Variation of extrachromosomal circular DNA in cancer cell lines

Carl Rung dos Santos1, Lasse Bøllehuus Hansen1, Astrid Zedlitz Johansen2, Mirna Perez-Moreno3, Birgitte Regenberg1‡.

 

Affiliations

1 Ecology and Evolution, Department of Biology, University of Copenhagen, Denmark

2 Department of Oncology, Copenhagen University Hospital - Herlev and Gentofte, DK-2730 Herlev, Denmark

3 Cell biology and Physiology, Department of Biology, University of Copenhagen, Denmark

‡Corresponding author: Birgitte Regenberg. E-mail: bregenberg@bio.ku.dk

Abstract

The presence of oncogene carrying eccDNAs is strongly associated with carcinogenesis and poor patient survival. Tumour biopsies and in vitro cancer cell lines are frequently utilized as models to investigate the role of eccDNA in cancer. However, eccDNAs are often lost during the in vitro growth of cancer cell lines, questioning the reproducibility of studies utilizing cancer cell line models. Here, we conducted a comprehensive analysis of eccDNA variability in seven cancer cell lines (MCA3D, PDV, HaCa4, CarC, MIA-PaCa-2, AsPC-1, and PC-3). We compared the content of unique eccDNAs between triplicates of each cell line and found that the number of unique eccDNA is specific to each cell line, while the eccDNA sequence content varied greatly among triplicates (~ 0 – 1 % eccDNA coordinate commonality). In the PC-3 cell line, we found that the large eccDNA (ecDNA) with MYC is present in high-copy number in an NCI cell line isolate but not present in ATCC isolates. Together, these results reveal that the sequence content of eccDNA is highly variable in cancer cell lines. This highlights the importance of testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.

Keywords: cancer cell lines; reproducibility; eccDNA; ecDNA; double minute

1.     Introduction

Extrachromosomal circular DNAs (eccDNAs) are acentromeric circular DNA structures found in all eukaryotes and tissues1–4. eccDNAs originate from chromosomal DNA and the size can vary from a few hundred basepairs to megabases5,6. As such, eccDNAs are often subcategorized based on their size as either microDNA (<104 base pairs) or ecDNA (104-107 base pairs)4. eccDNAs can carry full-length protein coding genes that, when expressed, can provide phenotypic advantages to host cells and allow them to adapt to unfavorable growth conditions3. This has been demonstrated in plants and in the single-celled eukaryotic model organism, Saccharomyces cerevisiae, where eccDNAs were shown to play a role in adaptation and survival7,8.

 

In cancer, oncogene-carrying eccDNAs can be a source of oncogene amplification across multiple cancer types and correlates with poor patient survival4,6,9. For example, MYCN has been found to be highly amplified via eccDNA in neuroblastoma10,11, EGFR in glioblastoma10–12, and ERBB2 in esophageal cancers13. The amplification of oncogenes is thought to provide a selective advantage to cancer cells over healthy cells by stimulating their growth and proliferation, fostering tumour development and chemotherapeutic resistance10,14. The acentromeric nature of eccDNAs allows for unequal segregation during mitosis, in accordance with a Gaussian distribution, leading to the rapid generation of genetic heterogeneity in a dividing cell population10,15,16. As a result, there is a significant variation in the number of oncogene carrying eccDNAs in individual cancer cells, even in homogeneously defined cancer cell lines6,15. Therefore, eccDNA has been investigated as a drug target, using cancer cell line models that recurrently harbour oncogene carrying eccDNAs, such as the PC-3 prostate cancer cell line, reported to harbour eccDNAs carrying the MYC gene resulting in its amplification6,10,17,18.

 

However, loss and variations in eccDNA content among cancer cell lines poses a challenge to reproducibility in research studies. As such, we aimed to address a key question: To what extent are cancer cell lines affected by variations in eccDNA content? The presence of an eccDNA in an eukaryotic cell is expected to be determined by five factors3: I) eccDNA formation rate, II) replication during mitosis, III) loss or elimination rate, IV) segregation during mitosis and, V) selective growth advantages provided to the host cells. Studies comparing eccDNA content in patient-derived cancer cell lines and matched tumours suggest that eccDNAs are often lost during prolonged in vitro growth of three weeks or more19. Earlier efforts at establishing stable eccDNA harbouring cancer cell lines by de novo cutting and ligating a chromosomal region together to form eccDNA, have failed because the cell lines gradually lose eccDNAs20. When cancer cell lines are propagated in culture, some eccDNAs tend to reintegrate into the chromosomes instead of staying extrachromosomal21. Finally, cancer cell lines are genetically unstable22, and DNA replication stress has been shown to promote both the formation and loss of eccDNA23,24. Therefore, we hypothesize that cancer cell lines exhibit a high turnover rate of eccDNA, leading to significant variations in eccDNA content.

 

We tested the hypothesis using two approaches: First, eccDNA was purified and sequenced from triplicates of seven cancer cell lines and identified using the Circle-Map pipeline25. This was done to assess inter-triplicate variations in the content of all unique eccDNAs in a cancer cell line population. Second, we applied the AmpliconArchitect pipeline26 to assess variations in high-copy number eccDNAs, which are likely to be maintained in a cancer cell line population due to the selective advantage these eccDNAs provide to host cells. We find substantial inter-triplicate variations in the content of eccDNA and cell line isolate specific variations in high copy-number eccDNAs. These variations could have implications for the reproducibility and reliability of studies that use cancer cell lines to study the role of eccDNAs in cancer.

1.     Materials and methods

2.1 Cell culture.

Mouse cancer cell lines MCA3D, PDV, HaCa4, and CarC27–29 were kind gifts of Dr. Amparo Cano (Instituto de Investigaciones Biomédicas Alberto Sols, Madrid, Spain). Human cancer cell lines MIA-PaCa-2, AsPC-1, and PC3 were supplied by Herlev Hospital and purchased from ATCC. Cells were cultured from a frozen stock (-80oC) until confluence was reached. Then, ~300.000 cells were transferred to new petri dishes in triplicates and cultured until confluence was reached. Finally, ~106 cells were pelleted. Triplicates were collected on the same day. PC3 and AsPC-1 cells were cultured in RPMI 1640, GlutaMAX, HEPES (cat.no. 72400021, Gibco), supplemented with 10 % fetal bovine serum (FBS) (cat.no. 10500064, Gibco), and 1 % penicillin/streptomycin (P/S) (cat.no. 15140122, Gibco). MCA3D, PDV, and HaCa4 cells were cultured in Ham’s F-12 Nutrient Mixture (cat.no. 11765054, Gibco), supplemented with 10 % FBS, and 1 % P/S. CarC and MIA-PaCa-2 cells were cultured in DMEM, GlutaMAX (cat.no. 11594446, Gibco), supplemented with 10 % FBS, and 1 % P/S. DPBS (cat.no. 14190169, Gibco) was used to wash adherent cells prior to detachment with trypsin-EDTA (0.05 %) (cat.no. 25300054, Gibco). All cells were cultured at 37 oC with 5 % CO­2.

 

2.2 Circle-Pure eccDNA extraction, purification and amplification

eccDNA was extracted and purified using Circle-Pure (cat.no. 1001-24, CARE-DNA), that is based on the Circle-Seq method30. 1) A cell pellet of ~106 cells was resuspended in 100 µl DNase/RNase free H2O (cat.no. 11538646, Invitrogen). Then, 750 µl α-buffer (CARE-DNA), 20 µl RNase A (cat.no. 19101, Qiagen), and 50 µl Proteinase K (cat.no. EO0492, Thermo Scientific) was added and the suspension was incubated for 30 minutes at room temperature (25 oC). Thereafter, 552 µl AMPure XP beads (0.6X current volume) (cat.no. A63881, Beckman Coulter) and 1400 µl β-buffer (CARE-DNA) was mixed with the sample by pipetting. Beads were aggregated on a magnetic rack and washed twice in 3000 µl 80 % ethanol. Then, beads were resuspended in 55 µl Elution Buffer (CARE-DNA), incubated at 50 oC for 5 minutes, and beads were aggregated to elute the DNA suspension. This was repeated once more to reach a final volume of 110 µl total DNA. 2) To remove linear DNA, 40 µg total DNA was mixed with 10 µl NEBuffer 4 (10 X), 3 µl Exonuclease V (10,000 U/ml) (cat.no. M0345L, NEB), 10 µl ATP (10 mM), and nuclease-free H2O to reach a final volume of 100 µl. This was incubated for 3 days at 37 oC. Every 24 hours additional 1.4 µl NEBuffer 4 (10 X), 3 µl Exonuclease V (10,000 U/ml), and 10 µl ATP were added to the reaction. The reaction was inactivated by incubation at 70 oC for 30 minutes. Thereafter, mtDNA was linearized using CRISPR-Cas9 with sgRNAs targeting two mtDNA positions31. To remove linearized mtDNA and residual linear DNA, another 4 days of exonuclease treatment were performed. mtDNA and linear DNA removal was confirmed by PCR. Purified eccDNA was amplified using TruePrime Phi29 rolling circle amplification (cat.no. 390100, 4basebio) for 48 hours.

 

2.3 mtDNA linearization

Targeted removal of mitochondrial DNA from eccDNA was performed according to previously published protocol31 using the Cas9 Nuclease, S. pyogenes (cat.no. M0386M, NEB). sgRNAs were synthesized using the EnGen sgRNA Synthesis Kit, S. pyogenes (cat.no. E3322S, NEB) according to the manufacturer’s protocol. Mouse-sgRNA1:  GTAGCATGAACGGCTAAACGA. Mouse-sgRNA2: GGCCTGATAATAGTGACGCT. Human-sgRNA1: GGCTTGGATTAGCGTTTAGA. Human-sgRNA2: GCGTAGGGGCCTACAACGTTG.

 

2.4 Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was carried out using the CHEF-DR II Pulsed Field Electrophoresis System (Bio-Rad). A 1 % agarose gel was cast using Pulsed Field Certified Agarose (cat.no. 1620137, Bio-Rad) in fresh 0.5 X TBE buffer. A CHEF DNA Size Marker, 0.2–2.2 Mb, S. cerevisiae Ladder (cat.no. 1703605, Bio-Rad) agarose plug was loaded in the first lane as a size reference. One µg total DNA with a volume of 40 µl was loaded in each well. The PFGE ran for 40 hours at 14 oC, at 5 V/cm, initial SW: 47 seconds, and final SW: 170 seconds. The gel was post-stained in a 3 X GelRed solution for 30 minutes.

 

2.5 PCR, gel electrophoresis and Sanger sequencing

Thermo Scientific DreamTaq PCR Master Mix (2 X) (K1071) was used for all PCR reactions. PCR products ran on a 0.7-2 % agarose gel in 1 X Bionic buffer (Sigma-Aldrich) to verify that the PCR reaction progressed as expected. The percentage of agarose was determined by how large a PCR product was expected. Monarch® DNA Gel Extraction Kit Protocol (NEB #T1020) was used to extract DNA from a gel slice and purify the PCR product for Sanger sequencing. Sanger sequencing was performed by Eurofins Genomics.

 

2.6 Whole-genome sequencing

DNA sequencing libraries were prepared using NEBNext Multiplex Oligos for Illumina (cat.no. E6440S, NEB) and NEBNext Ultra II DNA Library Prep Kit for Illumina (cat.no. E7645L, NEB). 500 ng amplified eccDNA/total DNA was fragmented by sonication to obtain fragment lengths of ~ 400 bp. AMPure XP beads (cat.no. A63881, Beckman Coulter) were used for size-selection of adaptor-ligated DNA fragments to reach ~ 400 - 600 bp. DNA libraries were pooled and paired-end (150 bp) sequenced on an Illumina NovaSeq 6000. Sequence reads were trimmed for adaptor content with BBduk (version 38.90). Reads were quality checked with FASTQC (version 0.11.9), and aligned against the mouse reference genome mm10 (GCA_000001635.2) or human reference GRCh38 (GCA_000001405.15) with BWA MEM (version 0.7.17). Genomic features were annotated with Bedtools intersect (version 2.30.0). The gencode.vM10.annotation.gtf.gz file was used to annotate genomic features on eccDNA coordinates in mouse cancer cell lines. The gencode.v42.annotation.gtf.gz file was used to annotate genomic features on eccDNA coordinates in human cancer cell lines. SAMtools (version 1.9) was used to sort .bam files and calculate sequencing read depth. Picard-tools (version 2.26.10) were used to mark duplicate reads and perform downsampling. CNVkit (version 0.9.9) was used to find copy-number variants based on sequencing read depth32.

 

2.7 Data acquisition

WGS data on PC-3 from Seim et al. was acquired from bioproject PRJNA361315 run SRR5196724. WGS data on PC-3 from Turner et al. was acquired from bioproject PRJNA338012 run SRR4009277. sratoolkit (version 2.11.3) https://github.com/ncbi/sra-tools was used for downloading the data.         

 

2.8 Identification of eccDNA with Circle-map

eccDNA sequencing data from samples purified with Circle-Pure was analyzed using the Circle-Map Realign25 bioinformatics pipeline to map eccDNA coordinate as detailed at: https://github.com/iprada/Circle-Map.

 

2.9 Identification of eccDNA with AmpliconArchitect

WGS data from total DNA of PC-3 extracted with Circle-Pure, from Seim et al, and Turner et al. was analyzed with AmpliconArchitect to find focal amplifications in the form of eccDNA26. AmpliconArchitect analysis was performed following the pipeline with default setting as detailed at: https://github.com/virajbdeshpande/AmpliconArchitect. AmpliconClassifier (version 0.4.12)13 https://github.com/jluebeck/AmpliconClassifier was used to determine whether amplicons were classified as eccDNA. CycleViz https://github.com/jluebeck/CycleViz was used to visualize amplicons classified as eccDNA identified by AmpliconArchitect.

3.     Results

3.1 The number of unique eccDNA is cancer cell line specific. To investigate the variation of eccDNA in cancer cell lines, we cultured seven cancer cell lines of different origin in triplicates and collected cell pellets consisting of ~106 cells. We extracted, purified, and amplified eccDNA from the cell pellets using the Circle-Pure method. Linear chromosomal DNA removal and mtDNA reduction was verified by PCR (Supplementary Fig. 1a-b). The amplified eccDNA was then sequenced and the bioinformatics pipeline Circle-Map25 was applied to identify every unique eccDNA present in each cancer cell line (Fig. 1a). The presence of each eccDNA identified in the mouse cancer lines (MCA3D, PDV, HaCa4, and CarC) was required to be supported by at least 1 soft-clipped read, 1 discordant read pair, and have at least 90 % read coverage to be considered valid (Supplementary Fig. 2a). Similarly, the presence of each eccDNA identified in the human cancer lines (MIA-PaCa-2, AsPC-1, and PC-3) was required to be supported by at least 4 soft-clipped reads, 4 discordant read pairs, and have at least 99 % read coverage to be considered valid (Supplementary Fig. 2b). We found that the number of unique eccDNA is specific to each cancer cell line, independent of sequencing depth (Fig. 1b, Table. 1). We downsampled the sequencing reads of the cell line replicates with the highest number of eccDNA/million mapped  reads. Downsampling indicated that sufficient read depth was achieved to capture most of the unique eccDNAs from an extract of ~106 cells of all cell lines investigated except for MIA-PaCa-2 (Fig. 1c). The kink seen in the downsampling curve from 90 % - 100 % relative read count (%), is a result of chromosomal regions covered by reads that support smaller eccDNAs being merged by Circle-map into a single larger eccDNA. It has previously been postulated that only small eccDNA (up to tens of kilobases) can be extracted using in-solution magnetic-bead-based DNA extraction methods33. However, we find eccDNAs extracted with Circle-Pure larger than 100 kilobases in the mouse and human cancer cell lines (Supplementary Fig. 2c). Furthermore, we demonstrate that with the Circle-Pure method, intact ultrahigh-molecular weight (UHMW) total DNA can be extracted (Fig. 1d). Altogether, our findings demonstrate that the number of unique eccDNA is specific to each cancer cell line and that eccDNAs of all sizes can be extracted from a mammalian cell pellet using the Circle-Pure method.

Figure 1. Workflow and summary of eccDNA extracted with Circle-Pure identified with Circle-Map. A) Schematic representation of the study workflow. B) Mean number of unique eccDNA (n = 3) identified by Circle-Map. The presence of all eccDNAs reported in the mouse cancer cell lines is supported by at least 1 soft-clipped read, 1 discordant read-pair, and at least 90% read coverage to be considered valid. The presence of all eccDNAs reported in human cancer cell lines is supported by at least 4 soft-clipped reads, 4 discordant read-pairs, and at least 99% read coverage to be considered valid. C) Saturation plot of relative read count percentage against the eccDNA count of the cell line replicates with highest eccDNA count/million mapped reads. D) A PFGE gel of total DNA extracted from MIA-PaCa-2 (rep 3) and AsPC-1 (rep 3) with Circle-Pure. L = CHEF DNA Size Marker, 0.2–2.2 Mb, S. cerevisiae Ladder.

Table 1. Summary of sequencing quality and eccDNAs identified and in the mouse and human cancer cell lines.

3.2 eccDNA content vary substantially among cancer cell line triplicates. To determine the degree of eccDNA variation in cancer cell lines, we annotated genetic features on the eccDNA chromosomal coordinates and compared the triplicates of each cell line. Of the total number of full-length protein-coding genes located on eccDNA, between 0 – 2 % reoccur in triplicates of each investigated cancer cell line (Fig. 2a, Supplementary Fig. 3). None of these recurring genes have been correlated with carcinogenesis, cell proliferation, or were related to growth (Fig. 2a, Supplementary Fig. 3). However, in the MCA3D cell line, multiple cancer-associated genes were found on eccDNAs in individual replicates. In replicate 1, individual eccDNAs carrying Rac3, Jun, Gstm5, and Fzd1 were identified. In replicate 2, individual eccDNAs carrying Casp3, Gadd45a, and Wnt16 were identified. In replicate 3, individual eccDNAs carrying Mapk3, Myc, Nfkb2, and Gnb2 were identified. We verified the presence of nine of these by outwards PCR spanning the eccDNA junction site (Fig. 2b). Further, we Sanger sequenced the PCR product of the [Rac3circle], [Wnt16circle], and [Nfkb2circle] to confirm that the outwards PCR specifically amplified the eccDNA junction. This revealed that these eccDNAs were formed from regions of microhomology (Fig. 2c-d). To have a non-genic measure of eccDNA variance, we examined how many unique eccDNAs the cancer cell line triplicates have in common based on their chromosomal coordinates +/- 200 basepairs in both start and end coordinate. Out of the total number of unique eccDNAs identified in the cancer cell lines, between 0.1 - 0.3 % are found in two replicates of the same cell line. When comparing triplicates, the eccDNA commonality is almost negligible (Table. 2). Taken together, these results demonstrate that the eccDNA content in individual cultures of the same cancer cell line population is highly variable.

Figure 2. Inter-replicate eccDNA variation. A) Venn diagram of unique full-length protein-coding genes located on eccDNA in each replicate (designated 1, 2, and 3) of the MCA3D cell line. Full-length protein coding genes located on eccDNA found in three replicate: Krtap10-4, Gm10840, Psmg3. B) Outwards PCR spanning the junction site of 11 eccDNAs identified in MCA3D that carry a known oncogene. Amplified eccDNA was used as the template DNA for the PCR reaction. The expected band size ranged from 0.8kb - 1.2kb. L = GeneRuler 1Kb DNA ladder. N = negative control. Lane numbers refer to replicate numbers. Stars indicate correct size of PCR product and orange boxes refer to the gel slices that were extracted for Sanger sequencing. C) eccDNA read coverage plot of Rac3circle, Wnt16circle and Nfkb2circle. D) Sanger sequencing results of the PCR bands highlighted in orange in B.

Table 2. Number of unique eccDNA with same chromosomal coordinates allowing 200 basepairs +/- overlap in both start and end eccDNA coordinate.

3.3 The presence of MYC eccDNA in PC-3 cells is isolate specific. To assess the variability in the presence of maintained eccDNAs in cancer cell lines, we whole-genome sequenced total DNA from triplicates of the PC-3 cell line (Fig. 1a). Prior studies have reported MYC amplification through eccDNA in this cell line6,10,17,18. We investigated copy-number variations (CNV) and applied the AmpliconArchitect26 (AA) pipeline to identify eccDNA focal amplifications in our own PC-3 triplicates and two publicly available PC-3 WGS datasets from Seim et al.34 and Turner et al.6. While MYC carrying eccDNA was identified in the WGS data from Turner et al., resulting in its amplification, our own and the Seim et al. WGS did not show such amplification (Fig 3, Supplementary Fig. 4a). We compared the global CNV profile in the five datasets, and found that the Turner et al. PC-3 isolate differs from ours and that from Seim et al. (Supplementary Fig. 4b). Both ours and the Seim et al. PC-3 isolate were purchased from the American Type Culture Collection (ATCC), whereas the isolate from Turner et al. was given as a gift by the National Cancer Institute (NCI). The publications describing MYC eccDNA in PC-3 are all based on the isolate from the NCI10,19,20. As such, we find that the presence of MYC carrying eccDNAs is specific to the NCI PC-3 isolate.

Figure 3. Copy-number variation and eccDNA identification in PC-3. A) CNVkit scatterplot of chr8:120Mb-140Mb of the PC-3 cell line isolate from this study (left), Seim et al. (middle), and Turner et al. (Right). The y-axis describes the log(2) copy ratio reported by CNVkit and represents the deviation in copy number of each genomic segment in the sample relative to the expected copy number based on the reference genome32. The vertical purple line represents the location of the MYC gene (~127Mb). B) AmpliconArchitect output of the amplicon that included the MYC gene in the PC-3 cell line isolate from this study (left), Seim et al. (Middle), and Turner et al. (Right). Read coverage is represented as grey coverage bars and absolute copy-number is represented as horizontal orange lines. The location of the MYC and PVT1 gene is shown as purple lines underneath the graphs. C) No MYC-carrying eccDNAs were identified in the PC-3 cell line isolate from this study (left) or with data from Seim et al. (middle). With the Turner et al. WGS data, MYC and PVT1 carrying eccDNAs were identified by AmpliconClassifier and visualized with CycleViz (right).

4. Discussion

Here we show that the eccDNA sequence content in cancer cell lines is highly variable and the number of unique eccDNA is specific to each cancer cell line investigated. These results suggest that the variability comes from a continuously high rate of eccDNA formation and loss, specific to each cell line3. Our findings also revealed, that the presence of MYC carrying eccDNAs in the PC-3 cell line is specific to the NCI isolate and is not identified in the ATCC isolate, most likely due to the loss of MYC amplification on eccDNA during the propagation of the ATCC cell line isolate. It has previously been shown that cancer cells propagated in a selective environment accumulate eccDNAs that enable them to adapt to such an environment. Furthermore, when propagated in a non-selective environment the number of these eccDNAs remain low10. This has also been observed in S. cerevisiae8,35. Loss of genetic driver alterations and large inter-laboratory genetic and transcriptional differences that result in deviations in drug response, have previously been observed in multiple cancer cell lines22,36. Therefore, we reason that the loss of MYC eccDNA in the PC-3 cell line occurred due to a combination of microenvironmental changes in selective pressure and due unequal segregation of eccDNA. As such, this suggests that eccDNAs expected to be maintained in a cancer cell line due to the selective growth advantage they provide to host cells, can be lost when cancer cell lines are propagated in vitro.

 

Understanding the role of eccDNAs in cancer is crucial for the development of effective cancer therapies. Therefore, cancer cell lines that consistently harbour the same oncogene-carrying eccDNA in many copies, as a result of the advantage it provides to the host, continue to be a valuable resource for studying the fundamental biology of eccDNAs in cancer. It is important to acknowledge that the eccDNA variability observed here is not an error, but rather a consequence of the inherent nature of eccDNA to undergo changes. Therefore, we advise testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.

Data availability

Sequencing data are publicly available as of the date of publication at the SRA database under BioProject number PRJNA960955.

 

Acknowledgements

We thank professor Julia Sidenius Johansen for hosting us in the cell line facilities at Herlev Gentofte hospital. We thank Anna Cazzola for assistance with cell lines. We thank professor Michael Lisby for the use of the CHEF-DR PFGE apparatus.

 

Funding

We had funding from the Novo Nordisk Foundation (NNF21OC0072023) to B.R., M.P.M., and C.R.d.S.. European Union’s Horizon 2020 research and innovation action under the FET-Open Programme (899417—CIRCULAR VISION) to B.R. and A.Z.J.. Innovation Fund Denmark under the Grand Solutions programme (8088-00049B CARE DNA) to B.R., A.Z.J., and L.B.H..

 

Author’s contribution

C.R.d.S. and B.R. designed and conceived the project. C.R.d.S. and A.Z.J. performed the experiments. C.R.d.S. analyzed the data and prepared figures. C.R.d.S, B.R., L.B.H., and M.P.M. designed the experiments. B.R. and L.B.H. supervised the study. C.R.d.S. and B.R. wrote the manuscript, and all authors edited the manuscript. All authors have read and approved the final version.

 

Conflict of interest

B.R. is cofounder of CARE-DNA. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Supplementary material

Supplementary figure 1. Linear and mtDNA removal confirmation. A) PCR with primers that amplify a region of ~1.5 Kb in the COX5B gene on chr2 in humans and chr1 in mice. T = Total DNA. E = Exonuclease treated DNA. L = GeneRuler 1Kb Plus DNA ladder. B) PCR with primers that amplify a region in the ChrM of ~400bp in mice and ~200bp in humans. T = Total DNA. E = CRISPR linearized mtDNA and exonuclease treated DNA for linear DNA removal. In leftmost gel L = GeneRuler 1Kb Plus DNA ladder, in rightmost gel L = GeneRuler 1Kb DNA ladder.

Supplementary figure 2. Integrative Genome Viewer read mapping examples. A) A 50 Kb window of the MCA3D (2) .bam file loaded in Integrative Genome Viewer (IGV) to display where reads align in the reference genome. Green reads represent soft-clipped reads. Dark blue reads represent discordantly mapped reads. The underlying graph denotes the position of specific genomic features. B) a 100 Kb window of the PC-3 (2) bam file loaded in IGV. C) Violin plots of eccDNA size in the cancer cell lines investigated. The y-axis represents the log10 eccDNA size (bp) and the x-axis represents the cancer cell lines.