A programmable method for massively parallel targeted sequencing
| Autor: | Hanlee P. Ji, Weiva Sieh, John Bell, Erik S. Hopmans, Susan M. Grimes, Georges Natsoulis |
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| Rok vydání: | 2014 |
| Předmět: |
Genomic Structural Variation
Hybrid genome assembly Genomics Biology Genome Polymorphism Single Nucleotide Deep sequencing 03 medical and health sciences Chromosome Breakpoints 0302 clinical medicine Neoplasms Genetics Humans Genotyping Exome sequencing Alleles 030304 developmental biology DNA Primers 0303 health sciences Massive parallel sequencing Genome Human High-Throughput Nucleotide Sequencing Sequence Analysis DNA 030220 oncology & carcinogenesis Mutation Methods Online |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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