Specific inherited mutations in BRCA1 and BRCA2 increase the risk of female breast and ovarian cancers. The BRCA genes have unusually long exons, some even as large as 3.5Kb and 5Kb, and contain regions that are exceptionally high in GC content limiting the coverage of many commercially available enrichment technologies. 

The TargetRich BRCA1 and BRCA2 sequencing panel provides 100% coverage of the 50 exons comprising both genes (18Kb total design space, 158 amplicons).  Target enrichment was validated using buccal swab gDNA and  sequenced with 150 base pair, paired end reads on a MiSeq¢ç. The amplicons targeting  BRCA1 and BRCA2 demonstrated high uniformity, 97% alignment and high depth of coverage across the three buccal swab samples.

• High uniformity across amplicons
• High alignment to targeted regions
• High reproducibility across buccal swab samples
• High coverage of amplicons across replicates

The BRCA1 and BRCA2  Sequencing Panel has been validated using  3 replicate gDNA samples from buccal swabs (250 ng input each).  DNA was enriched and sequenced on a MiSeq (150 base pairs, paired end).  Results are summarized in the box plots.  The bold horizontal line represents the median read depth across 158 amplicons.  Replicate samples show high reproducibility and uniform coverage of amplicons.

Superior Design Coverage Capabilities

By utilizing Nested Patch PCR technology and a proprietary design algorithm, custom panels can be developed with maximum coverage of desired regions.  We can provide coverage for regions where others have failed. Comparison of our design coverage versus competitors¡¯ for BRCA2 (> 10,000 bp).  Our designs covered 100% of the coding regions for BRCA2, whereas AmpliSeq Designs covered 94.3% (no coverage for 600 bp, using default settings).  34% coverage for BRCA2 using Illumina¡¯s Design Studio (data not shown). 

CRX Panel Shows High Reproducibility Across Samples and Consistency Even at Low Input from FFPE Samples

Tumor samples often provide limited, low quality DNA resulting in poor sequence depth of coverage and inconsistent results for genetic variant detection.  The TargetRich CRX Sequencing Panel covers 99.8% of the 10 of the most commonly studied cancer-related genes* including: BRAF, EGFR, FLT3, JAK2, KIT, KRAS, PIK3CA, PTEN, TP53 and VEGFA (155 exons, 23,155 bp total).  The panel is sensitive and reliable across HMW gDNA replicates and with low input, FFPE treated samples. 
 

• Sensitivity with low input FFPE
• High read depth of amplicons regardless of sample type or input amount 
• High reproducibility across gDNA replicate samples
• Superior design coverage

Sensitive Detection with Low Input FFPE Samples

Reproducibility Across gDNA Samples

The CRX Sequencing Panel has been validated using 10 ng to 100 ng of gDNA  from FFPE-treated samples and 3 replicate gDNA samples (250 ng input each).  DNA was enriched and sequenced on a MiSeq (150 base pairs, paired end).  Results are summarized in the box plots.  The bold horizontal line represents the median read depth across 262 amplicons.  Replicate samples show high reproducibility and lower inputs did not result in a loss of coverage  across the amplicons.


* Based on the Catalogue of somatic mutations in cancer ("COSMIC database," 2012)

 

 

 

TargetRich Workflow and Panel Components

Flexible design capability allows highly specific targeting of regions of interest. With two very specific target enrichment rounds; the designs are able to enrich the regions of interest with precision; maximizing the on-target enrichment and minimizing off-target coverage. TargetRich has the flexibility to design against many types of regions e.g. exons, SNPs and hotspots; and requires only the gene symbol, SNP ID, chromosome range of interest and whether UTRs are to be included. Once the panel is designed, a sequencing quality check run is performed to ensure its performance before delivery.


All components are pre-validated and necessary to perfom the TargetRich single tube workflow for target enrichment for sequencing. This includes Primers, Master Mixes for the two PCR enrichment steps, Primer Cleavage Master Mixes, Enzyme for Nucleotide Inactivation, Patch Ligation Master Mix, Enzyme Master Mixes for the two enzymatic cleanup steps as well as Read and Index  primers for the NGS run.
Single Tube Additive Workflow with minimal hands on time.

Custom panel advantages:

Validated:  Custom panels are validated using NGS analysis to optimize target coverage and representation of each amplicon.  Two samples of genomic DNA will be tested using the standard single tube protocol.

Single tube:  The Nested Patch PCR methodology uses an additive single-tube protocol.  Each component is added to the reaction without transfers or centrifugation.

Design coverage:  Nested Patch PCR and a proprietary design algorithm enable design flexibility with maximum coverage for regions of interest.  The custom panel shown below represents 99.6% coverage of 1215 amplicons spanning approximately 121 kb.

Specificity:  The two rounds of target-specific enrichment used in Nested Patch PCR provide high percentage alignment to targeted regions.  Sequencing results from the custom panel shown resulted in > 72% on target alignment. 

Sensitivity: High sensitivity of a targeted enrichment assay will improve the variant detection rate.  On average over 97% of known variants in the 121 kb of targeted sequence was detected. 


Summary of custom panel validation

Performance Across Samples Maximizes Sequencing Capacity

High Specificity Provides High Read Coverage From Across the PanelConsistent

A custom panel designed for 65 genes (targeting 815 exons and 120,651 base pairs) with 1215 amplicons was validated on 8 gDNA samples.  Samples were enriched and sequenced on a MiSeq (150 base pairs, paired end).  On-target sequencing results are summarized in the table above by P1, P10, P25 and P100 reads for the 8 samples.  Amplicon read depth across the replicate samples is shown by box blot where the bold horizontal line represents median read depth across the panel of 1215 amplicons.