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.
Targeted sequencing with PacBio long reads provides single-molecule observations of genomic regions linked to human disease. The result is continuous sequence data through 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 – Enrich target regions of interest
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| SMRT Sequencing with PacBio Systems
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 | Data Analysis with SMRT Analysis and PacBio DevNet
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Spotlight: SMRT Sequencing uncovers causative mutation within GC-rich region
PacBio long-read sequencing enabled scientists to detect a single causative mutation for autosomal dominate tubulointerstitial kidney disease (ADTKD) in MUC1 gene residing in a GC-rich variable number tandem repeat region (VNTR) that was previously intractable. Explore this research further:
Wenzel, A. et al., 2018. Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations. Scientific Reports, 8(1), p.4170.
References
- 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.
Selected Resources
- Cumming, Sarah A et al. (2018) De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1. European Journal of Human Genetics
- Prokopuk, Lexie et al. (2018) Loss of maternal EED results in postnatal overgrowth. Clinical Epigenetics
- Höijer, Ida et al. (2018) Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing. Human Mutation
- Song, Janet et al. (2018) Characterization of a human-specific tandem repeat associated with bipolar disorder and schizophrenia BioRxiv
- Bultitude, W P et al. (2018) KIR2DL1 allele sequence extensions and discovery of 2DL1*0010102 and 2DL1*0010103 alleles by DNA sequencing. HLA
- Cousin, Margot A et al. (2018) Utility of DNA, RNA, protein, and functional approaches to solve cryptic immunodeficiencies. Journal of Clinical Immunology
- Wenzel, Andrea et al. (2018) Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations. Scientific Reports
- Borel, Florie et al. (2018) Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema. Proceedings of the National Academy of Sciences of the United States of America
- Aneichyk, Tatsiana et al. (2018) Dissecting the causal mechanism of X-linked Dystonia-Parkinsonism by integrating genome and transcriptome assembly. Cell
- Turner, T R et al. (2018) Single molecule real-time (SMRT®) DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines. HLA
- Reiner, Jennifer et al. (2018) Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet-Biedl Syndrome 9 (BBS9) deletion. NPJ Genomic Medicine
- Tsai, Yu-Chih et al. (2017) Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions BioRxiv
- Gonçalves, Ana et al. (2017) Exonization of an intronic LINE-1 element causing Becker muscular dystrophy as a novel mutational mechanism in dystrophin gene. Genes
- Wilbe, Maria et al. (2017) A novel approach using long-read sequencing and ddPCR to investigate gonadal mosaicism and estimate recurrence risk in two families with developmental disorders. Prenatal Diagnosis
- Schüle, Birgitt et al. (2017) Parkinsons disease associated with pure ATXN10 repeat NPJ Parkinson's Disease
- Albrecht, V et al. (2017) Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles. HLA
- Borràs, Daniel M et al. (2017) Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing. Human Mutation
- Roe, D. et al. (2017) Revealing complete complex KIR haplotypes phased by long-read sequencing technology Genes and Immunity
- Yang, Yao et al. (2017) Sequencing the CYP2D6 gene: from variant allele discovery to clinical pharmacogenetic testing. Pharmacogenomics
- Ardui, Simon et al. (2017) Detecting AGG Interruptions in Male and Female FMR1 Premutation Carriers by Single-Molecule Sequencing. Human Mutation
- Seo, Jieun et al. (2016) Tofacitinib relieves symptoms of stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy caused by 2 de novo variants in TMEM173. The Journal of Allergy and Clinical Immunology
- Brykczynska, Urszula et al. (2016) CGG repeat-induced FMR1 silencing depends on the expansion size in human iPSCs and neurons carrying unmethylated full mutations. Stem Cell Reports
- 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
- Vossen, Rolf H A M et al. (2016) Full-length mitochondrial-DNA sequencing on the PacBio RSII. Methods in Molecular Biology
- 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
- Sheppard, Anna E et al. (2016) Nested Russian doll-like genetic mobility drives rapid dissemination of the Carbapenem resistance gene blaKPC Antimicrobial Agents and Chemotherapy
- Zhang, Huibin et al. (2016) Comprehensive mutagenesis of the fimS promoter regulatory switch reveals novel regulation of type 1 pili in uropathogenic Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America
- 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
- Poster: Larrea, Andres et al. (2018) A simple segue from Sanger to high-throughput SMRT Sequencing with a M13 barcoding system
- Poster: McLaughlin, Ian et al. (2018) No-amp targeted SMRT sequencing using a CRISPR-Cas9 enrichment method
- Poster: Ekholm, J. et al. (2018) Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions
- Poster: Ranade, S. et al. (2018) High-throughput SMRT Sequencing of clinically relevant targets
- Poster: Clark, Tyson A. et al. (2018) Amplification-free targeted enrichment and SMRT Sequencing of repeat-expansion genomic regions
- Poster: Chakraborty, Shreyasee et al. (2018) Mitochondrial DNA sequencing using PacBio SMRT technology
- Poster: McLaughlin, I., et al. (2017) Targeted sequencing using a long-read sequencing technology
- Poster: Ekholm, J. et al. (2017) Screening for causative structural variants in neurological disorders using long-read sequencing
- Poster: Baybayan, P. et al. (2017) Detection of low-frequency somatic variants using Single-Molecule, Real-Time Sequencing
- Poster: Kujawa, Steve et al. (2017) A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing
- 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, T. A. 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: Kujawa, S. et al. (2016) A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing
- 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: Ekholm, Jenny et al. (2016) Candidate gene screening using long-read sequencing
- Poster: Sethuraman, Anand et al. (2016) Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing
- Poster: Ekholm, Jenny et al. (2016) Enrichment of unamplified DNA and long-read SMRT Sequencing in unlocking the underlying biological disease mechanisms of repeat expansion disorders
- Poster: Baybayan, Primo et al. (2016) Application specific barcoding strategies for SMRT 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: Baybayan, Primo et al. (2015) SMRT Sequencing of DNA and RNA samples extracted from formalin-fixed and paraffin embedded tissues using adaptive focused acoustics by Covaris.
- Korlach, Jonas (2017) ASHG PacBio Workshop: PacBio applications updates & future roadmap
- Ranade, Swati (2017) Webinar: A paradigm shift in HLA sequencing: from exons to high-resolution allele-level HLA yyping
- Korlach, Jonas (2017) Webinar: Addressing “NGS Dead Zones” with third generation PacBio sequencing
- Kujawa, Steve (2017) Webinar: PacBio Targeted Sequencing of Long Amplicons Using PCR or Hybrid Capture
- Turner, Stephen (2017) Webinar: An introduction to PacBio’s long-read sequencing & how it has been used to make important scientific discoveries
- Ameur, Adam (2017) AGBT Virtual Poster: Analysis method for amplification-free SMRT sequencing and assessment on repeat expansions in Huntington’s disease
- Clark, T. A. and Tsai, Y. and Greenberg, D. (2017) AGBT Virtual Poster: Targeted SMRT Sequencing of difficult regions of the genome using a Cas9, non-amplification based method
- (2017) Tutorial: Long amplicon analysis application [SMRT Link v5.0.0]
- 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
- 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
- Gibbs, Richard (2015) ASHG PacBio Workshop: Medical diagnostic challenges and structural variation detection using the PacBio Platform
- Marsh, Steven (2015) Podcast: Long-read sequencing dramatically improves blood matching – Steven Marsh
- Mayor, Neema (2015) Webinar: 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
- Application Brief: Targeted Sequencing of Amplicons – Best Practices (2018)
- Application Brochure: No-Amp targeted sequencing. (2018)
- Application Note: Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies (2016)
- Product Note: Multiplexing Amplicons Up to 10 kb (2015)