Reduce Background for Results You Can Trust

ThruPLEX Tag-seq libraries were prepared from 30 ng of the 1% Horizon cfDNA standard. Following Agilent SureSelect target enrichment and sequencing to achieve ~10,000X coverage, data was analyzed with the Curio Genomics bioinformatics platform. Using the raw reads, the EGFR mutation (yellow dot) is obscured by background errors (left). The mutation is readily identified when UMTs are utilized in data processing to correct for errors (right).

Dramatically Increase the Signal-to-Noise Ratio to Discover More

Data from either the 30 ng Horizon cfDNA library (previous slider) (A) or a ThruPLEX Tag-seq library prepared from 10 ng Horizon cfDNA standard, enriched with a custom 240 KB panel (NimbleGen SeqCapEZ) and sequenced on a HiSeq 2500 to achieve ~5,000X coverage. Analysis was performed using the Curio Genomics bioinformatics platform.

Single-Tube, Three Step Workflow Helps your Precious Samples Go Further

Starting with 1 to 50 ng of DNA, ThruPLEX Tag-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 Technology Generates High Quality libraries the first time, every time

ThruPLEX technology is a 3-step reaction that starts with fragmented double-stranded DNA or cell-free DNA 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 adapter-adapter ligation.

Time Saving Bioinformatics Solutions

Reach results quickly with the Curio Genomics cloud-based software (www.curiogenomics.com) or Connor, an open-source tool available for data processing (https://github.com/umich-brcf-bioinf/Connor).

  • Results you can trust. ThruPLEX Tag-seq technology provides up to 16 million molecular tags to correct sequencing errors providing confident variant detection.
  • Discover more, cost effectively. Use with hybridization-based target enrichment systems to examine hundreds of genes, including mutations and structural variants, simultaneously.
  • High quality libraries the first time, every time. Unparalleled ease of use reduces user error and contamination with our single-tube, 2-hour, 3-step workflow.
  • Time-saving bioinformatics solutions. Reach results quickly with cloud-based software from Curio Genomics or open-source scripts available for data processing.
  • Precious samples go further. Use less DNA to analyze samples once too low to detect. ThruPLEX Tag-seq works with input amounts from 1 ng to 50 ng of cfDNA or fragmented dsDNA to accommodate a wide range of samples.

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

Store at –20 °C.

Ordering Information

CAT. NO. R400584 ThruPLEX Tag-seq 6S  Kit (12 reactions, 6 single indexes)

CAT. NO. R400585 ThruPLEX Tag-seq 48S Kit (48 reactions, 48 single indexes)

CAT. NO. R400586 ThruPLEX Tag-seq 96D Kit (96 reactions, 96 dual indexes)

ThruPLEX Tag-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 Tag-seq? 

ThruPLEX® Tag-seq Kit is designed to generate DNA libraries for Illumina® platforms from both cell-free DNA and fragmented, double-stranded DNA. The ThruPLEX Tag-seq chemistry produces diverse libraries with broad GC coverage, providing reproducible sequencing results from 1 to 50 ng of DNA.

  1. What are the major technical differences between the new ThruPLEX Tag-seq Kit and other ThruPLEX Kits?

ThruPLEX Tag-seq Kit contains proprietary ThruPLEX stem-loop adapters that have been redesigned to include unique molecular tags. Each kit contains more than 16 million molecular tags used to tag individual DNA fragments prior to amplification, providing tracking of the fragments through the library preparation, target enrichment and data analysis to detect low-frequency alleles at high sensitivity and specificity.

  1. How do the unique molecular tags (UMTs) work?

In ThruPLEX Tag-seq library preparation, UMTs are incorporated during the ligation step to uniquely label the starting DNA molecules. This allows the sequencing reads to be grouped by amplification families according to their UMTs during data processing. During data processing, the reads within each amplification family are compared to remove amplification and sequencing errors, and a consensus sequence is formed to provide confident variant detection.

  1. How many unique molecular tags (UMTs) does ThruPLEX Tag-seq have?

A 6nt degenerate (random) sequence is incorporated to each side of the ThruPLEX Tag-seq library structure through ligation of the ThruPLEX stem-loop adapters. Each 6nt sequence provides 4^6 or 4,096 combinations. In combination, the 6nt sequences on both sides of the library structure provide more than 16 million (4,096 x 4,096) molecular tags to ensure unique labeling of the input DNA molecules.

  1. How much input DNA is required to detect variants present at 1% allele frequency?

An appropriate input amount of DNA should be used to ensure sufficient variant copies are available for the library preparation process to achieve the desirable detection sensitivity. In general, detection of alleles present at lower frequencies requires higher input amounts of DNA. For example, a 10 ng DNA sample would contain a sufficient number of copies for  1% allele frequency detection. For more information, refer to Section C.III. in the ThruPLEX Tag-seq Kit Instruction Manual.

  1. How deep should I sequence my ThruPLEX Tag-seq libraries?

Sufficient coverage is required to utilize the unique molecular tags in ThruPLEX Tag-seq libraries to build consensus sequences. In general, detection of alleles present at lower frequencies requires sequencing to a higher depth. For example, when using a target peak amplification family size of 10 reads per unique molecule and 3 unique molecules to make a confident call, a sequencing depth of at least 600X would be required to call variants at 5% allele frequency. Similarly, 1% allele frequency would need 3,000X coverage, and 0.5% allele frequency, 6,000X coverage. For more information, refer to Section C.III. in the ThruPLEX Tag-seq Kit Instruction Manual.

  1. Is ThruPLEX Tag-seq Kit compatible with any target enrichment systems?

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

  1. Can I sequence ThruPLEX Tag-seq with other Illumina NGS libraries on the same run?

Yes, ThruPLEX Tag-seq libraries can be sequenced with other Illumina NGS libraries on the same sequencing run. However, care must be taken to ensure the samples carry unique and compatible sample indexes and receive adequate sequencing coverage. To realize the benefits of the unique molecular tags (UMTs), ThruPLEX Tag-seq libraries are usually target enriched and sequenced to a relatively high depth.

  1. How do I process my sequencing data obtained from ThruPLEX Tag-seq libraries?

To benefit from the information provided by the unique molecular tags (UMTs) in ThruPLEX Tag-seq libraries, the sequencing data must be processed to group reads into amplification families and construct consensus reads. Two bioinformatics solutions designed specifically for processing data from ThruPLEX Tag-seq libraries are available:

Curio

Curio is a cloud-based bioinformatics platform available through Curio Genomics. Sequencing data from ThruPLEX Tag-seq libraries can be easily uploaded, processed and visualized. The platform features an ultra-fast and user-friendly interface and a powerful alignment viewer. The platform is also equipped with modules for performing variant detection, analysis and visualization. For more information, visit www.curiogenomics.com.

Connor

Connor is an open-source bioinformatics tool hosted on GitHub (https://github.com/umich-brcf-bioinf/Connor) for processing sequencing data generated from ThruPLEX Tag-seq libraries. Connor takes an aligned BAM file as input, processes the UMT information and generates a processed BAM file containing consensus sequences. The output BAM file can be used with variant callers, such as FreeBayes, VarScan2 or GATK HaplotypeCaller.

  1. I have my own bioinformatic analysis pipeline. How can I modify it to process sequencing data obtained from ThruPLEX Tag-seq libraries?

Since each pipeline is different, we encourage you to take advantage of Connor, the open source bioinformatics tool created for  use with ThruPLEX Tag-seq.  Should you want to modify your existing pipeline the  information that follows  may help.  Each ThruPLEX Tag-seq library molecule has a 6nt unique molecular tag (UMT) on each side. The UMT is the first sequence read by either Read 1 or Read 2, followed by an 8 to 11nt dephasing stem sequence and then the template DNA sequence. For more information on the library structure, refer to the ThruPLEX Tag-seq Kit Instruction Manual (specific link and section).

Performance

Reduce Background for Results You Can Trust

ThruPLEX Tag-seq libraries were prepared from 30 ng of the 1% Horizon cfDNA standard. Following Agilent SureSelect target enrichment and sequencing to achieve ~10,000X coverage, data was analyzed with the Curio Genomics bioinformatics platform. Using the raw reads, the EGFR mutation (yellow dot) is obscured by background errors (left). The mutation is readily identified when UMTs are utilized in data processing to correct for errors (right).

Dramatically Increase the Signal-to-Noise Ratio to Discover More

Data from either the 30 ng Horizon cfDNA library (previous slider) (A) or a ThruPLEX Tag-seq library prepared from 10 ng Horizon cfDNA standard, enriched with a custom 240 KB panel (NimbleGen SeqCapEZ) and sequenced on a HiSeq 2500 to achieve ~5,000X coverage. Analysis was performed using the Curio Genomics bioinformatics platform.

Single-Tube, Three Step Workflow Helps your Precious Samples Go Further

Starting with 1 to 50 ng of DNA, ThruPLEX Tag-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 Technology Generates High Quality libraries the first time, every time

ThruPLEX technology is a 3-step reaction that starts with fragmented double-stranded DNA or cell-free DNA 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 adapter-adapter ligation.

Time Saving Bioinformatics Solutions

Reach results quickly with the Curio Genomics cloud-based software (www.curiogenomics.com) or Connor, an open-source tool available for data processing (https://github.com/umich-brcf-bioinf/Connor).

Advantages

  • Results you can trust. ThruPLEX Tag-seq technology provides up to 16 million molecular tags to correct sequencing errors providing confident variant detection.
  • Discover more, cost effectively. Use with hybridization-based target enrichment systems to examine hundreds of genes, including mutations and structural variants, simultaneously.
  • High quality libraries the first time, every time. Unparalleled ease of use reduces user error and contamination with our single-tube, 2-hour, 3-step workflow.
  • Time-saving bioinformatics solutions. Reach results quickly with cloud-based software from Curio Genomics or open-source scripts available for data processing.
  • Precious samples go further. Use less DNA to analyze samples once too low to detect. ThruPLEX Tag-seq works with input amounts from 1 ng to 50 ng of cfDNA or fragmented dsDNA to accommodate a wide range of samples.

Specifications

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

Store at –20 °C.

Ordering Information

CAT. NO. R400584 ThruPLEX Tag-seq 6S  Kit (12 reactions, 6 single indexes)

CAT. NO. R400585 ThruPLEX Tag-seq 48S Kit (48 reactions, 48 single indexes)

CAT. NO. R400586 ThruPLEX Tag-seq 96D Kit (96 reactions, 96 dual indexes)

ThruPLEX Tag-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 Tag-seq? 

ThruPLEX® Tag-seq Kit is designed to generate DNA libraries for Illumina® platforms from both cell-free DNA and fragmented, double-stranded DNA. The ThruPLEX Tag-seq chemistry produces diverse libraries with broad GC coverage, providing reproducible sequencing results from 1 to 50 ng of DNA.

  1. What are the major technical differences between the new ThruPLEX Tag-seq Kit and other ThruPLEX Kits?

ThruPLEX Tag-seq Kit contains proprietary ThruPLEX stem-loop adapters that have been redesigned to include unique molecular tags. Each kit contains more than 16 million molecular tags used to tag individual DNA fragments prior to amplification, providing tracking of the fragments through the library preparation, target enrichment and data analysis to detect low-frequency alleles at high sensitivity and specificity.

  1. How do the unique molecular tags (UMTs) work?

In ThruPLEX Tag-seq library preparation, UMTs are incorporated during the ligation step to uniquely label the starting DNA molecules. This allows the sequencing reads to be grouped by amplification families according to their UMTs during data processing. During data processing, the reads within each amplification family are compared to remove amplification and sequencing errors, and a consensus sequence is formed to provide confident variant detection.

  1. How many unique molecular tags (UMTs) does ThruPLEX Tag-seq have?

A 6nt degenerate (random) sequence is incorporated to each side of the ThruPLEX Tag-seq library structure through ligation of the ThruPLEX stem-loop adapters. Each 6nt sequence provides 4^6 or 4,096 combinations. In combination, the 6nt sequences on both sides of the library structure provide more than 16 million (4,096 x 4,096) molecular tags to ensure unique labeling of the input DNA molecules.

  1. How much input DNA is required to detect variants present at 1% allele frequency?

An appropriate input amount of DNA should be used to ensure sufficient variant copies are available for the library preparation process to achieve the desirable detection sensitivity. In general, detection of alleles present at lower frequencies requires higher input amounts of DNA. For example, a 10 ng DNA sample would contain a sufficient number of copies for  1% allele frequency detection. For more information, refer to Section C.III. in the ThruPLEX Tag-seq Kit Instruction Manual.

  1. How deep should I sequence my ThruPLEX Tag-seq libraries?

Sufficient coverage is required to utilize the unique molecular tags in ThruPLEX Tag-seq libraries to build consensus sequences. In general, detection of alleles present at lower frequencies requires sequencing to a higher depth. For example, when using a target peak amplification family size of 10 reads per unique molecule and 3 unique molecules to make a confident call, a sequencing depth of at least 600X would be required to call variants at 5% allele frequency. Similarly, 1% allele frequency would need 3,000X coverage, and 0.5% allele frequency, 6,000X coverage. For more information, refer to Section C.III. in the ThruPLEX Tag-seq Kit Instruction Manual.

  1. Is ThruPLEX Tag-seq Kit compatible with any target enrichment systems?

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

  1. Can I sequence ThruPLEX Tag-seq with other Illumina NGS libraries on the same run?

Yes, ThruPLEX Tag-seq libraries can be sequenced with other Illumina NGS libraries on the same sequencing run. However, care must be taken to ensure the samples carry unique and compatible sample indexes and receive adequate sequencing coverage. To realize the benefits of the unique molecular tags (UMTs), ThruPLEX Tag-seq libraries are usually target enriched and sequenced to a relatively high depth.

  1. How do I process my sequencing data obtained from ThruPLEX Tag-seq libraries?

To benefit from the information provided by the unique molecular tags (UMTs) in ThruPLEX Tag-seq libraries, the sequencing data must be processed to group reads into amplification families and construct consensus reads. Two bioinformatics solutions designed specifically for processing data from ThruPLEX Tag-seq libraries are available:

Curio

Curio is a cloud-based bioinformatics platform available through Curio Genomics. Sequencing data from ThruPLEX Tag-seq libraries can be easily uploaded, processed and visualized. The platform features an ultra-fast and user-friendly interface and a powerful alignment viewer. The platform is also equipped with modules for performing variant detection, analysis and visualization. For more information, visit www.curiogenomics.com.

Connor

Connor is an open-source bioinformatics tool hosted on GitHub (https://github.com/umich-brcf-bioinf/Connor) for processing sequencing data generated from ThruPLEX Tag-seq libraries. Connor takes an aligned BAM file as input, processes the UMT information and generates a processed BAM file containing consensus sequences. The output BAM file can be used with variant callers, such as FreeBayes, VarScan2 or GATK HaplotypeCaller.

  1. I have my own bioinformatic analysis pipeline. How can I modify it to process sequencing data obtained from ThruPLEX Tag-seq libraries?

Since each pipeline is different, we encourage you to take advantage of Connor, the open source bioinformatics tool created for  use with ThruPLEX Tag-seq.  Should you want to modify your existing pipeline the  information that follows  may help.  Each ThruPLEX Tag-seq library molecule has a 6nt unique molecular tag (UMT) on each side. The UMT is the first sequence read by either Read 1 or Read 2, followed by an 8 to 11nt dephasing stem sequence and then the template DNA sequence. For more information on the library structure, refer to the ThruPLEX Tag-seq Kit Instruction Manual (specific link and section).

Overview

ThruPLEX Tag-seq combines molecular tags with ThruPLEX chemistry to construct molecularly tagged and sample-indexed Illumina NGS libraries. Each kit contains more than 16 million unique sequences used to tag individual DNA fragments prior to amplification, providing tracking of the fragments through the library preparation, target enrichment and data analysis processes to detect low-frequency alleles or count individual fragments. The ThruPLEX chemistry is engineered and optimized to produce highly diverse libraries with reproducible sequencing performance from 1 to 50 ng of DNA. The entire three-step workflow takes place in a single tube or well in about 2 hours. No intermediate purification steps and no sample transfers are necessary, which prevent handling errors and loss of valuable samples. ThruPLEX Tag-seq Kit includes all necessary reagents including indexes for multiplexing up to 96 samples. Once purified and quantified, the resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocol.

Bioinformatics

To benefit from the information provided by the unique molecular tags (UMT) in ThruPLEX Tag-seq libraries, the sequencing data must be processed to group reads into amplification families and construct consensus reads. Two bioinformatics solutions designed specifically for processing data from ThruPLEX Tag-seq libraries are available:

 

Curio-Logo

 

 

Curio is a cloud-based bioinformatics platform available through Curio Genomics. Sequencing data from ThruPLEX Tag-seq libraries can be easily uploaded, processed and visualized. The platform features an ultra-fast and user-friendly interface and a powerful alignment viewer. The platform is also equipped with modules for performing variant detection, analysis and visualization. For more information, visit www.curiogenomics.com.

 

Connor-Logo-Mock- CAPS

 

 

Connor is an open-source bioinformatics tool hosted on GitHub (https://github.com/umich-brcf-bioinf/Connor) for processing sequencing data generated from ThruPLEX Tag-seq libraries. Connor takes an aligned BAM file as input, processes the UMT information and generates a processed BAM file containing consensus sequences. The output BAM file can be used with variant callers, such as FreeBayes, VarScan2 or GATK HaplotypeCaller.

 

ThruPLEX® Tag-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 Tag-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 Tag-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.