ThruPLEX®-FD Prep Kit

Single Tube Library Preparation Kit for Illumina® NGS Platforms

thruplex_product

“Our experience with the ThruPLEX library prep has been extremely positive. Both at Rubicon and in our hands it has proven able to generate high quality, diverse libraries from 10x less input than other kits we have used. For us this technology truly enables the study of samples that simply weren’t possible before adopting it.” 

—Henry Long, Ph.D., Associate Director, Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute

Overview

The ThruPLEX-FD Prep Kit* represents a breakthrough in library preparation that streamlines the Illumina Next Generation Sequencing protocol starting with fragmented DNA or cDNA. ThruPlex employs patented technology* based on stem loop adaptors and couples this with a high fidelity polymerase to deliver a highly efficient and user-friendly process. The entire reaction takes place in a single tube in less than 2 hours and eliminates purifications/clean-up steps. Efficiencies, sensitivities and reaction rates are all improved. Regardless of the NGS application– DNA-seq, RNA-seq, ChiP–seq-the ThruPLEX-FD Prep Kit provides consistent and reliable results.

*Protected by US patent 7,803,550 and EP1924704

Workflow

thruplex_workflow_illumina_tube
Learn more about ThruPLEX technology.

Shortest and Easiest Workflow

Shortest and Easiest Workflow
Shortest and Easiest Workflow

ThruPLEX-FD Prep Kit's simple one-tube workflow requires less hands-on and total time than any kit on the market.

Library Diversity & Sensitivity

Library Diversity & Sensitivity
Library Diversity & 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 10 ng diversity). These metrics can be evaluated at <300K reads.

Uniform Coverage Over a Wide Dynamic Range of Input DNA

Uniform Coverage Over a Wide Dynamic Range of Input DNA
Uniform Coverage Over a Wide Dynamic Range of Input DNA

Data at low resolution across the entire human chr 1 demonstrates the difference in coverage with different products and input amounts. ThruPlex preps performed well with fragmented DNA at 0.2 ng and 20 ng. This metric can be qualitatively evaluated with less than 300K reads. (Data courtesy of Washington University and the Broad Institute)

Uniform Coverage in CpG Islands

Uniform Coverage in CpG Islands
Uniform Coverage in CpG Islands

When compared to the Illumina PCR-free prep, the ThruPLEX-FD prep has strong representation across 4 RASSF1 CpG islands. The TruSeq prep shows significant systematic dips in representation across the same CpG island. Note the difference in input DNA levels. All data was analyzed at low coverage.

Low PCR Background

Low PCR Background
Low PCR Background

A 200 pg sample of Covaris-fragmented DNA was over-amplified by 10 PCR cycles above the suggested protocol. NTC (no template control) shows PCR background about >100X lower than the sample.

Low Resolution Coverage is Insensitive to Number of PCR Cycles

Low Resolution Coverage is Insensitive to Number of PCR Cycles
Low Resolution Coverage is Insensitive to Number of PCR Cycles

A 200 pg sample of Covaris-fragmented DNA was over-amplified in Step 3 of the ThruPLEX protocol by 10 cycles (above the 15 cycles required for minimal sequencing) without affecting low-resolution coverage. (Data calculated by DNAnexus.)

Library Diversity & Unmapped Reads are Insensitive to PCR Cycles

Library Diversity & Unmapped Reads are Insensitive to PCR Cycles
Library Diversity & Unmapped Reads are Insensitive 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 did not change significantly by adding 10 cycles of PCR.

GC Representation is Insensitive to PCR Cycles

GC Representation is Insensitive to PCR Cycles
GC Representation is Insensitive 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.)

ChIP Results Show Concordance with TruSeq Standard Prep

ChIP Results Show Concordance with TruSeq Standard Prep
ChIP Results Show Concordance with TruSeq Standard Prep

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. Data provided by Baylor Medical College.

Workflow Advantage

Workflow Advantage
Workflow Advantage

ThruPLEX-FD Kit has a simple workflow that eliminates transfers and reduces time to results.

Store at -20 °C. Guaranteed for 9 months at -20C in a constant temperature freezer.

ThruPLEX-FD Prep Kit is designed to work on a wide variety of DNA samples with amounts varying between 50 pg and 50 ng.  To ensure that your experiment delivers quality data, the table below provides a range of recommended input amounts of  human DNA based on (1) your application and (2) the source material.  The actual amount that you chose to use may vary from those suggested below.  Note that as the amount of input DNA decreases, the diversity of the library may also decrease

Application Input sample amount (50 – 500 bp ds DNA) Recommended input amount
WGS, WES, SNV, indels, SNP, STR gDNA 10 – 50 ng
FFPE DNA 20 – 50 ng
plasma DNA 1 – 50 ng
CNV plasma, gDNA or FFPE DNA 50 pg – 50 ng*
ChIP-seq ChIP DNA 50 pg – 50 ng*
RNA-seq cDNA 50 pg – 50 ng*

*Use of  less than 50 pg has been cited in scientific publications.

FAQs

Read all FAQ’s on ThruPLEX-FD Prep Kit

1. Is ThruPLEX®-FD Prep Kit compatible with SureSelect (Agilent Technologies) enrichment?
Yes, it is compatible with SureSelect enrichment, although ThruPLEX-FD libraries must first be amplified before undergoing the enrichment. Also, it might be necessary to optimize the amount of amplified ThruPLEX-FD library to be used as input to SureSelect, and/or optimize the concentration of bait.

2. Can ThruPLEX-FD Kit be run without a real time PCR machine? If so, how do I know when to stop my run?
Yes, the kit can be used even if a lab does not have access to a real time PCR machine. Use the table on page 6 of the ThruPLEX-FD Prep Kit manual as an approximation guide to the number of PCR cycles that should be run for one’s particular DNA input amount.

3. What DNA fragmentation size do you recommend?
Average fragment size of 300 bp is recommended; although this kit can accommodate a range of 50 – 1000 bp DNA input. Additionally, depending on the desired sequencing read length, the DNA needs to be fragmented to a specific average size.

4. While using ThruPLEX-FD, a problem with the size of the insert/fragment output (using Bioanalyzer)?
Unexpected fragment sizes post the prep (fragments were expected to be larger, but the end-result seemed to be as if only 60 bp were added onto their fragments and not the expected 120 bp), may be a result of incorrrect use of thermal cyclers  during the library prep.  These instruments do not  support the required 75-µl volume, and thus the reactions are not properly cycled.  Please make sure the instrumentation used during the prep is appropriate, can support 75-µl volume reactions, and the amplification is monitored in real time to ensure proper library preparation and amplification.

5. Is a high-fidelity enzyme used in the amplification reaction?
Yes, a high-fidelity, high-processivity, low-bias DNA polymerase is used in the amplification reaction.

6. What is the recommended elution/resuspension buffer for DNA sample(s) that will be used in Template Preparation step (step A.1 of the ThruPLEX-FD Prep Kit manual)? Are there any DNA purity considerations?
The DNA sample(s) used with this technology must be eluted/resuspened in a low-salt and low-EDTA, buffered solution, such as TE buffer, or TE with reduced EDTA content, prior to ThruPLEX-FD usage.

7. Is it recommended to perform a cleanup on my DNA sample(s) after shearing the DNA?
No. In order to preserve your total DNA and the overall diversity of the library, take a 10 μl aliquot of the sheared DNA sample and go to Template Preparation step (Step A.1 of the ThruPLEX-FD Prep Kit manual).

8. What is the recommended fluorescent dye for real time PCR monitoring?
EvaGreen® fluorescent dye (Biotium, Catalog No: 31000) is recommended, due to its high sensitivity and low interference with the amplification chemistry.

9. Is it possible to order more indices than the 12 provided in the ThruPLEX-FD Prep Kit? Alternatively, can I prepare them myself?
The current ThruPLEX-FD 48-rxn Prep Kit, just like the 12-rxn product, comes equipped with 12 indices. Unfortunately, Rubicon Genomics cannot support any substitution of index primers in this kit.

10. What concentration of ThruPLEX-FD library should be loaded onto the flow cell?
Please follow Illumina’s recommendations for optimal loading concentration specific to the version of flow cell you are using.

 

ThruPLEX®-FD Prep Kit (48 rxn, 12 indexes for Illumina® HiSeq/GA/MiSeq)   CAT. NO. R40048.

ThruPLEX®-FD Prep Kit (12 rxn, 12 indexes for Illumina® HiSeq/GA/MiSeq)    CAT. NO. R40012

Each kit has everything required for library preparation when starting with fragmented DNA or cDNA including 12 Illumina indices.

Order directly from our STORE.

 

For RNA-seq work, we recommend the Clontech SMARTer®  Universal Low Input RNA Kit.

Performance

Shortest and Easiest Workflow

Shortest and Easiest Workflow
Shortest and Easiest Workflow

ThruPLEX-FD Prep Kit's simple one-tube workflow requires less hands-on and total time than any kit on the market.

Library Diversity & Sensitivity

Library Diversity & Sensitivity
Library Diversity & 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 10 ng diversity). These metrics can be evaluated at <300K reads.

Uniform Coverage Over a Wide Dynamic Range of Input DNA

Uniform Coverage Over a Wide Dynamic Range of Input DNA
Uniform Coverage Over a Wide Dynamic Range of Input DNA

Data at low resolution across the entire human chr 1 demonstrates the difference in coverage with different products and input amounts. ThruPlex preps performed well with fragmented DNA at 0.2 ng and 20 ng. This metric can be qualitatively evaluated with less than 300K reads. (Data courtesy of Washington University and the Broad Institute)

Uniform Coverage in CpG Islands

Uniform Coverage in CpG Islands
Uniform Coverage in CpG Islands

When compared to the Illumina PCR-free prep, the ThruPLEX-FD prep has strong representation across 4 RASSF1 CpG islands. The TruSeq prep shows significant systematic dips in representation across the same CpG island. Note the difference in input DNA levels. All data was analyzed at low coverage.

Low PCR Background

Low PCR Background
Low PCR Background

A 200 pg sample of Covaris-fragmented DNA was over-amplified by 10 PCR cycles above the suggested protocol. NTC (no template control) shows PCR background about >100X lower than the sample.

Low Resolution Coverage is Insensitive to Number of PCR Cycles

Low Resolution Coverage is Insensitive to Number of PCR Cycles
Low Resolution Coverage is Insensitive to Number of PCR Cycles

A 200 pg sample of Covaris-fragmented DNA was over-amplified in Step 3 of the ThruPLEX protocol by 10 cycles (above the 15 cycles required for minimal sequencing) without affecting low-resolution coverage. (Data calculated by DNAnexus.)

Library Diversity & Unmapped Reads are Insensitive to PCR Cycles

Library Diversity & Unmapped Reads are Insensitive to PCR Cycles
Library Diversity & Unmapped Reads are Insensitive 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 did not change significantly by adding 10 cycles of PCR.

GC Representation is Insensitive to PCR Cycles

GC Representation is Insensitive to PCR Cycles
GC Representation is Insensitive 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.)

ChIP Results Show Concordance with TruSeq Standard Prep

ChIP Results Show Concordance with TruSeq Standard Prep
ChIP Results Show Concordance with TruSeq Standard Prep

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. Data provided by Baylor Medical College.

Workflow Advantage

Workflow Advantage
Workflow Advantage

ThruPLEX-FD Kit has a simple workflow that eliminates transfers and reduces time to results.

Specifications

Store at -20 °C. Guaranteed for 9 months at -20C in a constant temperature freezer.

ThruPLEX-FD Prep Kit is designed to work on a wide variety of DNA samples with amounts varying between 50 pg and 50 ng.  To ensure that your experiment delivers quality data, the table below provides a range of recommended input amounts of  human DNA based on (1) your application and (2) the source material.  The actual amount that you chose to use may vary from those suggested below.  Note that as the amount of input DNA decreases, the diversity of the library may also decrease

Application Input sample amount (50 – 500 bp ds DNA) Recommended input amount
WGS, WES, SNV, indels, SNP, STR gDNA 10 – 50 ng
FFPE DNA 20 – 50 ng
plasma DNA 1 – 50 ng
CNV plasma, gDNA or FFPE DNA 50 pg – 50 ng*
ChIP-seq ChIP DNA 50 pg – 50 ng*
RNA-seq cDNA 50 pg – 50 ng*

*Use of  less than 50 pg has been cited in scientific publications.

Resources

FAQs

FAQs

Read all FAQ’s on ThruPLEX-FD Prep Kit

1. Is ThruPLEX®-FD Prep Kit compatible with SureSelect (Agilent Technologies) enrichment?
Yes, it is compatible with SureSelect enrichment, although ThruPLEX-FD libraries must first be amplified before undergoing the enrichment. Also, it might be necessary to optimize the amount of amplified ThruPLEX-FD library to be used as input to SureSelect, and/or optimize the concentration of bait.

2. Can ThruPLEX-FD Kit be run without a real time PCR machine? If so, how do I know when to stop my run?
Yes, the kit can be used even if a lab does not have access to a real time PCR machine. Use the table on page 6 of the ThruPLEX-FD Prep Kit manual as an approximation guide to the number of PCR cycles that should be run for one’s particular DNA input amount.

3. What DNA fragmentation size do you recommend?
Average fragment size of 300 bp is recommended; although this kit can accommodate a range of 50 – 1000 bp DNA input. Additionally, depending on the desired sequencing read length, the DNA needs to be fragmented to a specific average size.

4. While using ThruPLEX-FD, a problem with the size of the insert/fragment output (using Bioanalyzer)?
Unexpected fragment sizes post the prep (fragments were expected to be larger, but the end-result seemed to be as if only 60 bp were added onto their fragments and not the expected 120 bp), may be a result of incorrrect use of thermal cyclers  during the library prep.  These instruments do not  support the required 75-µl volume, and thus the reactions are not properly cycled.  Please make sure the instrumentation used during the prep is appropriate, can support 75-µl volume reactions, and the amplification is monitored in real time to ensure proper library preparation and amplification.

5. Is a high-fidelity enzyme used in the amplification reaction?
Yes, a high-fidelity, high-processivity, low-bias DNA polymerase is used in the amplification reaction.

6. What is the recommended elution/resuspension buffer for DNA sample(s) that will be used in Template Preparation step (step A.1 of the ThruPLEX-FD Prep Kit manual)? Are there any DNA purity considerations?
The DNA sample(s) used with this technology must be eluted/resuspened in a low-salt and low-EDTA, buffered solution, such as TE buffer, or TE with reduced EDTA content, prior to ThruPLEX-FD usage.

7. Is it recommended to perform a cleanup on my DNA sample(s) after shearing the DNA?
No. In order to preserve your total DNA and the overall diversity of the library, take a 10 μl aliquot of the sheared DNA sample and go to Template Preparation step (Step A.1 of the ThruPLEX-FD Prep Kit manual).

8. What is the recommended fluorescent dye for real time PCR monitoring?
EvaGreen® fluorescent dye (Biotium, Catalog No: 31000) is recommended, due to its high sensitivity and low interference with the amplification chemistry.

9. Is it possible to order more indices than the 12 provided in the ThruPLEX-FD Prep Kit? Alternatively, can I prepare them myself?
The current ThruPLEX-FD 48-rxn Prep Kit, just like the 12-rxn product, comes equipped with 12 indices. Unfortunately, Rubicon Genomics cannot support any substitution of index primers in this kit.

10. What concentration of ThruPLEX-FD library should be loaded onto the flow cell?
Please follow Illumina’s recommendations for optimal loading concentration specific to the version of flow cell you are using.

 

Ordering Information

ThruPLEX®-FD Prep Kit (48 rxn, 12 indexes for Illumina® HiSeq/GA/MiSeq)   CAT. NO. R40048.

ThruPLEX®-FD Prep Kit (12 rxn, 12 indexes for Illumina® HiSeq/GA/MiSeq)    CAT. NO. R40012

Each kit has everything required for library preparation when starting with fragmented DNA or cDNA including 12 Illumina indices.

Order directly from our STORE.

 

For RNA-seq work, we recommend the Clontech SMARTer®  Universal Low Input RNA Kit.