons; D015723:Gene Library; D018095:Germ-Line Mutation; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017354:Point Mutation; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*