Target your critical genomic regions
Targeted sequencing optimizes resources by focusing data collection on the most relevant genomic regions. However, neither time nor money is saved if the information you need isn’t captured. Homopolymeric stretches, repetitive elements, or short tandem repeats (STRs) within genes interfere with short-read approaches when targeted regions are prone to structural variation. Similarly, haplotype reconstruction from short reads relies on imputation, which is not always reliable — particularly when structural variants or de novo mutations are a factor1.
Resolve complex genomics underlying human disease
Targeted sequencing with PacBio long reads provides single-molecule observations of genomic regions linked to human disease. The result is continuous sequence data though polymorphic or repetitive regions and in-depth information on complex mixtures. These reads deliver standalone de novo solutions, but also resolve ambiguities in short-read data sets.
Single Molecule, Real-Time (SMRT) Sequencing enables a wide range of applications, giving you the ability to:
- Definitively phase polymorphisms over entire genes
- Sequence through STRs and other low-complexity regions within genes
- Detect common or de novo structural variants and map breakpoints with precision
- Fully characterize minor variants in polyclonal samples
Workflow: from targeted region to accurate variant or haplotype
- Library preparation
- SMRT Sequencing with PacBio Systems
- Take advantage of the Sequel System to reduce project costs and generate 7X more reads compared with the PacBio RS II
- Achieve ~10 kb average read lengths, with some reads as long as 60 kb
- Adjust run times (0.5 to 6 hours) to maximize sample throughput and turn-around time
- Obtain consensus accuracies > 99.999% by avoiding mapping and systematic errors
- Produce high single-molecule consensus accuracies through multiple observations of single circularized templates for complex population resolution
- Data analysis with SMRT Analysis or PacBio DevNet
- Long Amplicon Analysis (LAA) for generation of reference-free de novo haplotype sequences from pooled amplicons
- Minor Variant Analysis for detection and quantitation of single nucleotide polymorphisms
- ClusterConsensus for reference-free deconvolution of genomes in a complex mixture
- HGAP for comprehensive de novo assembly of BACs or fosmids
Featured research: targeting structural variants in Potocki-Lupski Sydrome
“Interestingly, when all of the junctions are examined, 3 of the 5 breakpoints resulted in Alu-Alu chimeras, which may be difficult to detect with shorter reads. Additionally, in a cohort of 123 PTLS (Potocki-Lupski syndrome) patients, about 60% of the non-recurrent rearrangements have one or more breakpoint within an LCR (low copy repeat)2.”
Explore this research further.
To learn more about how to access the full spectrum of genetic variation with SMRT Sequencing, contact us.
- Willems, T., et al., (2014) The landscape of human STR variation. Genome Research. 24(11), 1894-1904.
- Wang, M., et al., (2015) PacBio-LITS: a large-insert targeted sequencing method for characterization of human disease-associated chromosomal structural variations. BMC Genomics. 16, 214.
- Pham, Thang T et al. (2016) Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications. Molecular Genetics and Genomics
- Gowda, Malali et al. (2016) Comparative analyses of low, medium and High-resolution HLA typing technologies for human populations Journal of Clinical & Cellular Immunology
- De Ravin, Suk See et al. (2016) Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nature Biotechnology
- Pradhan, Barun et al. (2016) Detection and screening of chromosomal rearrangements in uterine leiomyomas by long-distance inverse PCR. Genes, Chromosomes & Cancer
- Yang, Jie et al. (2016) The dentin phosphoprotein repeat region and inherited defects of dentin. Molecular Genetics & Genomic Medicine
- Qiao, Wanqiong et al. (2015) Long-read Single-Molecule Real-Time (SMRT) full gene sequencing of cytochrome P450-2D6 (CYP2D6). Human Mutation
- Lee, Stella Suyong et al. (2015) ATM kinase is required for telomere elongation in mouse and human cells. Cell Reports
- Hargreaves, Chantal E et al. (2015) Fc? receptors: genetic variation, function, and disease. Immunological Reviews
- Shukla, Sachet A et al. (2015) Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes. Nature Biotechnology
- Hosomichi, Kazuyoshi et al. (2015) The impact of next-generation sequencing technologies on HLA research. Journal of Human Genetics
- McFarland, Karen N. et al. (2015) SMRT Sequencing of long tandem nucleotide repeats in SCA10 reveals unique insight of repeat expansion structure. PLoS One
- Chen, Hsuan-Yu et al. (2015) R331W Missense Mutation of Oncogene YAP1 Is a Germline Risk Allele for Lung Adenocarcinoma With Medical Actionability. Journal of Clinical Oncology
- Agarwal, Prasoon et al. (2015) CGGBP1 mitigates cytosine methylation at repetitive DNA sequences. BMC Genomics
- Mayor, Neema P et al. (2015) HLA typing for the next generation. PLoS One
- Wang, Min et al. (2015) PacBio-LITS: a large-insert targeted sequencing method for characterization of human disease-associated chromosomal structural variations. BMC Genomics
- Orkunoglu-Suer, Funda et al. (2015) Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome. BMC Genomics
- Poster: Ekholm, JM et al. (2016) Resolving KIR genotypes and haplotypes simultaneously using Single Molecule, Real-Time Sequencing
- Poster: Ranade, Swati et al. (2016) Immune regions are no longer incomprehensible with SMRT Sequencing
- Poster: Hon, Lawrence S. et al. (2016) Targeted sequencing and chromosomal haplotype assembly using TLA and SMRT Sequencing
- Poster: Pollard, Martin O. et al. (2015) HLA variant identification techniques
- Poster: Ranade, S. et al. (2015) Access full spectrum of polymorphisms in HLA class I & II genes, without imputation for disease association and evolutionary research.
- Poster: Lang, Kathrin et al. (2015) Full-length sequencing of HLA class I genes of more than 1000 samples provides deep insights into sequence variability
- Poster: Lang, Kathrin et al. (2015) Phased full-length SMRT Sequencing of HLA DPB1
- Poster: Hall, Richard J. et al. (2015) Assembly of complete KIR haplotypes from a diploid individual by the direct sequencing of full-length fosmids.
- Poster: Pyo, Chul-Woo et al. (2015) Complete resequencing of extended genomic regions using fosmid target capture and single molecule real-time (SMRT) long read sequencing technology.
- Poster: Ranade, Swati et al. (2015) Multiplexing human HLA class I & II genotyping with DNA barcode adapters for high throughput research.
- Poster: Kujawa, Steve et al. (2015) Targeted SMRT Sequencing and phasing using Roche NimbleGens SeqCap EZ enrichment
- Hon, Lawrence (2016) AGBT Virtual Poster: Targeted sequencing and chromosomal haplotype assembly using TLA and SMRT Sequencing
- Ranade, Swati (2016) AGBT Virtual Poster: Immune regions are no longer incomprehensible with SMRT Sequencing
- Geraghty, Dan (2015) AGBT Virtual Poster: Insight into MHC and KIR genomic regions associated with autoimmune disease
- Mayor, Neema (2015) Seminar: Benefits of SMRT Sequencing for HLA typing at Anthony Nolan
- Korlach, Jonas (2015) ASHG PacBio Workshop: Going beyond the $1,000 genome – the future of high quality de novo human genomes, epigenomes and transcriptomes
- Marsh, Steven (2015) Mendelspod: Long-read sequencing dramatically improves blood matching – Steven Marsh
- Gibbs, Richard (2015) ASHG PacBio Workshop: Medical diagnostic challenges and structural variation detection using the PacBio Platform
- Application Note: Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies (2016)
- Application Notes: Targeted sequencing and chromosomal haplotype assembly using Cergentis TLA technology with SMRT Sequencing (2016)
- Targeted Sequencing Application Brochure: Capture your regions of interest in high resolution (2015)
- Immunology Brochure: Invaluable insights into immunology (2015)
- HLA Sequencing Application Brochure: Fully phased, allele-specific HLA sequencing – the perfect pair (2015)
- Product Note: Barcoded adapters and barcoded universal primers. (2015)