ThruPLEX® DNA-seq Kit

Higher Multiplexing Capability with Improved Performance for Illumina® NGS Platforms

ThruPLEX-DNA-seq-Kit-Box-for-Web

 

“We are very happy with the results and we plan to start using ThruPLEX DNA-seq as a part of our capture workflows. Variants are called with very equal distribution (only a small fraction of low/high coverage extremes). In our classic sample preps, the coverage is not this homogenous, and low tail is much longer. Our lab staff was pleased with the convenience of the ThruPLEX workflow (less manual steps).

- Pekka Ellonen, Head of DNA Sequencing Laboratory, Institute for Molecular Medicine Finland FIMM

 

 

Overview

Create more libraries with greater sensitivity!  ThruPLEX DNA-seq builds on the innovative ThruPLEX chemistry to generate DNA libraries with expanded multiplexing capability and even greater diversity with lower duplication rates. Kits contain either 48 or 96 dual read Illumina-compatible indexes pre-dispensed and sealed in barcoded microplates. Starting with as little as 50 pg of fragmented double-stranded DNA or cDNA, the entire ThruPLEX DNA-seq workflow is still performed in a single tube or well about 2 hours and requires no purification steps or sample transfers. With a low input requirement, ThruPLEX DNA-seq produces uniform GC coverage and improves molecular complexity yielding reproducible sequencing results. ThruPLEX DNA-seq can be used in any DNA-seq application, RNA-seq, ChIP-seq and offers robust target enrichment performance with all of the leading platforms.

ThruPLEX DNA-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.

  • Expanded multiplexing capability – up to 96 indexes
  • High performance from lowest input – 50 pg to 50 ng
  • Fast and simple workflow – 3 steps in a single tube or well in about 2 hours, no transfers necessary
  • Complete package for creating libraries- optimized indexing and adaptor reagents, enzymes, buffers and nuclease-free water
  • Compatible with major target enrichment platforms – Agilent SureSelect®, Roche NimbleGen® SeqCap® EZ, and IDT xGen® Lockdown® probes

Workflow

ThruPLEX DNA-seq_Single-Tube 060415

 

ThruPLEX DNA-seq Technology

ThruPLEX DNA-seq Technology
ThruPLEX DNA-seq Technology

ThruPLEX technology is a 3-step reaction that starts with fragmented double-stranded DNA (0.05 ng to 50 ng) which is repaired in a highly efficient process. Background is reduced using double-stranded adaptors with no single-stranded tails. Blunt end ligation occurs with high-efficiency. Blocked 5’ends reduce adaptor-adaptor ligation. Background is further reduced by destroying unused adaptors after ligation

ThruPLEX DNA-seq Outperforms Other NGS LIbrary Prep Kits

ThruPLEX DNA-seq Outperforms Other NGS LIbrary Prep Kits
ThruPLEX DNA-seq Outperforms Other NGS LIbrary Prep Kits

NGS libraries were prepared from the indicated amount of DNA using the library preparation kits indicated and sequenced on a MiSeq, v3 flow cell. (Left) Note that in every case, both ThruPLEX–FD (light blue) and ThruPLEX DNA-seq (gold) yielded more diverse libraries with less input DNA than any of the other kits. (Middle) ThruPLEX libraries had fewer duplicates at 25M read pairs than other kits. (Right) Both ThruPLEX kits have a lower and broader recommended input range than the other kits.

ThruPLEX DNA-seq Minimizes GC Bias

ThruPLEX DNA-seq Minimizes GC Bias
ThruPLEX DNA-seq Minimizes GC Bias

Relative coverage of the human genome as a function of %GC (50 ng input DNA). (Left) ThruPLEX DNA-seq has excellent coverage throughout the entire range. (Right) ThruPLEX DNA-seq (gold) performs as well as or better than other kits tested. Note that the optimal coverage should be equal to 1 in these graphs. GC composition distribution of the human genome is represented by the red bars.

ThruPLEX DNA-seq Workflow Advantage: High Quality NGS Libraries in About 2 Hours

ThruPLEX DNA-seq Workflow Advantage: High Quality NGS Libraries in About 2 Hours
ThruPLEX DNA-seq Workflow Advantage: High Quality NGS Libraries in About 2 Hours

ThruPLEX DNA-seq converts double-stranded DNA samples to indexed libraries in 3 simple steps: end repair, adapter ligation, and high-fidelity library amplification. The streamlined workflow prevents sample loss and handling errors and is performed in a single tube or well in less than 2 hours.

ThruPLEX with SureSelectXT2

ThruPLEX with SureSelectXT2
ThruPLEX with SureSelectXT2

ThruPLEX DNA-seq can be easily integrated with Agilent SureSelectXT2 by utilizing the reagents as outlined in the table.

ThruPLEX with SeqCap EZ

ThruPLEX with SeqCap EZ
ThruPLEX with SeqCap EZ

ThruPLEX DNA-seq can be easily integrated with Roche NimbleGen SeqCap EZ by utilizing the reagents as outlined in the table.

Store at -20 °C.

Guaranteed for 12 months at -20°C in a constant temperature freezer.

ThruPLEX DNA-seq  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.

Click here to read all FAQ’s on ThruPLEX DNA-seq Kit

1. What are the major differences between ThruPLEX®-FD Prep Kit and the new ThruPLEX DNA-seq Kit?

  • Increased multiplexing capability from 12 6nt single indexes to up to 96 8nt dual indexes (Illumina TruSeq HT) or up to 48 8nt single indexes (Sanger, see Appendix 1 of the ThruPLEX DNA-seq Kit Instruction Manual)
  • Total reaction volume lowered from 75 µL to 50 µL to accommodate more thermal cyclers
  • Protocol modified to account for reduced volume
  • Fewer PCR amplification cycles during Stage 5 of the Library Amplification Step
  • Indexing reagents for kits containing 48 or 96 indexes are pre-dispensed into a 96-well plate

2. Is ThruPLEX DNA-seq Kit compatible with any target enrichment systems?

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

3. I purchased a ThruPLEX DNA-seq 48D kit and have been using it for 4-5 samples at a times. Why am I running out of reagents?  

ThruPLEX DNA-seq Kit reagents contain enough volume to prepare master mixes FOUR times, coincident with the number of times the indexing reagents should be used (up to 4 freeze/thaw cycles).  When accessing the kit more than the four times, we cannot guarantee that enough reagent volume will be available.

4. Does ThruPLEX DNA-seq have enough overfill to be used on my automation device?

Each automation platform has its own dead volume and reagent requirements; hence the volume of reagents in ThruPLEX DNAs-seq Kit may not provide the expected number of reactions that would be performed manually.

5. Do I need to perform cleanup on my DNA sample(s) after shearing?

No, simply proceed to the Template Preparation Step (Section D.I.) in the ThruPLEX DNA-seq Kit Instruction Manual in order to preserve your total DNA and the overall diversity of the library.

6. What types of DNA samples can be used with ThruPLEX DNA-seq Kit?

  • Fragmented, double-stranded DNA, such as that from:
  • Mechanically-sheared DNA
  • Enzymatically-fragmented DNA
  • Low molecular weight cell-free DNA from plasma, urine, and other biofluids
  • Double-stranded cDNA
  • Chromatin Immunoprecipitated (ChIP) DNA
  • Sonicated DNA from formalin-fixed material (FFPE)

7. Can RNA be used with ThruPLEX DNA-seq Kit?

Yes, but RNA needs to be converted into double-stranded cDNA and fragmented to proper size prior to preparation with ThruPLEX DNA-seq Kit.

8. Can plasma, urine, or other biofluids be directly used with ThruPLEX DNA-seq Kit?

No, the template DNA must be first extracted with a DNA extraction/purification kit. Since DNA from these sources is normally degraded/fragmented, additional fragmentation is unnecessary. The volume of the sample used in the
ThruPLEX DNA-seq Kit must be no more than 10 µL.

9. Can I prepare samples for both single- and paired-end NGS sequencing?

Yes, ThruPLEX DNA-seq Kit is compatible with both single- and paired-end sequencing. The final amplified libraries will contain Illumina-compatible index sequences.

10. Is there a required DNA sample input volume for ThruPLEX DNA-seq Kit?

Yes, the required DNA sample volume is 10 µL.

ThruPLEX DNA-seq contains everything needed, including indexes in a barcoded 96-well plate.

CAT. NO. R400406 ThruPLEX DNA-seq 48D Kit (48 reactions, 48 dual indexes)

CAT. NO. R400407 ThruPLEX DNA-seq 96D Kit (96 reactions, 96 dual indexes)

CAT. NO. R400427 ThruPLEX DNA-seq 48S Kit (48 reactions, 48 single indexes)

CAT. NO. R400428 ThruPLEX DNA-seq 12S (48) Kit (48 reactions, 12 single indexes)

CAT. NO. R400429 ThruPLEX DNA-seq 12S  Kit (12 reactions, 12 single indexes)

Order directly from our STORE.

Performance

ThruPLEX DNA-seq Technology

ThruPLEX DNA-seq Technology
ThruPLEX DNA-seq Technology

ThruPLEX technology is a 3-step reaction that starts with fragmented double-stranded DNA (0.05 ng to 50 ng) which is repaired in a highly efficient process. Background is reduced using double-stranded adaptors with no single-stranded tails. Blunt end ligation occurs with high-efficiency. Blocked 5’ends reduce adaptor-adaptor ligation. Background is further reduced by destroying unused adaptors after ligation

ThruPLEX DNA-seq Outperforms Other NGS LIbrary Prep Kits

ThruPLEX DNA-seq Outperforms Other NGS LIbrary Prep Kits
ThruPLEX DNA-seq Outperforms Other NGS LIbrary Prep Kits

NGS libraries were prepared from the indicated amount of DNA using the library preparation kits indicated and sequenced on a MiSeq, v3 flow cell. (Left) Note that in every case, both ThruPLEX–FD (light blue) and ThruPLEX DNA-seq (gold) yielded more diverse libraries with less input DNA than any of the other kits. (Middle) ThruPLEX libraries had fewer duplicates at 25M read pairs than other kits. (Right) Both ThruPLEX kits have a lower and broader recommended input range than the other kits.

ThruPLEX DNA-seq Minimizes GC Bias

ThruPLEX DNA-seq Minimizes GC Bias
ThruPLEX DNA-seq Minimizes GC Bias

Relative coverage of the human genome as a function of %GC (50 ng input DNA). (Left) ThruPLEX DNA-seq has excellent coverage throughout the entire range. (Right) ThruPLEX DNA-seq (gold) performs as well as or better than other kits tested. Note that the optimal coverage should be equal to 1 in these graphs. GC composition distribution of the human genome is represented by the red bars.

ThruPLEX DNA-seq Workflow Advantage: High Quality NGS Libraries in About 2 Hours

ThruPLEX DNA-seq Workflow Advantage: High Quality NGS Libraries in About 2 Hours
ThruPLEX DNA-seq Workflow Advantage: High Quality NGS Libraries in About 2 Hours

ThruPLEX DNA-seq converts double-stranded DNA samples to indexed libraries in 3 simple steps: end repair, adapter ligation, and high-fidelity library amplification. The streamlined workflow prevents sample loss and handling errors and is performed in a single tube or well in less than 2 hours.

ThruPLEX with SureSelectXT2

ThruPLEX with SureSelectXT2
ThruPLEX with SureSelectXT2

ThruPLEX DNA-seq can be easily integrated with Agilent SureSelectXT2 by utilizing the reagents as outlined in the table.

ThruPLEX with SeqCap EZ

ThruPLEX with SeqCap EZ
ThruPLEX with SeqCap EZ

ThruPLEX DNA-seq can be easily integrated with Roche NimbleGen SeqCap EZ by utilizing the reagents as outlined in the table.

Specifications

Store at -20 °C.

Guaranteed for 12 months at -20°C in a constant temperature freezer.

ThruPLEX DNA-seq  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

Click here to read all FAQ’s on ThruPLEX DNA-seq Kit

1. What are the major differences between ThruPLEX®-FD Prep Kit and the new ThruPLEX DNA-seq Kit?

  • Increased multiplexing capability from 12 6nt single indexes to up to 96 8nt dual indexes (Illumina TruSeq HT) or up to 48 8nt single indexes (Sanger, see Appendix 1 of the ThruPLEX DNA-seq Kit Instruction Manual)
  • Total reaction volume lowered from 75 µL to 50 µL to accommodate more thermal cyclers
  • Protocol modified to account for reduced volume
  • Fewer PCR amplification cycles during Stage 5 of the Library Amplification Step
  • Indexing reagents for kits containing 48 or 96 indexes are pre-dispensed into a 96-well plate

2. Is ThruPLEX DNA-seq Kit compatible with any target enrichment systems?

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

3. I purchased a ThruPLEX DNA-seq 48D kit and have been using it for 4-5 samples at a times. Why am I running out of reagents?  

ThruPLEX DNA-seq Kit reagents contain enough volume to prepare master mixes FOUR times, coincident with the number of times the indexing reagents should be used (up to 4 freeze/thaw cycles).  When accessing the kit more than the four times, we cannot guarantee that enough reagent volume will be available.

4. Does ThruPLEX DNA-seq have enough overfill to be used on my automation device?

Each automation platform has its own dead volume and reagent requirements; hence the volume of reagents in ThruPLEX DNAs-seq Kit may not provide the expected number of reactions that would be performed manually.

5. Do I need to perform cleanup on my DNA sample(s) after shearing?

No, simply proceed to the Template Preparation Step (Section D.I.) in the ThruPLEX DNA-seq Kit Instruction Manual in order to preserve your total DNA and the overall diversity of the library.

6. What types of DNA samples can be used with ThruPLEX DNA-seq Kit?

  • Fragmented, double-stranded DNA, such as that from:
  • Mechanically-sheared DNA
  • Enzymatically-fragmented DNA
  • Low molecular weight cell-free DNA from plasma, urine, and other biofluids
  • Double-stranded cDNA
  • Chromatin Immunoprecipitated (ChIP) DNA
  • Sonicated DNA from formalin-fixed material (FFPE)

7. Can RNA be used with ThruPLEX DNA-seq Kit?

Yes, but RNA needs to be converted into double-stranded cDNA and fragmented to proper size prior to preparation with ThruPLEX DNA-seq Kit.

8. Can plasma, urine, or other biofluids be directly used with ThruPLEX DNA-seq Kit?

No, the template DNA must be first extracted with a DNA extraction/purification kit. Since DNA from these sources is normally degraded/fragmented, additional fragmentation is unnecessary. The volume of the sample used in the
ThruPLEX DNA-seq Kit must be no more than 10 µL.

9. Can I prepare samples for both single- and paired-end NGS sequencing?

Yes, ThruPLEX DNA-seq Kit is compatible with both single- and paired-end sequencing. The final amplified libraries will contain Illumina-compatible index sequences.

10. Is there a required DNA sample input volume for ThruPLEX DNA-seq Kit?

Yes, the required DNA sample volume is 10 µL.

Ordering Information

ThruPLEX DNA-seq contains everything needed, including indexes in a barcoded 96-well plate.

CAT. NO. R400406 ThruPLEX DNA-seq 48D Kit (48 reactions, 48 dual indexes)

CAT. NO. R400407 ThruPLEX DNA-seq 96D Kit (96 reactions, 96 dual indexes)

CAT. NO. R400427 ThruPLEX DNA-seq 48S Kit (48 reactions, 48 single indexes)

CAT. NO. R400428 ThruPLEX DNA-seq 12S (48) Kit (48 reactions, 12 single indexes)

CAT. NO. R400429 ThruPLEX DNA-seq 12S  Kit (12 reactions, 12 single indexes)

Order directly from our STORE.