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
- 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
- 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
- Turner, T R et al. (2017) Single molecule real-time (SMRT®) DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines. HLA
- 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
- 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
- Fraiture, Marie-Alice et al. (2017) An integrated strategy combining DNA walking and NGS to detect GMOs. Food Chemistry
- 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
- 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
- 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
- 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
- Fischer, Matthias G et al. (2016) Host genome integration and giant virus-induced reactivation of the virophage mavirus. Nature
- 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
- Giolai, Michael et al. (2016) Targeted capture and sequencing of gene-sized DNA molecules. BioTechniques
- Eudes, Aymerick et al. (2016) Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast. Microbial Cell Factories
- Oren, Matan et al. (2016) Short tandem repeats, segmental duplications, gene deletion, and genomic instability in a rapidly diversified immune gene family. BMC Genomics
- 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
- Dowell, Noah L et al. (2016) The deep origin and recent loss of venom toxin genes in rattlesnakes. Current Biology
- 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
- Palmer, William H et al. (2016) Variation and evolution in the glutamine-rich repeat region of Drosophila argonaute-2. G3
- Bellec, Arnaud et al. (2016) Long read sequencing technology to solve complex genomic regions assembly in plants Journal of Next Generation Sequencing & Applications
- Rafati, Nima et al. (2016) Large deletions at the SHOX locus in the pseudoautosomal region are associated with skeletal atavism in Shetland ponies. G3
- 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 Typing
- 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: Beginners Guide to PacBio SMRT Sequencing Data Analysis
- Turner, Stephen (2017) Webinar: An Introduction to PacBios 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
- Clark, T. A. and Tsai, Y. and Greenberg, D. (2017) AGBT Conference: Targeted SMRT Sequencing of difficult regions of the genome using a Cas9, non-amplification based method
- Ameur, Adam (2017) AGBT Virtual Poster: Analysis method for amplification-free SMRT sequencing and assessment on repeat expansions in Huntington’s disease
- (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)