Simple multiplexed pcr-based barcoding of dna for ultrasensitive mutation detection by next-generation sequencing

ABSTRACT 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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS SELECTIVE MULTIPLEXED ENRICHMENT FOR THE DETECTION AND QUANTITATION OF LOW-FRACTION DNA

VARIANTS VIA LOW-DEPTH SEQUENCING Article 03 May 2021 DETECTION OF LOW-FREQUENCY DNA VARIANTS BY TARGETED SEQUENCING OF THE WATSON AND CRICK STRANDS Article 03 May 2021 ADAPTOR TEMPLATE

OLIGO-MEDIATED SEQUENCING (ATOM-SEQ) IS A NEW ULTRA-SENSITIVE UMI-BASED NGS LIBRARY PREPARATION TECHNOLOGY FOR USE WITH CFDNA AND CFRNA Article Open access 04 February 2021 REFERENCES * ten

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circulating DNA. _PLoS One_ 6, e23418 (2011). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS 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 Author

notes * Anders Ståhlberg and Paul M Krzyzanowski: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Pathology and Genetics, Sahlgrenska Cancer Center,

Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden Anders Ståhlberg & Stefan Filges * Ontario Institute for Cancer Research, MaRS Centre,

Toronto, Ontario, Canada Paul M Krzyzanowski & Lincoln Stein * Department of Surgery, Boston University School of Medicine, Boston, Massachusetts, USA Matthew Egyud & Tony E Godfrey

Authors * Anders Ståhlberg View author publications You can also search for this author inPubMed Google Scholar * Paul M Krzyzanowski View author publications You can also search for this

author inPubMed Google Scholar * Matthew Egyud View author publications You can also search for this author inPubMed Google Scholar * Stefan Filges View author publications You can also

search for this author inPubMed Google Scholar * Lincoln Stein View author publications You can also search for this author inPubMed Google Scholar * Tony E Godfrey View author publications

You can also search for this author inPubMed Google Scholar CONTRIBUTIONS 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. ETHICS DECLARATIONS 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

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of DNA for ultrasensitive mutation detection by next-generation sequencing. _Nat Protoc_ 12, 664–682 (2017). https://doi.org/10.1038/nprot.2017.006 Download citation * Published: 02 March

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