Targeted resequencing strategies provide an efficient method to quickly interrogate particular genomic regions of interest, from single PCR products up to 50 Mb regions via hybridization capture technologies. The clonal nature of 454 Sequencing Systems allows unambiguous allele resolution of variation in complex regions of the genome, along with quantitative detection of variants present in less than 1% of a mixture. Perform haplotyping, genotyping, rare variant detection, structural variation detection, copy number variation analysis and heterozygote calling all using the same platform.
- Discover germline or somatic mutations (e.g., cancer and disease associated regions)
- Detect and quantify low-frequency variants such as rare drug-resistant viral mutations (e.g., HIV, HBV or microbial pathogens)
- Unambiguously resolve highly complex genomic regions (e.g., profile the human immune system including HLA and immunoglobulin complexity – Available now! GS GType HLA Primer Sets)
- Screen samples in GWAS follow-up to identify potentially causative mutations in disease-associated regions
- Validate mutations from whole genome approaches
- Sequence larger custom genomic regions or whole exomes with hybridization enrichment technologies
- Obtain more complete coverage of target regions: Discover gene loci for haplotyping of distantly linked variants through long amplicon lengths
- Easily transition capillary sequencing amplicons to 454 Sequencing Systems: Long read lengths >400 bp allow use of existing amplicon designs by simply adding adaptors
- Detect variations with high sensitivity: Use high-depth coverage to detect rare (<1%) genetic variants in mixed sample populations without individual sample cloning.
- Expand further into repetitive genomic regions: Long reads allow extension into regions adjacent to hybridization probes revealing variation in regions of the genome inaccessible to other technologies
- Multiplex samples more easily: Long read lengths mean that there is plenty of sequence available for regions of interest even after appending MIDs
Unambiguous haplotyping using 454 Sequencing Systems. Non-clonal sequencing of amplicons can lead to ambiguous results due to the difficulty in confidently haplotyping loci with more than one variant. For example, this short 14-base region of the HLA DQA1 exon 2 locus contains five variants resulting in 32 possible allelic combinations. Haplotyping using traditional approaches involves physical separation of the two alleles by cloning, a time-consuming and labor-intensive process.
In contrast, 454 Sequencing approach yields hundreds to thousands of clonal reads for each amplicon. Haplotyping can be accomplished completely in software without any need for additional sequencing. In addition, the large number of clonal reads also allows for confident calling of low-frequency variations.
454 Sequencing Systems support a variety of targeted resequencing approaches, including PCR amplicon sequencing, high-throughput PCR amplicon sequencing using the Fluidigm Access Array System or the RainDance System, or hybridization enrichment such as NimbleGen Sequence Capture.
Sequence amplicons by integrating 454-specific sequencing adaptors onto PCR products, either during PCR or by ligation. The use of 454 fusion primers in locus-specific amplifications results in amplicons that can proceed directly into emPCR amplification and sequencing without additional library preparation steps.
Take advantage of universal tailing strategies to cost-effectively analyze highly multiplexed sample pools. The GS FLX and GS Junior System enable independent sequencing of hundreds to thousands of copies of amplicons in a single run. Directionality can be maintained throughout sequencing and analysis.
Improve sample throughput and increase experimental flexibility by sequencing a wide range of primer-sample combinations per run (from a few to hundreds) using multiplexing options. Carefully designed Multiplex Identifiers (MIDs) maximize the utility of each sequencing run by combining multiple samples together for sequencing while allowing accurate sample identification through bioinformatic processing.
Fusion Primer Ordering
Order 454 fusion primers directly from Integrated DNA Technologies. Design, synthesis and purify primers for use with the GS FLX and GS Junior Systems > Learn more
A number of targeted sequencing technologies are available to streamline amplicon preparation prior to emPCR amplification and sequencing on the GS Junior or GS FLX Systems.
Fluidigm – The Fluidigm Access Array System is a benchtop system that performs automated processing of up to 48 unique samples against 48 unique amplicons on a single microfluidic chip with minimal hands-on time.
RainDance – RainDance’s automated microdroplet-based RainStorm technology enables high-throughput preparation of up to 2 million PCR reactions, interrogating up to 20,000 amplicons per sample.
Sequence capture is an efficient way to isolate large or highly dispersed regions of interest from a pool of DNA molecules.
Roche NimbleGen Sequence Capture
NimbleGen Sequence Capture technologies are optimized for use with 454 Sequencing Systems and offer targeted sequencing of custom regions up to 50 Mb or the whole human exome. NimbleGen GS FLX Titanium-Optimized Sequence Capture utilizes a 454-specific library preparation so that the captured products proceed directly into emPCR amplification, resulting in fewer steps, less amplification, and simpler, more robust protocols.
Two software solutions are available for analyzing target region data, depending on the sequencing methods and experimental design.
GS Amplicon Variant Analysis Software: Aligns PCR amplicon reads against a reference sequence, accurately detects and quantifies known variants in complex pools, and discovers novel variants.
GS Reference Mapper Software: Rapidly and accurately align reads to any reference genome and explore the full range of genomic variations: SNPs, insertions, deletions and structural variations.
PCR Amplicon Sequencing
- A multi-site study using high-resolution HLA genotyping by next generation sequencing. Holcomb CL et al. (2011) Tissue Antigens 77(3): 206-217.
- Diverse somatic mutation patterns and pathway alterations in human cancers. Kan Z t al. (2010) Nature 466(7308):869-73.
- Deep resequencing reveals excess rare recent variants consistent with explosive population growth. Coventry A et al. (2010) Nature Communications 1(8): 131.
- Low-abundance drug-resistant viral variants in chronically HIV-infected, antiretroviral treatment–naive patients significantly impact treatment outcomes. Simen B et al. (2009) Journal of Infectious Diseases 199(5): 693-701
- Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing. Boyd SD et al. (2009) Science Translational Medicine 1(12): 12ra23.
- Somatic mutation profiles of MSI and MSS colorectal cancer identified by whole exome next generation sequencing and bioinformatics analysis. Timmermann B et al. (2010). PloS One 5(12):e15661.
- A comprehensive resequence analysis of the KLK15-KLK3-KLK2 locus on chromosome 19q13.33. Parikh H et al. (2010) Human Genetics 127(1): 91-9.
- Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer. Chmielecki J et al. (2010) Nucleic Acids Research 38(20): 6985-6996.
- Exome sequencing of a multigenerational human pedigree. Hedges D et al. (2009) PLoS One 4(12): e8232.
- Application Note: Capturing Complex Human Genetic Variations using the GS FLX+ System
- Application Note: Parallel Copy Number Variation and Sequence Variation Detection Using Amplicon-based Sequencing on the GS Junior System
- Application Note: Targeted Sequencing of Leukemia-Associated Genes Using 454 Sequencing Systems
- Application Note: Screening Cancer Genes using NimbleGen Sequence Capture Arrays and the GS Junior Sequencing System
- Amplicon Sequencing with the GS FLX Titanium Series: High Sensitivity Targeted Resequencing