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Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing


Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

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Figure 1: Overview of the SiMSenSeq procedure.
Figure 2: Real-time adaptor PCR amplification testing.
Figure 3: Fragment analyzer examples for single- and multiplex libraries.
Figure 4: Use of Debarcer to inform raw and consensus sequencing depth.
Figure 5: Visualizing SiMSen-seq error correction with Debarcer.


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This work was supported by the National Institutes of Health (R21CA172999 to T.E.G.), the Swedish Cancer Society (to A.S.), the Swedish Childhood Cancer Foundation (to A.S.), the Sahlgrenska Academy at the University of Gothenburg (to A.S.).

Author information

Authors and Affiliations



A.S. and T.E.G. conceived and designed the protocol; P.M.K. and L.S. developed the Debarcer software for data analysis; and M.E. and S.F. contributed to protocol development. A.S., P.M.K., M.E., S.F. and T.E.G. wrote the manuscript.

Corresponding authors

Correspondence to Anders Ståhlberg or Tony E Godfrey.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Concept of barcoding to suppress error in sequencing.

Each template DNA molecule is barcoded with a random and unique sequence (marked as red, turquoise and green). All PCR generated molecules that are generated from the same original molecule receive the same barcode. If a polymerase induced error occurs (blue cross), only a fraction of all DNA molecules with the same barcode will amplify that specific error. Conversely, a template DNA molecule with mutation will generate PCR amplicons with that particular barcode and can therefore be called a true mutant.

Supplementary Figure 2 Organization of SiMSen-Seq analysis data produced by Debarcer.

The Debarcer output directory contains files in the main output directory (denoted ‘Top Level’) and two subdirectories found in this location (Tables directory and Figures directory). Files described by each unshaded box in the figure can be found in their respective directories.

Supplementary Figure 3 Fragment analysis of single-plex library at low DNA input.

Unpurified libraries were generated for a target amplicon in the CD1C gene using 10, 5 and 3 ng of high quality human genomic DNA (left side) or fragmented plasma DNA (right side) along with a no DNA control (bottom). Expected library products are indicated by the horizontal bar while all other products (<200bp) are non-specific. Clear library PCR products can be seen in all plots except the no DNA control.

Supplementary Figure 4 Fragment analysis of triplex library at low DNA input.

Unpurified libraries were generated for three target amplicons in the CD1C, ERBB4, and COL5A1 genes using 10, 5 and 3 ng of high quality human genomic DNA (left side) or fragmented plasma DNA (right side) along with a no DNA control (bottom). Expected library products are indicated by the horizontal bar while all other products (<200bp) are non-specific. Clear library PCR products can be seen in all plots except the no DNA control.

Supplementary Figure 5 Schematic representation of the first three rounds of PCR used to incorporate barcodes.

Figure shows PCR products that are formed in cycles 1, 2 and 3 of the barcoding PCR step. Note that only DNA strands with both forward and reverse adapter sequences (or their complement) can act as templates in the second round of PCR. These products are indicated with thicker lines.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1 (PDF 1118 kb)

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Ståhlberg, A., Krzyzanowski, P., Egyud, M. et al. Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing. Nat Protoc 12, 664–682 (2017).

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