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.

[1]  P. A. van der Kemp,et al.  dUTPase activity is critical to maintain genetic stability in Saccharomyces cerevisiae , 2006, Nucleic acids research.

[2]  J. R. Scotti,et al.  Available From , 1973 .

[3]  Thomas H Segall-Shapiro,et al.  Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome , 2010, Science.

[4]  Gang Bao,et al.  Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. , 2014, Cell reports.

[5]  H. Swerdlow,et al.  A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers , 2012, BMC Genomics.

[6]  David Baker,et al.  Massively parallel determination and modeling of endonuclease substrate specificity , 2014, Nucleic acids research.

[7]  M. Cappellini,et al.  Iron overload and toxicity: the hidden risk of multiple blood transfusions , 2009, Vox sanguinis.

[8]  David Dunson,et al.  Genetic heterogeneity of diffuse large B-cell lymphoma , 2013, Proceedings of the National Academy of Sciences.

[9]  Tomio S. Takahashi,et al.  Topoisomerase 1 provokes the formation of short deletions in repeated sequences upon high transcription in Saccharomyces cerevisiae , 2010, Proceedings of the National Academy of Sciences.

[10]  Matthew W. Snyder,et al.  Haplotype-resolved genome sequencing: experimental methods and applications , 2015, Nature Reviews Genetics.

[11]  Donald Sharon,et al.  A single-molecule long-read survey of the human transcriptome , 2013, Nature Biotechnology.

[12]  Nayun Kim,et al.  Role for topoisomerase 1 in transcription-associated mutagenesis in yeast , 2010, Proceedings of the National Academy of Sciences.

[13]  S. Jinks-Robertson,et al.  Molecular structures of crossover and noncrossover intermediates during gap repair in yeast: implications for recombination. , 2010, Molecular cell.

[14]  S. Jinks-Robertson,et al.  Roles of exonucleases and translesion synthesis DNA polymerases during mitotic gap repair in yeast. , 2013, DNA repair.

[15]  S. Turner,et al.  A flexible and efficient template format for circular consensus sequencing and SNP detection , 2010, Nucleic acids research.

[16]  Andrew W. Murray,et al.  Estimating the Per-Base-Pair Mutation Rate in the Yeast Saccharomyces cerevisiae , 2008, Genetics.

[17]  John L. Spouge,et al.  Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi , 2012, Proceedings of the National Academy of Sciences.