SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing.
Single-molecule real-time (SMRT) sequencing generates much longer reads than other widely used next-generation (next-gen) sequencing methods, but its application to whole genome/exome analysis has been limited. Here, we describe the use of SMRT sequencing coupled with barcoding to simultaneously analyze one or a small number of genomic targets derived from multiple sources. In the budding yeast system, SMRT sequencing was used to analyze strand-exchange intermediates generated during mitotic recombination and to analyze genetic changes in a forward mutation assay. The general barcoding-SMRT approach was then extended to diffuse large B-cell lymphoma primary tumors and cell lines, where detected changes agreed with prior Illumina exome sequencing. A distinct advantage afforded by SMRT sequencing over other next-gen methods is that it immediately provides the linkage relationships between SNPs in the target segment sequenced. The strength of our approach for mutation/recombination studies (as well as linkage identification) derives from its inherent computational simplicity coupled with a lack of reliance on sophisticated statistical analyses.
Graduate Program in Molecular Cancer Biology, Duke University, Durham, North Carolina 27710.
Xiaoge
X
Guo
University Program in Genetics and Genomics, Duke University, Durham, North Carolina 27710.
Kevin
K
Lehner
University Program in Genetics and Genomics, Duke University, Durham, North Carolina 27710.
Karen
K
O'Connell
University Program in Genetics and Genomics, Duke University, Durham, North Carolina 27710.
Jenny
J
Zhang
Duke Cancer Institute, Duke University, Durham, North Carolina 27710 Department of Medicine, Duke University, Durham, North Carolina 27710.
Sandeep S
SS
Dave
Duke Cancer Institute, Duke University, Durham, North Carolina 27710 Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina 27710 sue.robertson@duke.edu.
Sue
S
Jinks-Robertson
D000483:Alleles; D045744:Cell Line, Tumor; D058893:DNA Barcoding, Taxonomic / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods; D005800:Genes, Fungal; D008040:Genetic Linkage; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008223:Lymphoma / Q000235:genetics; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D015003:Yeasts / Q000145:classification / Q000235:genetics
PacBio; SNP linkage; barcoded sequencing; rare variant; single-molecule sequencing