Teng et al.

Genome Biology (2019) 20:132

https://doi.org/10.1186/s13059-019-1742-z

SHORT REPORT Open Access

CDetection: CRISPR-Cas12b-based DNA

detection with sub-attomolar sensitivity
and single-base specificity
Fei Teng1,2,3†, Lu Guo1,2,3†, Tongtong Cui1,2,3, Xin-Ge Wang1,2,3, Kai Xu1,2,3, Qingqin Gao1,2,3, Qi Zhou1,2,3* and
Wei Li1,2,3*

Abstract
CRISPR-based nucleic acid detection methods are reported to facilitate rapid and sensitive DNA detection. However,
precise DNA detection at the single-base resolution and its wide applications including high-fidelity SNP genotyping
remain to be explored. Here we develop a Cas12b-mediated DNA detection (CDetection) strategy, which shows higher
sensitivity on examined targets compared with the previously reported Cas12a-based detection platform. Moreover, we
show that CDetection can distinguish differences at the single-base level upon combining the optimized tuned guide
RNA (tgRNA). Therefore, our findings highlight the high sensitivity and accuracy of CDetection, which provides an
efficient and highly practical platform for DNA detection.

Background achieve dsDNA detection [5]. Though prior CRISPR-Cas-

Accurate, rapid, and economical DNA detection method is based nucleic acid detection platforms provide us with sen-
of great value in clinical diagnostics and quarantine inspec- sitive and specific diagnostic methods [2–6], they still re-
tion. Recently, CRISPR-Cas-based DNA detection plat- quire further improvement, and new platforms with
forms, including SHERLOCK (Cas13) [1–3], DETECTR prominent features are still desired. Inspired by the ssDNA
(Cas12a) [3, 4], and Cas14-DETECTR [5], were developed trans-cleavage activity of the Cas12b enzyme [4], we aim to
with high sensitivity and specificity. The SHERLOCK plat- develop a new dsDNA detection strategy combining with
form utilizes the RNA trans-cleavage activity of Cas13 en- our previously reported AaCas12b enzyme [7]. Since the
zymes and combines additional T7 in vitro transcription AaCas12b system cleaves dsDNA with high activity and
process, thereby achieving detection of RNA viruses, like specificity, we speculate that AaCas12b enables direct de-
Zika virus (ZIKA) and dengue virus (DENV) [2, 3, 6]. tection of dsDNA with high sensitivity and accuracy.
Meanwhile, through introducing synthetic mismatches in
the guide sequence, this SHERLOCK strategy can discrim-
inate SNPs in human genotyping and cell-free Results and discussion
DNA (cfDNA) detection [2, 3, 6]. The Cas12a-DETECTR To test the non-canonical trans-cleavage activity of
platform can detect double-stranded DNA (dsDNA) sam- Cas12b (Additional file 1: Figure S1a), we conducted the
ples directly using its ability to trans-cleave single-stranded in vitro ssDNA cleavage assay using Cas12b, Cas12a, and
DNA (ssDNA) reporter [3, 4], yet its accuracy still requires Cas9 with their cognate guide RNAs (gRNAs) (Add-
to be improved [4]. More recently, the Cas14-DETECTR itional file 2: Table S1). The results showed that
platform enables SNP detection in HERC2 gene [5]. But, AaCas12b [7] and three Cas12a nucleases (ArCas12a,
Cas14 only binds and cleaves ssDNA, thus requiring an HkCas12a, and PrCas12a) [8] could induce rapid degrad-
extra step to generate ssDNA from dsDNA to successfully ation of the single-stranded M13 DNA phage, while
SpCas9 could not [4] (Additional file 1: Figure S1b and
* Correspondence: [email protected]; [email protected] Additional file 2: Table S1). Meanwhile, AaCas12b and
†
Fei Teng and Lu Guo contributed equally to this work. Cas12a proteins degraded M13 phage in the presence of a
1
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of
Zoology, Chinese Academy of Sciences, Beijing 100101, China non-target gRNA (NT-gRNA) and its complementary
Full list of author information is available at the end of the article ssDNA “activator” that shares no sequence homology to
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Teng et al. Genome Biology (2019) 20:132 Page 2 of 7

the M13 phage genome (Additional file 1: Figure S1c and (Additional file 1: Figure S4b-d), but also distinguish two
Additional file 2: Table S1). The non-canonical collateral dsDNA viruses, HPV16 and HPV18, in identical group
ssDNA cleavage activity was abolished with catalytically (Additional file 1: Figure S4c, d). Compared with Cas12a-
inactive variants, indicating a RuvC domain-dependent based DNA detection, AaCas12b exhibited higher detection
cleavage activity (Additional file 1: Figure S2). Collectively, sensitivity than Cas12a did at both detected sites, as CDe-
these results reveal that AaCas12b-single guide RNA tection generated a higher signal level and a lower back-
(sgRNA) complex can acquire RuvC domain-dependent ground level (Fig. 1c and Additional file 1: Figure S4e). To
non-specific ssDNA trans-cleavage activity once triggered further enhance its sensitivity, we performed isothermal
by RNA-guided DNA binding. amplification by recombinase polymerase amplification
Previous study in SHERLOCK [3] indicates that Cas12b (RPA). CDetection combined with RPA enabled substrate
might also exhibit biases to different homopolymeric re- detection at 1 attomole (åM) (Fig. 1d and Additional file 1:
porters and different buffer composition. So to optimize Figure S4f), which shows higher sensitivity than the
the Cas12b-mediated DNA detection system, we next pro- Cas12a-based detection platform did [3, 4] (Fig. 1e). Then
filed the cleavage preference of AaCas12b-sgRNA complex we chose 12 previously reported targeting sites for compre-
on fluorophore quencher (FQ)-labeled homopolymer re- hensive and unbiased comparison of the sensitivity between
porters. The results showed that AaCas12b preferred thy- AaCas12b- and LbCas12a-based DNA detection (Additional
mine polymer (ploy T) as well as poly A and poly C, file 2: Table S1). The averaged results (8 of 12 sites) show
whereas poly G could hardly be cleaved (Additional file 1: that CDetection platform possessed a higher sensitivity
Figure S3a and Additional file 2: Table S1). Meanwhile, the when compared with LbCas12a-based detection platform
cleavage efficiency could be optimized in NEBuffer™ 2 except for one site (DNMT1 site 2) and three sites (EMX1
(Additional file 1: Figure S3b and Additional file 2: Table site 2, DNMT1 site 3, and BRCA1_3232G) with poor or un-
S2). Afterwards, we performed AaCas12b-mediated cleav- detectable signals (Additional file 1: Figure S5).
age assay using poly T reporter in NEBuffer™ 2. Using To explore the applications of CDetection in molecu-
sgRNA-complementary on-target ssDNA (OT-ssDNA) lar diagnostics, we tested its feasibility in infectious virus
and OT-dsDNA, and sgRNA-non-complementary non- detection. After diluting HPV dsDNAs with human gen-
target ssDNA (NT-ssDNA) and NT-dsDNA as activators omic DNAs, we showed that CDetection could identify
separately, we found OT-ssDNA and OT-dsDNA were able the diluted substrate at the sub-attomolar magnitude
to trigger AaCas12b to cleave the FQ reporter, though OT- (0.1 åM) (Additional file 1: Figure S6a). The high sensi-
dsDNA activator was less efficient than OT-ssDNA (Add- tivity and longevity of AaCas12b in human plasma [7]
itional file 1: Figure S3c and Additional file 2: Table S1). then urged us to test the application of CDetection in
We next tested the specificity of trans-cleavage activation cell-free DNA (cfDNA)-based non-invasive diagnoses.
using either ssDNA or dsDNA activators bearing various Prior SHERLOCK strategy has achieved cfDNA detec-
mismatches (Additional file 2: Table S1). The results tion in a simulated condition [2] and using isolated sam-
showed that the PAM sequence is critical for the dsDNA ples [3], but the Cas13-based SHERLOCK requires a T7
activator to trigger AaCas12b trans-cleavage activity and is in vitro transcription step to achieve DNA detection. To
dispensable for the ssDNA activator, and mismatches in indicate the advantage of CDetection platform in cfDNA
dsDNA rather than ssDNA would impede or even abolish detection, we diluted HPV dsDNAs into human plasma
the trans-cleavage activity of AaCas12b (Fig. 1a, b). Next, and examined the sensitivity of this newly established
we determined the detection sensitivity of AaCas12b- method. The results showed that CDetection could dir-
sgRNA-activator system. Without pre-amplification, ectly detect the existence of HPV DNAs in human
AaCas12b did not produce a detectable signal when sub- plasma at the concentration of 1 åM (Fig. 1f and Add-
strate concentrations were lower than 1.6 nM for ssDNA itional file 1: Figure S6b), indicating the possibility to
activator and lower than 8 nM for dsDNA activator, re- rapidly detect infectious viruses in only one drop of
spectively (Additional file 1: Figure S4a). Since dsDNA acti- blood. Together, these results demonstrate that CDetec-
vator possessed a higher specificity (Fig. 1a, b), we tion is able to detect target DNA sequences with higher
engineered the AaCas12b-sgRNA-dsDNA-activator system specificity and sensitivity.
as a Cas12b-based DNA detection (CDetection) platform. To further expand the applications of CDetection in ac-
To explore the sensitivity of CDetection, we further synthe- curate diagnostics, we designed experiments using three
sized one Cauliflower mosaic virus (CaMV) dsDNA and targeting sgRNA and corresponding dsDNA activators
two pairs of human papillomavirus (HPV) dsDNAs (on- versus off-activator) to identify six common human
(HPV16 and HPV18, site 1 and site 2) [4] as activator for ABO alleles [9] (Additional file 2: Table S1). Theoretically,
detection reaction (Additional file 2: Table S1). When the CDetection carrying each of the three sgRNAs can identify
input concentration of activator is equal or greater than 10 O01, O02/O03, and B101, respectively. And if no fluores-
nM, CDetection could not only produce detectable signals cent signal can be detected for all sgRNAs, the allele should
Teng et al. Genome Biology (2019) 20:132 Page 3 of 7

a b

c d

e f

Fig. 1 Specificity and sensitivity of Cas12b-mediated DNA detection. a, b (Upper) Schematics showing mismatched position in the targeting
sequence. PAM sequences are colored in red, protospacers are colored in blue, SNPs are colored in pink. (Lower) Characterization of trans-cleavage
activity of AaCas12b using ssDNA or dsDNA activator with indicated a single mismatch or b continuous mismatches. Error bars indicate standard
errors of the mean (s.e.m.)., n = 3. RFU, relative fluorescence units; PT, perfect target; mPAM, mutated PAM. Two-tailed Student’s t test is used for
significance analysis between each mutated sample and the PT sample. c Comparison of the specificity among AaCas12b, PrCas12a, and LbCas12a in
dsDNA distinguishability using synthetic HPV16 activator (site 2). Error bars indicate s.e.m., n = 3. d Comparison of trans-cleavage activity and pre-
amplification enhanced trans-cleavage activity (CDetection) for AaCas12b using dsDNA activator. AaCas12b is incubated with a sgRNA targeting a
synthetic dsDNA 1. Error bars indicate s.e.m., n = 3. RPA, recombinase polymerase amplification. Two-tailed Student’s t test is used for significance
analysis between the two samples in each group. e Maximum fluorescence signal obtained from AaCas12b-, PrCas12a-, and LbCas12a-based DNA
detection with RPA pre-amplification. Cas12 proteins are incubated with their cognate guide RNA (gRNA) targeting a synthetic HPV16 dsDNA (site 2)
mixed with background genome. Error bars indicate s.e.m., n = 3. Two-tailed Student’s t test is used for significance analysis among each other in the
group. f Fluorescence timecourses obtained from AaCas12b- and LbCas12a-based DNA detection with RPA pre-amplification. Cas12 is incubated with
a cognate gRNA targeting a synthetic HPV16 dsDNA (site 2) diluted in human plasma with a final concentration of 10−18 M. Error bars indicate s.e.m.,
n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance

be A101/A201 (Additional file 1: Figure S7a). Our results state of two similar targets differed by a single base into the
showed that CDetection failed to distinguish different ABO distinguishable state (Additional file 1: Figure S7c). To
alleles as it produced indistinguishable fluorescent signals elucidate the feasibility of this enhanced CDetection, we re-
between the on- and off-dsDNA activator groups (Add- peated the ABO blood genotyping test. As our results
itional file 1: Figure S7b). To improve the specificity of indicated, CDetection with tgRNAs could determine the
CDetection, we introduced tuned guide RNA (tgRNA) blood types with high accuracy in an antigen-antibody-
containing a single-nucleotide mismatch in the spacer independent and blood-free manner (Fig. 2b and Add-
sequence [10], which transforms the undistinguishable itional file 1: Figure S6c).
Teng et al. Genome Biology (2019) 20:132 Page 4 of 7

Disease-associated point mutations were usually detected tgRNA exhibited mismatch tolerance pattern (Add-
by sequencing and probe detection [11]. However, sequen- itional file 1: Figure S9a, b) consistent with above results
cing is costly and time-consuming, and its sensitivity is (Fig. 1a, b) and previous studies [4]. To eliminate the
dependent on sequencing depth, while probe-based off-target-associated undesired false positive of CDetec-
methods perform poor sensitivity, particularly for single- tion, we indicated that a specific RPA pre-amplification
nucleotide variation [11]. We sought to use CDetection to was necessary (Additional file 1: Figure S9c, d).
detect single-base mutations in the human genome. We se- Taken together, we establish a CDetection method based
lected the cancer-related TP53 856G>A mutation to test on the non-canonical ssDNA trans-cleavage properties of
the feasibility. The results showed that CDetection could the Cas12b nuclease triggered by targeted dsDNA, which
accurately distinguish the point mutated allele from the enables DNA detection with sub-attomolar sensitivity. The
wild-type allele using selected tgRNAs (Additional file 1: CDetection strategy can perform better than the Cas12a-
Figure S8a and Additional file 2: Table S1). Furthermore, DETECTR in sensitivity assay. Meanwhile, the CDetection
we applied CDetection platform in detecting two hotspots is able to detect dsDNA directly without an extra step re-
in breast cancer-related BRCA1 gene (3232A>G and 353 quiring in Cas13-SHERLOCK or Cas14-DETECTR plat-
7A>G) [12]. CDetection with selected tgRNAs (tgRNA- form [2, 3, 5, 6]. Moreover, we demonstrate that
3232-1 and tgRNA-3537-4) performed excellently to dis- CDetection enables to achieve single-nucleotide sensitivity
criminate point mutations while sgRNAs could hardly sup- in DNA detection coupled with optimized tgRNA, which
port point mutation detection (Fig. 2c, d, Additional file 1: has been applied extendedly in other CRISPR-based detec-
Figure S8b, c and Additional file 2: Table S1). As a proof of tion platforms [2, 6, 24]. As a proof of concept, we use
principle, we aimed to detect the human BRCA1 gene, mock samples composed of diluted synthetic DNA and hu-
which contains a SNP (3232A>G) responsible for breast man plasma to demonstrate the capability of CDetection to
cancer [12]. We amplified the BRCA1 wild-type allele and perform direct cfDNA detection. However, the detection of
3232A>G allele from DNAs in human breast cancer cell clinical specimens may face bigger challenges due to the
lines MDA-MB-468 (ATCC® HTB-132™) and SUM1315 poor purity and integrity. We anticipate that CDetection
MO2 (BioIVT) respectively, using the RPA amplification will be used for the detection of cfDNA in clinical speci-
approach. And tgRNA-programmed CDetection achieved mens in the future. Besides, the intrinsic features of
to discriminate the SNP with strong fluorescent signal for CRISPR-Cas systems possessing mismatch tolerance [7,
3232A>G SNP while with near-background signal for the 14–22] and our results indicate that RPA pre-amplification
wild-type allele (Fig. 2d and Additional file 1: Figure S8d) process is necessary to produce strong signal and avoid
with allelic fractions as low as 1% (Additional file 1: Figure false positive for CRISPR-Cas-based nucleic acid detection
S8e). Meanwhile, the results also indicated that the targeted platforms. During the preparation process of this manu-
genomic DNAs without pre-amplification are not able to script, an independent study reported a less sensitive nu-
unleash the trans-cleavage activity of Cas12b enzyme (Add- cleic acid detection platform [25] using previously reported
itional file 1: Figure S8d). And the reason is possibly that AacCas12b protein [4] with loop-mediated isothermal
Cas12b-RNA remains tightly bound to the cleave genomic amplification (LAMP). In their works, the authors generate
DNAs, thus failing to enable the ssDNA substrate access ssDNA using asymmetric PCR or dsDNA using LAMP as
and turnover [4, 13]. Furthermore, to mock the early clin- an activator to perform SNP discrimination in the tested
ical detection of primary diseases using cfDNA by CDetec- site [25], which is different from our strategy in principle.
tion, we diluted BRCA1 3232A>G dsDNAs into human Our work, together with the AacCas12b-based work [25],
plasma. Our results demonstrated that CDetection could demonstrates that Cas12b-based nucleic acid detection
achieve point mutation detection at the single-base reso- method should be a feasible platform for molecular diag-
lution (Fig. 2e and Additional file 1: Figure S8f). These re- nostics. This rapid and price-competitive DNA detection
sults demonstrate that CDetection with tgRNAs is able to platform may have wide applications in molecular diagnos-
achieve rapid DNA detection at the single-base resolution tics and clinical research (Additional file 1: Figure S10). For
in clinical research. instance, we estimate that over 20,000 known human
The intrinsic features of base-pairing between spacer disease-associated point mutations could be detected by
sequences and the corresponding target sequences make CDetection (Additional file 1: Figure S11).
CRISPR-Cas systems exist potential off-target effect as
previous works indicated [7, 14–22]. So we assessed the Methods
possible false positive of CDetection platform using po- Protein purification
tential off-targets containing 1 to 3 mismatches in their SpCas9 and LbCas12a proteins were commercially pur-
protospacer sequences predicted from targeting sgRNAs chased (NEB). AaCas12b, ArCas12a, HkCas12a, and
or tgRNAs using Cas-OFFinder [23] (Additional file 2: PrCas12a proteins were purified according to our previous
Table S1). The results showed that both sgRNA and report [7]. Briefly, BPK2014-Cas12-His10 plasmid was
Teng et al. Genome Biology (2019) 20:132 Page 5 of 7

b c

d e

Fig. 2 (See legend on next page.)

Teng et al. Genome Biology (2019) 20:132 Page 6 of 7

(See figure on previous page.)

Fig. 2 Broad applications for CDetection. a (Left) Schematics showing the sequence variation within ABO genes and corresponding sgRNAs and
tgRNAs. PAM sequences are colored in red, protospacers are colored in blue, SNPs are colored in pink, and base substitution in tgRNAs are
colored in orange. (Right) CDetection combined with tgRNA achieve ABO blood genotyping detection with a single-nucleotide-resolution
specificity. Error bars indicate standard errors of the mean (s.e.m.), n = 3. RFU, relative fluorescence units; tgRNA, tuned guide RNA. Two-tailed
Student’s t test is used for significance analysis among each other in the group. b, c (Upper) Schematics showing the sequence variation within
BRCA1 gene and targeting sgRNA and tgRNAs. (Lower) Maximum fluorescence signal showing the specificity of CDetection without RPA for
human BRCA1 b 3232A>G and c 3537A>G mutation detection using sgRNA and tgRNA. Error bars indicate s.e.m., n = 3. Two-tailed Student’s t test
is used for significance analysis among each other in the group. d Fluorescence timecourses showing the sensitivity and specificity of CDetection
with RPA for human BRCA1 3232A>G mutation detection using tgRNA (3232-1). BRCA1 wild-type genomic DNAs or 3232A>G mutant genomic
DNAs are extracted from cell lines. Error bars indicate s.e.m., n = 3. e Fluorescence timecourses showing the sensitivity and specificity of
CDetection with RPA for human BRCA1 3232A>G mutation detection using tgRNA (3232-1). BRCA1 wild-type or 3232A>G dsDNAs are diluted in
human plasma with a final concentration of 10−18 M. Error bars indicate s.e.m., n = 3. **P < 0.01; ****P < 0.0001; ns, no significance

transformed into E. coli strain BL21 (λDE3) and protein ex- Corning® 384-well Polystyrene NBS Microplate. Reactions
pression was induced with 0.5 mM IPTG at 16 °C for 16 h. were incubated at 37 °C for indicated timecourse in a fluor-
Cell pellets were harvested and lysed, followed by washing escence plate reader (BioTek Synergy 4) with fluorescent
and elution using His60 Ni Superflow Resin (Takara). Puri- kinetics measured every 5 min (λex = 485 nm; λem = 528 nm,
fied Cas12 proteins were dialyzed, concentrated, and finally transmission gain = 61). The fluorescence results were ana-
quantitated using the BCA Protein Assay Kit (Thermo lyzed by SigmaPlot software.
Fisher).
Recombinase polymerase amplification (RPA) reactions
Nucleic acid preparation
Recombinase polymerase amplification (RPA) reactions
DNA oligos were commercially purchased (Genscript).
were proceeded using TwistAmp Basic (TwistDx) ac-
Double-stranded DNA activators were obtained by PCR
cording to the manufacturer’s protocol. The 50-μl RPA
reaction and purified using Oligo Clean & Concentrator
reaction system containing varying amounts of DNA in-
Kit (ZYMO Research). In order to avoid false positive re-
put was incubated in 37 °C for 10 min. Sixteen microli-
sults caused by target strand (TS) ssDNA, we took non-
ters of RPA product was directly transferred to the 20-μl
target strand (NTS) ssDNA as PCR template. PCR
detection assay as above mentioned.
primers and ssDNA templates were listed in Add-
itional file 2: Table S1.
Guide RNAs (gRNAs: sgRNAs or crRNAs) were in Statistical analysis
vitro transcribed using HiScribe™ T7 High Yield RNA Syn- All the replicate experiments in this study consisted of
thesis Kit (NEB) and purified using MicroElute RNA Clean three repeats. Uncertainties in the reported mean values
Up Kit (Omega). Targeting gRNAs containing a T7 pro- are indicated as standard errors of the mean (s.e.m.). Stat-
moter were used as transcription template. AaCas12b istical analysis was performed in SigmaPlot (version 12.0).
sgRNA (AasgRNA) and SpCas9 sgRNA templates for in
vitro RNA transcription were PCR amplified using primers Additional files
bearing a T7 promoter (Additional file 2: Table S1). Cas12a
crRNA templates were obtained by annealing oligos con- Additional file 1: Figure S1. Non-specific DNase activity is conserved
taining a T7 promoter (Additional file 2: Table S1). across Cas12 proteins. Figure S2. The RuvC domain is responsible for
ssDNA trans-cleavage. Figure S3. Preference for Cas12b-mediated trans-
Background genomic DNAs used in indicated reac- activated cleavage of non-specific ssDNA. Figure S4. Sensitivity and spe-
tions were crudely purified from human embryonic kid- cificity of AaCas12b-mediated DNA detection. Figure S5. Comparison of
ney 293T cells using Mouse Direct PCR Kit (Bimake). sensitivity of AaCas12b- and LbCas12a-based DNA detection. Figure S6.
CDetection achieves sub-attomolar sensitivity in DNA detection. Figure
To mimic the cell-free DNA (cfDNA), dsDNAs were di- S7. Develop CDetection platform. Figure S8. Accurate DNA detection
luted into human plasma (Thermo Fisher) at indicated using CDetection platform. Figure S9. Potential off-target analysis of
concentrations. CDetection. Figure S10. Rapid and accurate diagnostic applications of
CDetection. Figure S11. Genetic variants from ClinVar that, in principle,
can be detected by CDetection platform. (PDF 2218 kb)
Fluorophore quencher (FQ)-labeled reporter assays Additional file 2: Table S1. Nucleic acids used in this study. Table S2.
Detection assays were performed with 30 nM Cas12, 36 The components of buffers tested for Cas12b-mediated DNA detection in
nM gRNA, 40 nM activator (unless otherwise indicated) this study. (PDF 293 kb)
mixed in 40 ng background genomic DNAs (in indicated Additional file 3: Review history. (DOCX 1323 kb)
reaction), 200 nM custom synthesized homopolymer
ssDNA FQ reporter (Additional file 2: Table S1) and NEB- Review history
uffer™ 2 (unless otherwise indicated) in a 20-μl reaction in a The review history is available as Additional file 3.
Teng et al. Genome Biology (2019) 20:132 Page 7 of 7

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1
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