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Tech Note

Analysis of low-frequency mutations in cell-free DNA using unique molecular tags

Introduction  

Liquid biopsies are rapidly becoming an invaluable method to understand cancer, with cell-free DNA (cfDNA) providing a non-invasive tool for diagnosis and continued monitoring of cancer. The ability to accurately detect low-frequency mutations in cell-free DNA (cfDNA) is critical to advancing oncology research, but, due to the rarity of these mutations, it can be difficult to distinguish true mutations from errors in library preparation, amplification, and sequencing steps. The ThruPLEX Tag-seq Kit resolves these challenges by providing up to 16 million molecular tags that reduce false positives, identify sequencing errors, and provide confident variant detection. In this experiment, we demonstrate the strength of our ThruPLEX Tag-seq Kit in preparing sequencing libraries from low quantities (between 1 and 50 ng) of cfDNA.

Results  

cfDNA sequencing results using the ThruPLEX Tag-seq Kit

Sequencing libraries were generated with the ThruPLEX Tag-seq Kit using 50 ng each of 0.1% and 1% Multiplex I cfDNA Reference Standards (Horizon). These standards represent a solution of cfDNA containing eight known mutations adjusted to 0.1% or 1% allele frequency. Libraries were subjected to enrichment using IDT xGen Lockdown Probes and then sequenced. Our kit demonstrated robust mapping efficiency with a high number of unique on-target reads, good on-target rate, and in-depth coverage coupled with a low error rate (Table I). 

Total number of reads Reads mapped (%) On-target rate (%) Number of unique reads on target Average coverage Error rate
ThruPLEX + IDT target enrichment kit 80,206,918 99.87 43.78 3,349,740 1,993 0.125

Table I. Sequencing metrics from libraries generated with the ThruPLEX Tag-seq Kit using cfDNA input (1% Multiple I cfDNA Reference Standard).

ThruPLEX base coverage in targeted regions of cfDNA

Our ThruPLEX kit Tag-seq Kit demonstrated robust coverage of a targeted region in cfDNA across a wide range of sequencing depths, with >90% of reads containing the target in up to 1,500X coverage and >70% in 2,000X coverage (Figure 1). This strength was further validated when the coverage was analyzed as a function of read depth. Saturation of coverage was accomplished with only ~20 million reads (Figure 2), indicating the high efficiency of the ThruPLEX Tag-seq Kit.

Figure 1. Coverage of target base compared to total coverage.

Figure 2. Rapid saturation of coverage as a function of sequencing depth.

Detection of low-frequency mutant alleles in cfDNA

Following the generation of sequencing data, cfDNA libraries generated using the ThruPLEX Tag-seq Kit were analyzed for the detection of mutant alleles. 8 out of 8 variants were detected using the 1% Multiplex I cfDNA Reference Standard, while 6 of 8 (75%) were detected using the 0.1% reference standard. This data demonstrates the remarkable accuracy and sensitivity of our ThruPLEX technology. 

Sample Information 1.0% Multiplex I cfDNA Reference Standard 0.1% Multiplex I cfDNA Reference Standard
Variant REF ALT AF REF ALT AF
EGFR (L858R) 2,192 20 0.91% 1,977 2 0.10%
EGFR (ΔE746 - A750) 1,966 9 0.46% 1,869 0 0.00%
EGFR (T790M) 2,163 30 1.39% 2,160 3 0.14%
EGFR (V769 - D770Ins) 1,997 9 0.45% 2,092 0 0.00%
KRAS (G12D) 1,320 19 1.44% 1,179 2 0.17%
NRAS (Q61K) 4,093 40 0.98% 3,505 2 0.06%
NRAS (A59T) 4,158 42 1.01% 3,594 8 0.22%
PIK3CA (E545K) 1,309 15 1.15% 1,112 1 0.09%
Number of detected mutations
(detection sensitivity)		 8 (100%) 6 (75%)

Table II. Reference and mutant allele detection in libraries generated with the ThruPLEX Tag-seq Kit. REF = reference allele; ALT = mutant allele; AF = mutation frequency (calculated as the ratio of ALT in all reads).

Conclusions  

Our ThruPLEX Tag-seq Kit generates robust sequencing libraries using cfDNA as an input. It is highly sensitive, helps to eliminate sequencing errors, and provides confident detection—even of rare variants. Moving forward, the ability to accurately detect low-frequency mutations in challenging samples such as cfDNA will be critical to understanding cancer. Our ThruPLEX Tag-seq Kit meets this challenge and represents a reliable, high-quality method to accelerate and advance cancer research.

Methods  

Sequencing libraries were generated using our ThruPLEX Tag-seq Kit following manufacturer's instructions. 50 ng each of 0.1% and 1% Multiplex I cfDNA Reference Standards (Horizon, Cat. No. HD780) were used as input. Following preparation, libraries were subjected to enrichment using IDT xGen Lockdown Probes targeting genes that comprise a total of 160 kb of the human genome, and then sequenced on an Illumina HiSeq® 2500 using 100-bp paired-end reads. Following sequencing, data was mapped and aligned using the Burrows-Wheel Aligner MEM (BWA-MEM) software, and barcode analysis/mutation calling was performed using a proprietary development pipeline.

Related Products

Cat. # Product Size Price License Quantity Details
R400586 ThruPLEX® Tag-seq 96D Kit 96 Rxns USD $4987.00

The ThruPLEX Tag-seq Kit includes all necessary reagents for generating and multiplexing DNA-seq libraries with the incorporation of Unique Molecular Indexes (UMIs), and includes 96 dual index PCR primer sets. Once purified and quantified, the resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocols. Only 50 pg to 50 ng of fragmented double-stranded DNA is required for library preparation. The entire three-step workflow takes place in a single tube or well in about two hours. No intermediate purification steps or sample transfers are necessary, preventing handling errors and loss of valuable samples. This kit includes reagents sufficient for 96 reactions with 96 dual-index primer sets.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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R400585 ThruPLEX® Tag-seq 48S Kit 48 Rxns USD $2833.00

The ThruPLEX Tag-seq Kit includes all necessary reagents for generating and multiplexing DNA-seq libraries with the incorporation of Unique Molecular Indexes (UMIs), and includes 48 unique single index PCR primer sets. Once purified and quantified, the resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocols. Only 50 pg to 50 ng of fragmented double-stranded DNA is required for library preparation. The entire three-step workflow takes place in a single tube or well in about two hours. No intermediate purification steps or sample transfers are necessary, preventing handling errors and loss of valuable samples. This kit includes reagents sufficient for 48 reactions with 48 single-index primer sets.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data
R400584 ThruPLEX® Tag-seq 6S (12) Kit 12 Rxns USD $850.00

The ThruPLEX Tag-seq Kit includes all necessary reagents for generating and multiplexing DNA-seq libraries with the incorporation of Unique Molecular Indexes (UMIs), and includes 6 unique single index PCR primer sets. Once purified and quantified, the resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocols. Only 50 pg to 50 ng of fragmented double-stranded DNA is required for library preparation. The entire three-step workflow takes place in a single tube or well in about two hours. No intermediate purification steps or sample transfers are necessary, preventing handling errors and loss of valuable samples. This kit includes reagents sufficient for 12 reactions with 6 single-index primer sets.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

See what our customers are saying about ThruPLEX Tag-seq technology!

"ThruPLEX Tag-seq was easy to use with its simple and straightforward protocol. The unique molecular tags reduced the false positive variant calls and enabled accurate detection of true mutations present at low frequencies."
—Jinglan Zhang, Ph.D., Technical Director for NGS, BAYLOR MIRACA GENETICS LABORATORIES

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