ThruPLEX-FD

Single tube sample preparation for Illumina® NGS Platforms for Nanogram to Picogram Inputs of Fragmented dsDNA

ThruPLEX technology is unlike anything on the market.  Different Biochemistry. Different components. Different Protocols.  The result is a breakthrough in sample preparation for Next Generation Sequencing.  Enzymes and reagents are carefully selected and matched to achieve a multiplexing of reactants. Efficiencies, sensitivities and reaction rates all improve. 

To the end user this means:

• Much smaller DNA input requirements
• Less contaminants, cleaner preps yielding lower backgrounds and better sequencing data
• Fewer processing steps
• Greater daily output
• High sensitivity results with greater uniformity.  

 

Product Sourcing

ThruPLEX-FD is sold directly through our online store. For commercial applications, special orders or volume discounts contact Sales at Rubicon Genomics.

 

Simple Fast Protocol - Easily Automated

ThruPLEX-FD Prep is a 1-tube, 2 hour, 3 step process for preparing 0.5 pg-50 ng of fragmented DNA for accurate sequencing, genotyping, and mutation detection. ThruPLEX-FD is 10-100X more sensitive than conventional NGS preps of plasma, FFPE, and ChIP DNA.

Increased Daily Output

One Lab Technician can prepare 192 indexed samples per day.

 

 

 

 

Metrics We Use to Evaluate the Performance of Our Technologies

1. Library diversity (for constant input)
2. NGS sensitivity (for constant diversity)
3. Low resolution coverage
4. Coverage of CpG islands and other difficult sequences
5. PCR background
6. Sensitivity of low resolution coverage to PCR cycles
7. Sensitivity of library diversity to PCR cycles
8. Sensitivity of unmapped reads to PCR cycles
9. Sensitivity of GC representation to PCR cycles
10. Concordance between biological information from functionally-enriched and sequence-enriched libraries from low amounts of input compared to “gold-standard” results from much larger inputs.

 

Metric #1: Library Diversity - Metric #2: NGS Sensitivity

Covaris-fragmented human DNA was prepared and sequenced by a university core lab using an NEBNext prep and ThruPLEX-FD prep. Library complexity as measured by DNAnexus “bottleneck score” or diversity calculations shows ThruPLEX has ~10X higher diversity (at 10 ng input) or 30X higher sensitivity (at 10e8 diversity). These metrics can be evaluated at <300K reads.

Metric #3: Low Resolution Uniformity of Coverage

Data across entire human chr 1 shows that different NGS preps give different coverage uniformity. This metric can be qualitatively evaluated with less than 300K reads.

Metric #4: Uniformity of Coverage in CpG Islands

Coverage across 4 RASSF1 CpG islands for three technologies is compared. Using the Illumina PCR-free prep data as the gold standard, ThruPLEX-FD at low coverage has strong representation across CpG islands. The TruSeq prep shown has significant systematic dips in representation across CpG islands. Evaluation of RASSF1 needs ~100M reads.

Metric #5: PCR Backgrounds

Library properties as a function of PCR cycles.  200 pg of Covaris-fragmented DNA was over-amplified by 10 PCR Cycles above that required for minimal sequencing, to achieve a “plateau.”  NTC (no template control) shows PCR background about >100X smaller than the 200 pg sample.

Metric #6: Sensitivity of Low Resolution Coverage to Number of PCR Cycles

200 pg of Covaris-fragmented DNA was over-amplified by 10 PCR Cycles above that required for minimal sequencing without affecting low-resolution coverage. (DNANexus)

Metric #7: Sensitivity of Library Diversity to PCR Cycles
Metric #8: Sensitivity of Unmapped Reads to PCR Cycles

200 pg of Covaris-fragmented DNA was over-amplified by 10 PCR Cycles above that required for minimal sequencing, to achieve a “plateau.”  The library diversity and the number of unmapped reads do not change significantly by adding 10 cycles of PCR.

Metric #9: Sensitivity of GC Representation to PCR Cycles

200 pg of Covaris-fragmented DNA was over-amplified by 10 PCR Cycles above that required for minimal sequencing, without serious changes in GC represention.  GC-representation calculated by DNANexus

Metric #10: Concordance Between Low Input ChIP Peaks and Gold Standard of High Input of Same Sample Into TruSeq Preparation

ThruPLEX-FD peaks from 50 - 200 pg ChIP DNA input were >92% concordant with peaks from 10 ng ChIP DNA from 300,000 cells precipitated with the same antibodies and prepped with Illumina ChIP-seq kit. However, TruSeq has lower % duplicates, because there are fewer reads on target (a case of pseudo-diversity). Data provided by Baylor Medical College.