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 SMARTer 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 SMARTer ThruPLEX Tag-seq Kit in preparing sequencing libraries from low quantities (between 1 and 50 ng) of cfDNA.
- Single-cell RNA- and DNA-seq
- Immune profiling
- Epigenetics and smRNA-seq
Analysis of low-frequency mutations in cell-free DNA using unique molecular tags
cfDNA sequencing results using the SMARTer ThruPLEX Tag-seq Kit
Sequencing libraries were generated with the SMARTer 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 SMARTer ThruPLEX Tag-seq Kit using cfDNA input (1% Multiple I cfDNA Reference Standard).
ThruPLEX base coverage in targeted regions of cfDNA
Our SMARTer 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 SMARTer ThruPLEX Tag-seq Kit.
Detection of low-frequency mutant alleles in cfDNA
Following the generation of sequencing data, cfDNA libraries generated using the SMARTer 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 SMARTer ThruPLEX technology.
|Sample Information||1.0% Multiplex I cfDNA Reference Standard||0.1% Multiplex I cfDNA Reference Standard|
|EGFR (ΔE746 - A750)||1,966||9||0.46%||1,869||0||0.00%|
|EGFR (V769 - D770Ins)||1,997||9||0.45%||2,092||0||0.00%|
|Number of detected mutations
|8 (100%)||6 (75%)|
Table II. Reference and mutant allele detection in libraries generated with the SMARTer ThruPLEX Tag-seq Kit. REF = reference allele; ALT = mutant allele; AF = mutation frequency (calculated as the ratio of ALT in all reads).
Our SMARTer 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 SMARTer ThruPLEX Tag-seq Kit meets this challenge and represents a reliable, high-quality method to accelerate and advance cancer research.
See what our customers are saying about SMARTer 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
Takara Bio USA, Inc.
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