A programmable method for massively parallel targeted sequencing.
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.
Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.
Erik S
ES
Hopmans
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Georges
G
Natsoulis
Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.
John M
JM
Bell
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Susan M
SM
Grimes
Department of Health Research and Policy, Stanford University School of Medicine, 259 Campus Drive, Stanford, CA 94305, USA.
Weiva
W
Sieh
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.
Hanlee P
HP
Ji
D000483:Alleles; D056905:Chromosome Breakpoints; D017931:DNA Primers; D015894:Genome, Human; D056914:Genomic Structural Variation; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers