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.
- Gasperini, Molly et al. (2016) Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements bioRxiv
- Grunert, Marcel et al. (2016) Comparative DNA methylation and gene expression analysis identifies novel genes for structural congenital heart diseases. Cardiovascular Research
- Ashley, Euan A et al. (2016) Towards precision medicine. Nature Reviews. Genetics
- Nuttle, Xander et al. (2016) Emergence of a Homo sapiens-specific gene family and chromosome 16p11.2 CNV susceptibility. Nature
- 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
- Gowda, Malali et al. (2016) Comparative analyses of low, medium and high-resolution HLA typing technologies for human populations Journal of Clinical & Cellular Immunology
- Pradhan, Barun et al. (2016) Detection and screening of chromosomal rearrangements in uterine leiomyomas by long-distance inverse PCR. Genes, Chromosomes & Cancer
- Qiao, Wanqiong et al. (2016) Long-read Single-Molecule Real-Time (SMRT) full gene sequencing of cytochrome P450-2D6 (CYP2D6). Human Mutation
- Kane, Megan S et al. (2016) Mitotic intragenic recombination: A mechanism of survival for several congenital disorders of glycosylation. American Journal of Human Genetics
- Yang, Jie et al. (2016) The dentin phosphoprotein repeat region and inherited defects of dentin. Molecular Genetics & Genomic Medicine
- Lee, Stella Suyong et al. (2015) ATM kinase is required for telomere elongation in mouse and human cells. Cell Reports
- Shukla, Sachet A et al. (2015) Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes. Nature Biotechnology
- Hargreaves, Chantal E et al. (2015) Fc? receptors: genetic variation, function, and disease. Immunological Reviews
- 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
- Hendel, Ayal et al. (2014) Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. Cell Reports
- 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
- Orkunoglu-Suer, Funda et al. (2015) Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome. BMC Genomics
- Wang, Min et al. (2015) PacBio-LITS: a large-insert targeted sequencing method for characterization of human disease-associated chromosomal structural variations. BMC Genomics
- Antonacci, Francesca et al. (2014) Palindromic GOLGA8 core duplicons promote chromosome 15q13.3 microdeletion and evolutionary instability. Nature Genetics
- Hüser, Daniela et al. (2014) Adeno-associated virus type 2 wild-type and vector-mediated genomic integration profiles of human diploid fibroblasts analyzed by third-generation PacBio DNA sequencing. Journal of Virology
- Davis, Caleb F et al. (2014) The somatic genomic landscape of chromophobe renal cell carcinoma. Cancer Cell
- Dickel, Diane E et al. (2014) Function-based identification of mammalian enhancers using site-specific integration. Nature Methods
- Fine, Eli J et al. (2014) An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage. Nucleic Acids Research
- Doi, Koichiro et al. (2014) Rapid detection of expanded short tandem repeats in personal genomics using hybrid sequencing. Bioinformatics
- Guo, Xueliang et al. (2014) Genome reference and sequence variation in the large repetitive central exon of human MUC5AC. American Journal of Respiratory Cell and Molecular Biology
- Huddleston, John et al. (2014) Reconstructing complex regions of genomes using long-read sequencing technology. Genome Research
- Loomis, Erick W et al. (2013) Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. Genome Research
- Poster: Ekholm, Jenny et al. (2017) Screening and characterization of causative structural variants for bipolar disorder in a significantly linked chromosomal region onXq24-q27 in an extended pedigree from a genetic isolate
- Poster: Clark, Tyson et al. (2017) Targeted SMRT Sequencing of difficult regions of the genome using a Cas9, non-amplification based method
- Poster: Eng, K. et al. (2016) Target enrichment using a neurology panel for 12 barcoded genomic DNA samples on the PacBio SMRT Sequencing platform
- Poster: McCalmon, S. et al. (2016) “SMRTer Confirmation”: Scalable clinical read-through variant confirmation using the Pacific Biosciences SMRT Sequencing platform
- Poster: Watson, C. T. et al. (2016) Characterizing haplotype diversity at the immunoglobulin heavy chain locus across human populations using novel long-read sequencing and assembly approaches
- Poster: Pollard, M. O. et al. (2016) The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations
- Poster: Sethuraman, A. et al. (2016) Characterization of the Poly-T variants in the TOMM40 gene using PacBio long reads
- Poster: Ekholm, J. et al. (2016) Enrichment of unamplified DNA and long-read SMRT Sequencing to unlock repeat expansion disorders
- Poster: Cereb, N et al. (2016) Whole gene sequencing of KIR-3DL1 with SMRT Sequencing and the distribution of allelic variants in different ethnic groups
- Poster: Jenny Gu, Kevin Eng, Anand Sethuraman, Steve Kujawa et al. (2016) Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies
- 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 NimbleGen’s SeqCap EZ enrichment
- Poster: Eng, Kevin et al. (2014) Unique haplotype structure determination in human genome using Single Molecule, Real-Time (SMRT) Sequencing of targeted full-length fosmids.
- Poster: Mayor, Neema P et al. (2014) Genomic DNA sequences of HLA class I alleles generated using multiplexed barcodes and SMRT DNA Sequencing technology.
- (2017) Tutorial: Long Amplicon Analysis application
- Korlach, Jonas (2016) ASHG PacBio Workshop: A future of high-quality genomes, transcriptomes, and epigenomes
- Ashley, Euan (2016) ASHG PacBio Workshop: Towards precision medicine
- Pollard, Martin (2016) ASHG Virtual Poster: The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations
- Ekholm, J. and Tsai, Y. and Greenberg, D. and Clark, T. (2016) ASHG Virtual Poster: Enrichment of unamplified DNA and long-read SMRT Sequencing to unlock repeat expansion disorders
- Ashby, Meredith (2016) Nature Webinar: Bringing hidden biology into focus with target capture and SMRT Sequencing
- Ashizawa, Tetsuo (2016) Nature Webinar: Large interrupted pentanucleotide repeats of SCA10
- Ranade, Swati (2016) AGBT Virtual Poster: Immune regions are no longer incomprehensible with SMRT Sequencing
- Hon, Lawrence (2016) AGBT Virtual Poster: Targeted sequencing and chromosomal haplotype assembly using TLA and SMRT Sequencing
- Korlach, Jonas (2015) ASHG PacBio Workshop: Going beyond the $1,000 genome? – the future of high quality de novo human genomes, epigenomes and transcriptomes?
- Gibbs, Richard (2015) ASHG PacBio Workshop: Medical diagnostic challenges and structural variation detection using the PacBio Platform
- Marsh, Steven (2015) Mendelspod: Long-read sequencing dramatically improves blood matching – Steven Marsh
- Mayor, Neema (2015) Seminar: Benefits of SMRT Sequencing for HLA typing at Anthony Nolan
- Geraghty, Dan (2015) AGBT Virtual Poster: Insight into MHC and KIR genomic regions associated with autoimmune disease
- Maiers, Martin (2014) ASHI PacBio Workshop: KIR haplotypes – The long and short of it
- Marsh, Steven (2014) ASHI PacBio Workshop: The challenge of HLA diversity in 2014
- Geraghty, Dan (2014) Mendelspod: Major sequencing projects should be done with long reads
- Application Note: Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies (2016)
- Application Note: Targeted sequencing and chromosomal haplotype assembly using Cergentis TLA technology with SMRT Sequencing (2016)
- HLA Sequencing Application Brochure: Fully phased, allele-specific HLA sequencing – the perfect pair (2015)
- Immunology Brochure: Invaluable insights into immunology (2015)
- Targeted Sequencing Application Brochure: Capture your regions of interest in high resolution (2015)
- Product Note: Barcoded adapters and barcoded universal primers. (2015)
- Featured Interview: “We’re Going to Find the Keys” – Dan Geraghty discusses an approach to understanding causal genetic variation. (2014)