Highest Diversity, Fewest Unmapped Reads from Cell-Free DNA

ThruPLEX Plasma-seq Kit generated quality libraries with high diversity and a low number of duplicates and unmapped reads. Cell-free DNA was extracted from 3 plasma samples, and libraries were prepared at the amounts indicated as measured by Qubit®. The amount of mononucleosomal DNA in each sample as measured by the Bioanalyzer® corresponded to 0.09 ng, 0.62 ng, and 15.44 ng. Pooled libraries were sequenced on an Illumina NextSeq® 500 as a paired-end run with 17M to 25M reads per library, Duplication rates were calculated after down-sampling the data to 17 M reads per library.

Outstanding Target Enrichment Peformance

ThruPLEX Plasma-seq Kit libraries were captured at high efficiency and generated data with deep coverage of the kinome for mutation detection. Libraries were prepared from 3 plasma samples at input amounts of 5 ng, 6.5 ng, and 10 ng in triplicate, and targeted sequencing was carried out on an Illumina MiSeq using samples enriched with the ClearSeq Human DNA Kinome Panel for SureSelectXT2. On average, 5M reads were generated per library. Selected bases were successfully captured bases that were in or within 250 bp of the baits.

Reproducible, Unbiased GC Coverage

ThruPLEX Plasma-seq Kit provided the most reproducible and unbiased GC coverage across the human genome. ThruPLEX libraries showed minimal variability across 9 individual plasma samples tested. Libraries were prepared from cell-free DNA isolated from 1 mL of plasma samples and sequenced on an Illumina NextSeq 500. Four separate plasma samples were used to construct the NEBNext Ultra libraries.

Evaluated at Karolinska Institute and CRUK

Illumina NGS libraries were prepared with 5 ng of cell-free DNA isolated from pooled plasma samples using ThruPLEX Plasma-seq Kit. Low-pass WGS was conducted on a HiSeq with approximately 12.5M reads per library. (Data courtesy of the Karolinska Institute, Sweden).

ThruPLEX Plasma-seq Kit Single-Tube Workflow

Starting with 1 to 30 ng of cell-free DNA, ThruPLEX Plasma-seq Kit creates indexed libraries in 3 simple steps: end repair, adapter ligation, and high-fidelity library amplification. No purification or sample transfer steps are required. The streamlined workflow is performed in 2 hours in a single tube or well, preventing sample loss and enhancing positive sample identification.

ThruPLEX Plasma-seq Kit Technology

ThruPLEX Plasma-seq Kit technology is a 3-step reaction that is optimized for cell-free DNA. Cell-free DNA (1 ng to 30 ng) is first repaired in a highly efficient process. Background is reduced using double-stranded adapters with no single-stranded tails. Blunt-end ligation occurs with high-efficiency. Blocked 5’ ends reduce adapter-adapter ligation. Background is further reduced by destroying unused adapters after ligation. A high-fidelity amplification completes the reaction to generate indexed Illumina libraries.

  • Designed for cell-free DNA: newly formulated repair and ligation reagents with optimized protocols
  • High performance NGS libraries: high diversity with broad and reproducible GC coverage
  • Variable sample input: <1 ng to 30 ng of cell-free DNA
  • Fast and simple workflow: 3 steps in a single tube or well in 2 hours with no purification or sample transfer steps
  • Research applications: libraries from samples where results require high sensitivity including liquid biopsies, ctDNA, and targeted sequencing
  • Automation-friendly: Beckman® FXP Workstation

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

Store at –20 °C.

Ordering Information

CAT. NO. R400490 ThruPLEX Plasma-seq 12S  Kit (12 reactions, 12 single indexes)

CAT. NO. R400491 ThruPLEX Plasma-seq 48S Kit (48 reactions, 48 single indexes)

CAT. NO. R400492 ThruPLEX Plasma-seq 96D Kit (96 reactions, 96 dual indexes)

ThruPLEX Plasma-seq contains all necessary reagents for preparing indexed Illumina NGS libraries, including optimized Illumina-compatible adapters and indexing reagents.

 

HOW TO BUY

1. What types of samples and inputs can be used with ThruPLEX Plasma-seq? 

ThruPLEX Plasma-seq Kit is designed to generate DNA libraries for Illumina NGS from cell-free DNA isolated from plasma. The ThruPLEX Plasma-seq chemistry is reformulated to produce diverse libraries with broad GC coverage, providing reproducible sequencing results from 1 to 30 ng* of cell-free DNA.

Application Input sample type Recommended input amount
Sequencing of DNA from Plasma & Liquid Biopsy; ctDNA Analysis, Targeted Sequencing Cell-free DNA isolated from plasma 1 ng – 30 ng*

*Libraries have been generated from input amounts less than 1 ng and over 30 ng.
Contact technical support for further information.

2. What are the major technical differences between ThruPLEX DNA-seq Kit and the new ThruPLEX Plasma-seq Kit?

  • ThruPLEX Plasma-seq Kit has been optimized for cell-free DNA isolated from plasma.
  • Shearing or fragmentation of the cell-free DNA is not necessary with ThruPLEX Plasma-seq Kit.
  • The protocol is different (see below).

3.  What are the protocol differences between the ThruPLEX Plasma-seq and ThruPLEX DNA-seq?

  • Template Preparation Step: volumes and holding temperature
  • Library Synthesis Step: volumes and incubation temperature
  • Library Amplification Step: volumes and number of amplification cycles

4.  Can the reagents from ThruPLEX Plasma-seq be interchanged with the reagents from ThruPLEX DNA-seq?

No, contents of ThruPLEX Plasma-seq Kit are not interchangeable with other Rubicon Genomics products.

5.  What type of indexes or barcodes do you have available?

ThruPLEX Plasma-seq 12S and 48S Kits are provided with Illumina-compatible 8nt single indexes dispensed in tubes or a 96-well plate. These index sequences were developed by the Wellcome Trust Sanger Institute in Cambridge, UK. Information about the Sanger index sequences can be found in Nature Methods 7, 111-118 (2010).

ThruPLEX Plasma-seq 96D Kit are provided with the Illumina TruSeq HT i5 and i7 dual indexes dispensed in a 96-well plate. The i5 indexes have the Illumina D501 to D508 sequences, and the i7 indexes have the D701 to D712 sequences.

For more information about the indexes, refer to Appendix 1 in the ThruPLEX Plasma-seq Kit Instruction Manual.

6.  What recommendations do you have for isolating the cell-free DNA from plasma?

ThruPLEX Plasma-seq was optimized with cell-free DNA prepared using the QIAamp® Circulating Nucleic Acid Kit (Qiagen, CAT. NO. 55114).

The concentrations of Tris and EDTA in the buffer containing the input DNA must not exceed 10 mM and 0.1 mM, respectively. Avoid phosphate containing buffers.

7.  What kind of collection tube should I use to draw blood from subjects?

The scientific staff at Rubicon Genomics uses blood collected in Vacutainer Venous blood Collection Tubes with K2 EDTA (Becton Dickinson, CAT. NO. 36643).

For detailed instructions, refer to the Blood Collection and Plasma Preparation Protocol at the ThruPLEX Plasma-seq Product Page, under the Resources tab:  http://rubicongenomics.com/products/thruplex-plasma-seq-kit/.

8.  How should I prepare plasma from blood before isolating the cell-free DNA?

Plasma should be separated from blood using a double-centrifugation protocol. Store processed plasma samples at –80° until cell-free DNA is to be isolated.

For detailed instructions, refer to the Blood Collection and Plasma Preparation Protocol at the ThruPLEX Plasma-seq Product Page, under the Resources tab:  http://rubicongenomics.com/products/thruplex-plasma-seq-kit/.

9.  Can cell-free DNA isolated from urine or other biofluids be used with ThruPLEX Plasma-seq Kit?

ThruPLEX Plasma-seq was optimized with cell-free DNA isolated from human plasma; however, the kit may work with degraded DNA from other biofluids. Cell-free DNA from other sample sources must first be extracted with a DNA extraction/purification kit. Since cell-free DNA from these sources is normally degraded/fragmented, additional fragmentation is unnecessary. The volume of input cell-free DNA for each reaction must not exceed 10 µL.

 10.  Is ThruPLEX Plasma-seq Kit compatible with any target enrichment systems?

Yes, ThruPLEX Plasma-seq Kit is compatible with the major exome and target enrichment products, including Agilent SureSelect®, Roche NimbleGen® SeqCap® EZ and custom panels. ThruPLEX Plasma-seq Kit target enrichment protocols and application notes can be accessed at: http://rubicongenomics.com/applications/enrichment/.

 

Performance

Highest Diversity, Fewest Unmapped Reads from Cell-Free DNA

ThruPLEX Plasma-seq Kit generated quality libraries with high diversity and a low number of duplicates and unmapped reads. Cell-free DNA was extracted from 3 plasma samples, and libraries were prepared at the amounts indicated as measured by Qubit®. The amount of mononucleosomal DNA in each sample as measured by the Bioanalyzer® corresponded to 0.09 ng, 0.62 ng, and 15.44 ng. Pooled libraries were sequenced on an Illumina NextSeq® 500 as a paired-end run with 17M to 25M reads per library, Duplication rates were calculated after down-sampling the data to 17 M reads per library.

Outstanding Target Enrichment Peformance

ThruPLEX Plasma-seq Kit libraries were captured at high efficiency and generated data with deep coverage of the kinome for mutation detection. Libraries were prepared from 3 plasma samples at input amounts of 5 ng, 6.5 ng, and 10 ng in triplicate, and targeted sequencing was carried out on an Illumina MiSeq using samples enriched with the ClearSeq Human DNA Kinome Panel for SureSelectXT2. On average, 5M reads were generated per library. Selected bases were successfully captured bases that were in or within 250 bp of the baits.

Reproducible, Unbiased GC Coverage

ThruPLEX Plasma-seq Kit provided the most reproducible and unbiased GC coverage across the human genome. ThruPLEX libraries showed minimal variability across 9 individual plasma samples tested. Libraries were prepared from cell-free DNA isolated from 1 mL of plasma samples and sequenced on an Illumina NextSeq 500. Four separate plasma samples were used to construct the NEBNext Ultra libraries.

Evaluated at Karolinska Institute and CRUK

Illumina NGS libraries were prepared with 5 ng of cell-free DNA isolated from pooled plasma samples using ThruPLEX Plasma-seq Kit. Low-pass WGS was conducted on a HiSeq with approximately 12.5M reads per library. (Data courtesy of the Karolinska Institute, Sweden).

ThruPLEX Plasma-seq Kit Single-Tube Workflow

Starting with 1 to 30 ng of cell-free DNA, ThruPLEX Plasma-seq Kit creates indexed libraries in 3 simple steps: end repair, adapter ligation, and high-fidelity library amplification. No purification or sample transfer steps are required. The streamlined workflow is performed in 2 hours in a single tube or well, preventing sample loss and enhancing positive sample identification.

ThruPLEX Plasma-seq Kit Technology

ThruPLEX Plasma-seq Kit technology is a 3-step reaction that is optimized for cell-free DNA. Cell-free DNA (1 ng to 30 ng) is first repaired in a highly efficient process. Background is reduced using double-stranded adapters with no single-stranded tails. Blunt-end ligation occurs with high-efficiency. Blocked 5’ ends reduce adapter-adapter ligation. Background is further reduced by destroying unused adapters after ligation. A high-fidelity amplification completes the reaction to generate indexed Illumina libraries.

Advantages

  • Designed for cell-free DNA: newly formulated repair and ligation reagents with optimized protocols
  • High performance NGS libraries: high diversity with broad and reproducible GC coverage
  • Variable sample input: <1 ng to 30 ng of cell-free DNA
  • Fast and simple workflow: 3 steps in a single tube or well in 2 hours with no purification or sample transfer steps
  • Research applications: libraries from samples where results require high sensitivity including liquid biopsies, ctDNA, and targeted sequencing
  • Automation-friendly: Beckman® FXP Workstation

Specifications

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

Store at –20 °C.

Ordering Information

CAT. NO. R400490 ThruPLEX Plasma-seq 12S  Kit (12 reactions, 12 single indexes)

CAT. NO. R400491 ThruPLEX Plasma-seq 48S Kit (48 reactions, 48 single indexes)

CAT. NO. R400492 ThruPLEX Plasma-seq 96D Kit (96 reactions, 96 dual indexes)

ThruPLEX Plasma-seq contains all necessary reagents for preparing indexed Illumina NGS libraries, including optimized Illumina-compatible adapters and indexing reagents.

 

HOW TO BUY

Resources

FAQs

1. What types of samples and inputs can be used with ThruPLEX Plasma-seq? 

ThruPLEX Plasma-seq Kit is designed to generate DNA libraries for Illumina NGS from cell-free DNA isolated from plasma. The ThruPLEX Plasma-seq chemistry is reformulated to produce diverse libraries with broad GC coverage, providing reproducible sequencing results from 1 to 30 ng* of cell-free DNA.

Application Input sample type Recommended input amount
Sequencing of DNA from Plasma & Liquid Biopsy; ctDNA Analysis, Targeted Sequencing Cell-free DNA isolated from plasma 1 ng – 30 ng*

*Libraries have been generated from input amounts less than 1 ng and over 30 ng.
Contact technical support for further information.

2. What are the major technical differences between ThruPLEX DNA-seq Kit and the new ThruPLEX Plasma-seq Kit?

  • ThruPLEX Plasma-seq Kit has been optimized for cell-free DNA isolated from plasma.
  • Shearing or fragmentation of the cell-free DNA is not necessary with ThruPLEX Plasma-seq Kit.
  • The protocol is different (see below).

3.  What are the protocol differences between the ThruPLEX Plasma-seq and ThruPLEX DNA-seq?

  • Template Preparation Step: volumes and holding temperature
  • Library Synthesis Step: volumes and incubation temperature
  • Library Amplification Step: volumes and number of amplification cycles

4.  Can the reagents from ThruPLEX Plasma-seq be interchanged with the reagents from ThruPLEX DNA-seq?

No, contents of ThruPLEX Plasma-seq Kit are not interchangeable with other Rubicon Genomics products.

5.  What type of indexes or barcodes do you have available?

ThruPLEX Plasma-seq 12S and 48S Kits are provided with Illumina-compatible 8nt single indexes dispensed in tubes or a 96-well plate. These index sequences were developed by the Wellcome Trust Sanger Institute in Cambridge, UK. Information about the Sanger index sequences can be found in Nature Methods 7, 111-118 (2010).

ThruPLEX Plasma-seq 96D Kit are provided with the Illumina TruSeq HT i5 and i7 dual indexes dispensed in a 96-well plate. The i5 indexes have the Illumina D501 to D508 sequences, and the i7 indexes have the D701 to D712 sequences.

For more information about the indexes, refer to Appendix 1 in the ThruPLEX Plasma-seq Kit Instruction Manual.

6.  What recommendations do you have for isolating the cell-free DNA from plasma?

ThruPLEX Plasma-seq was optimized with cell-free DNA prepared using the QIAamp® Circulating Nucleic Acid Kit (Qiagen, CAT. NO. 55114).

The concentrations of Tris and EDTA in the buffer containing the input DNA must not exceed 10 mM and 0.1 mM, respectively. Avoid phosphate containing buffers.

7.  What kind of collection tube should I use to draw blood from subjects?

The scientific staff at Rubicon Genomics uses blood collected in Vacutainer Venous blood Collection Tubes with K2 EDTA (Becton Dickinson, CAT. NO. 36643).

For detailed instructions, refer to the Blood Collection and Plasma Preparation Protocol at the ThruPLEX Plasma-seq Product Page, under the Resources tab:  http://rubicongenomics.com/products/thruplex-plasma-seq-kit/.

8.  How should I prepare plasma from blood before isolating the cell-free DNA?

Plasma should be separated from blood using a double-centrifugation protocol. Store processed plasma samples at –80° until cell-free DNA is to be isolated.

For detailed instructions, refer to the Blood Collection and Plasma Preparation Protocol at the ThruPLEX Plasma-seq Product Page, under the Resources tab:  http://rubicongenomics.com/products/thruplex-plasma-seq-kit/.

9.  Can cell-free DNA isolated from urine or other biofluids be used with ThruPLEX Plasma-seq Kit?

ThruPLEX Plasma-seq was optimized with cell-free DNA isolated from human plasma; however, the kit may work with degraded DNA from other biofluids. Cell-free DNA from other sample sources must first be extracted with a DNA extraction/purification kit. Since cell-free DNA from these sources is normally degraded/fragmented, additional fragmentation is unnecessary. The volume of input cell-free DNA for each reaction must not exceed 10 µL.

 10.  Is ThruPLEX Plasma-seq Kit compatible with any target enrichment systems?

Yes, ThruPLEX Plasma-seq Kit is compatible with the major exome and target enrichment products, including Agilent SureSelect®, Roche NimbleGen® SeqCap® EZ and custom panels. ThruPLEX Plasma-seq Kit target enrichment protocols and application notes can be accessed at: http://rubicongenomics.com/applications/enrichment/.

 

Overview

ThruPLEX® Plasma-seq is powered by ThruPLEX chemistry to generate high performance NGS libraries from cell-free DNA isolated from plasma.

The chemistry is optimized specifically for cell-free DNA to maximize the library complexity and to preserve the GC representation of the input DNA. Libraries can be used for CNV analysis, whole genome sequencing applications or enriched using your custom panels or leading target enrichment platforms, such as Agilent SureSelect® and Roche NimbleGen SeqCap® EZ.

Libraries prepared with ThruPLEX Plasma-seq provide added sensitivity to better understand genetic variations found within the plasma samples. Plasma, along with other liquid biopsies, has gained significant interest in the research community as a valuable and easily available sample type.  These communities include translational research in oncology and developmental research involving the fetal fraction derived from maternal plasma.

ThruPLEX® Plasma-seq Kit is intended for Research Use Only. It may not be used for any other purposes including, but not limited to, use in diagnostics, forensics, therapeutics, or in humans. ThruPLEX Plasma-seq may not be transferred to third parties, resold, modified for resale or used to manufacture commercial products without prior written approval of Rubicon Genomics, Inc.ThruPLEX Plasma-seq Kit  is protected by U.S. Patents 7,803,550; 8,071,312; 8,399,199; 8,728,737 and corresponding foreign patents. Additional patents are pending.