No-Amp targeted sequencing
Enrichment of hard-to-amplify genomic regions like repeat expansions is now possible with our no-amplification (No-Amp) targeted sequencing method utilizing the CRISPR/Cas9 system.
Unraveling repeat expansion disorders
Many of the disease-causative genes for repeat expansion disorders were mapped decades ago; however, the underlying disease mechanisms are still not fully understood. These expansions can be several kilobases in size which makes them inaccessible with base-level resolution to most technologies. Now, by combining No-Amp targeted enrichment using the CRISPR/Cas9 system with SMRT Sequencing, scientists can holistically in one experiment:
- Eliminate PCR bias and errors
- Sequence through entire repeat expansions with base-level resolution
- Quantify repeat numbers in normal- and mutant-expanded alleles
- Identify interruption sequences
- Characterize somatic mosaicism
Workflow: from gDNA to Complete Repeat Expansion Sequence
Sample and library prep
With our 2-day No-Amp protocol you can now enrich for your region of interest without amplification using the CRISPR-Cas9 system.
- Start with high-quality genomic DNA (~5-24 µg / SMRT Cell)
- Prepare SMRTbell libraries in 2-days with stream-lined protocol
- Block 5’ & 3’ ends to prevent off-target ligation
- Use custom-design guide RNAs to enrich for regions-of-interest
- Multiplex samples using barcoded adapters
- Remove off-target products with trypsin exonuclease digestion
- Explore recommendations for all SMRT Sequencing applications
Pacific Biosciences does not sell a kit for carrying out the overall No-Amp Targeted Sequencing method. Use of the method may require rights to third-party owned intellectual property.
Sequencing
Use the Sequel Systems to accurately sequence a human genome.
- Sequence multiplexed targets and/or samples on the Sequel Systems using the latest chemistry*
- Adjust movie time parameters based on repeat expansion size
- Run up to 48 samples and up to 15 targets per SMRT Cell on the Sequel II or IIe System
*Read lengths, reads/data per SMRT Cell and other sequencing performance results vary based on sample quality/type and insert size.
Data analysis
Analysis solutions for every user in the lab with SMRT Link and visualization tools.
- Use SMRT Link to recall asymmetric SMRTbell adapters for demultiplexing of samples and perform circular consensus sequencing (CCS) to generate highly accurate long reads (HiFi reads)
- Output data in FASTQ format for results summary reporting on repeat counts and on-target rates
- Visualize results with the IGV and command-line scripts for easy review of repeat count of both alleles, mosaic characterization, identification of interruption sequences and CRISPR-Cas9 off-targets
- Explore our Github resource for repeat analysis tools
Application Brief
Learn more about these best practices for No-Amp targeted sequencing.
Application Brief: Learn more about these best practices for No-Amp targeted sequencing.
Spotlight
No-Amp targeted sequencing advances Ataxia research
Scientists are exploring the genetic composition of complete repeat expansions to uncover novel phenotype-genotype correlations between Parkinson’s disease and the gene ATXN10. Explore this research further.