Capture regions of interest in high resolution
The discovery of genomic signatures of evolution and the improvement of plants and animals for agricultural purposes have relied on the identification of SNPs, which are associated with phenotypic differences. In order to identify the genes underlying specific phenotypes, the regions containing the informative SNPs must be fine-mapped, typically with targeted sequencing. Often, these regions are large — in the order of several kilobases — and may contain structural variations that are hard to identify with existing technologies.
Flexible targeting for all your regions of interest
Single Molecule, Real-Time (SMRT) Sequencing offers a flexible solution for targeting regions of interest, regardless of size, and delivers the most comprehensive view of the associated genes. It also delivers the read length and accuracy needed to improve fine mapping and simplify the assembly of large regions.
SMRT Sequencing gives you the ability to:
- Efficiently multiplex large amplicons for rapid screening and identification of variants
- Discover haplotype-specific markers for improved breeding of polyploids
- Accurately reassemble entire multi-megabase regions for the study of gene linkage
- Confirm insertion sites of transgenes and validate gene editing events
- Study informative regions to determine evolutionary or domestication history
- Capture complete genes of varying size for flexible and efficient experimental design in gene surveys
Workflow: from targeted region to base-level resolution
- 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: De Novo Assembly of Resistance Genes Using SMRT RenSeq
Using the resistance gene enrichment and sequencing method (RenSeq), scientists were able to identify a gene in a wild relative of potato that confers resistance to Phytophthora infestans, the pathogen responsible for late blight disease, which could be used to develop resistant potatoes (A). For this work the authors targeted the NLR genes responsible for activating plant defense mechanisms. The RenSeq method used in this study was refined to capture sequence fragments of up to 3.2 kb, the average NLR gene length, which were then subjected to SMRT Sequencing. Prior to this, assembly of R-genes was very difficult due to their highly repetitive nature; short-read sequencing did not provide adequate contiguity. The incorporation of SMRT Sequencing, however, resulted in de novo assembly of a single contig containing the gene and also captured >1 kb of the flanking promoter and terminator sequences (B).
Explore this research further.
To learn more about how SMRT Sequencing can give you the long view of your targeted regions, contact us.
- Witek K. et al. (2016) Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing. Nature Biotechnology. 34, 656-660.
- Poster: Ranade, Swati et al. (2016) Immune regions are no longer incomprehensible with SMRT Sequencing
- Presentation: Wing, Rod A et al. (2015) Old school/new school genome sequencing: One step backward — a quantum leap forward.
- Presentation: Berges, Helene et al. (2016) Long read sequencing technology to solve complex genomic regions assembly in plants
- Poster: Gu, Jenny et al. (2016) Targeted sequencing of genes from soybean using NimbleGen SeqCap EZ and PacBio SMRT Sequencing
- Berges, Hélène (2016) PAG Conference: Long reads sequencing technology to solve complex genomic regions assembly in plants
- Ranade, Swati (2016) AGBT Virtual Poster: Immune regions are no longer incomprehensible with SMRT Sequencing
- 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)
- Case Study: In the Netherlands, scientists at KeyGene crack tough plant genomes with PacBio Sequencer. (2015)