Sequence your targets
Targeted sequencing is a powerful way to increase the cost-effectiveness of variant discovery and detection. To generate a full view of relevant targets, you need complete and uniform coverage across regions of interest. Unfortunately, short-read sequencing data is prone to mis-mapping and often fails to span repeats. Additionally, PCR-related bias can result in insufficient coverage for variant calling in GC-rich regions1.
Single Molecule, Real-Time (SMRT) Sequencing combines single-molecule observation, long-read sequencing, and the lowest degree of bias to fully characterize genetic complexity — including structural variation, rare SNPs, indels, copy number variation, microsatellites, haplotypes, and phasing. Through targeted sequencing on the PacBio Systems, you move beyond simply cataloging SNPs and target all types of variation across relevant genomic regions, including low complexity regions like repeat expansions, promoters, and flanking regions of transposable elements.
PacBio sequencing hits the targets others miss
- Explore No-Amp targeted sequencing to enrich hard-to-amplify genomic regions
- Characterize complex regions underlying human genetic disease
- Perform candidate gene or domestication studies for plants and animals
- Resolve complex regions in microbial and infectious disease research
- Phase full-length HLA allele variation without imputation
- Characterize extended haplotypes of complex immune regions
- Fill knowledge gaps with BAC sequencing
Spotlight: Allelic origins of genes and transcripts
The causes of Alzheimer’s disease (AD) are genetically complex. This webinar details a probe-based targeted enrichment strategy that allows scientists to fully characterize genes, corresponding splice isoforms, and associated alleles. The power of this approach is demonstrated using a panel of probes targeting 35 AD candidate genes to determine their genetic profile.
References
Selected Resources
- Kosicki, Michael et al. (2018) Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nature Biotechnology
- Castellanos, Mildred et al. (2018) A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat. Molecular Microbiology
- 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
- Verhey, Theodore B et al. (2018) Antigenic variation in the lyme spirochete: Insights into recombinational switching with a suggested role for error-prone repair. Cell Reports
- 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
- Hoff, Siv Nam Khang et al. (2018) Long-read sequence capture elucidates the evolution of the hemoglobin gene clusters in codfishes BioRxiv
- 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
- Haig, Sarah-Jane et al. (2018) A high-throughput approach for identification of nontuberculous mycobacteria in drinking water reveals relationship between water age and Mycobacterium avium. mBio
- Ebbert, Mark T. W. et al. (2018) Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for genetic discovery efforts in human disease BioRxiv
- 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
- Zimmer, Christoph T et al. (2018) Neofunctionalization of duplicated P450 genes drives the evolution of insecticide resistance in the brown planthopper. Current Biology
- Verhey, Theodore B et al. (2017) Analysis of recombinational switching at the antigenic variation locus of the Lyme spirochete using a novel PacBio sequencing pipeline. Molecular Microbiology
- Tsai, Yu-Chih et al. (2017) Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions BioRxiv
- Fraiture, Marie-Alice et al. (2017) An integrated strategy combining DNA walking and NGS to detect GMOs. Food Chemistry
- 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
- Dara, Antoine et al. (2017) A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples. Malaria Journal
- Albrecht, V et al. (2017) Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles. HLA
- Vu, Giang T H et al. (2017) Endogenous sequence patterns predispose the repair modes of CRISPR/Cas9-induced DNA double strand breaks in Arabidopsis thaliana. The Plant Journal
- Novák, Karel et al. (2017) Diversity of the TLR4 immunity receptor in Czech native cattle breeds revealed using the Pacific Biosciences sequencing platform. Animal Biotechnology
- Borràs, Daniel M et al. (2017) Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing. Human Mutation
- D'Amore, Rosalinda et al. (2017) SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms. BioTechniques
- Vu, Giang T H et al. (2017) Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage. The New Phytologist
- 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
- Viluma, Agnese et al. (2017) Genomic structure of the horse major histocompatibility complex class II region resolved using PacBio long-read sequencing technology. Scientific Reports
- Hans, Jörg B et al. (2017) Gorilla MHC class I gene and sequence variation in a comparative context. Immunogenetics
- 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: Oh, S. et al. (2016) Minimization of chimera formation and substitution errors in full-length 16S PCR amplification
- Poster: Hall, R. et al. (2016) Workflow for processing high-throughput, Single Molecule, Real-Time Sequencing data for analyzing the microbiome of patients undergoing fecal microbiota transplantation
- 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: Hepler, N. Lance et al. (2016) An improved circular consensus algorithm with an application to detection of HIV-1 Drug-Resistance Associated Mutations (DRAMs)
- 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
- McFarland, Karen (2017) ASHG PacBio Workshop: Expansion sequence variations underlie distinct disease phenotypes in SCA10
- 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
- Singh, Siddharth (2017) Webinar: Beginner’s Guide to PacBio SMRT Sequencing Data Analysis
- Turner, Stephen (2017) Webinar: An introduction to PacBio’s long-read sequencing & how it has been used to make important scientific discoveries
- Cereb, Nezih (2017) AGBT PacBio Workshop: High-throughput HLA class I whole gene and HLA class II long range typing on PacBio RSII and Sequel Platforms
- 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) 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]
- Wing, Rod (2017) PAG PacBio Workshop: Introducing 5 new high-quality PacBio genome assemblies for rice to help solve the 10-billion people question
- 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
- Morey, Robert (2016) AGBT Virtual Poster: Using PacBio sequencing as a synthetic biology error analysis tool
- Berges, Hélène (2016) PAG Conference: Long reads sequencing technology to solve complex genomic regions assembly in plants
- Application Brochure: No-Amp targeted sequencing. (2018)
- 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)