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Nucleic Acids Res. 2014 Jun;42(10):e88. doi: 10.1093/nar/gku282. Epub 2014 Apr 29.

A programmable method for massively parallel targeted sequencing.

Author information

1
Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.
2
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
3
Department of Health Research and Policy, Stanford University School of Medicine, 259 Campus Drive, Stanford, CA 94305, USA.
4
Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA genomics_ji@stanford.edu.

Abstract

We have developed a targeted resequencing approach referred to as Oligonucleotide-Selective Sequencing. In this study, we report a series of significant improvements and novel applications of this method whereby the surface of a sequencing flow cell is modified in situ to capture specific genomic regions of interest from a sample and then sequenced. These improvements include a fully automated targeted sequencing platform through the use of a standard Illumina cBot fluidics station. Targeting optimization increased the yield of total on-target sequencing data 2-fold compared to the previous iteration, while simultaneously increasing the percentage of reads that could be mapped to the human genome. The described assays cover up to 1421 genes with a total coverage of 5.5 Megabases (Mb). We demonstrate a 10-fold abundance uniformity of greater than 90% in 1 log distance from the median and a targeting rate of up to 95%. We also sequenced continuous genomic loci up to 1.5 Mb while simultaneously genotyping SNPs and genes. Variants with low minor allele fraction were sensitively detected at levels of 5%. Finally, we determined the exact breakpoint sequence of cancer rearrangements. Overall, this approach has high performance for selective sequencing of genome targets, configuration flexibility and variant calling accuracy.

PMID:
24782526
PMCID:
PMC4041455
DOI:
10.1093/nar/gku282
[Indexed for MEDLINE]
Free PMC Article

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