A Microfluidic-Based SNP Genotyping Method for Hereditary Hearing

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A microfluidic-based SNP genotyping method for hereditary hearing-loss detection Ying Lu, Shan Chen, Li Wei, Lanhua Sun, Houming Liu, and Youchun Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00652 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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Analytical Chemistry

A microfluidic-based SNP genotyping method for hereditary hearing-loss detection Ying Lu, a# Shan Chen, b# Li Wei, c Lanhua Sun, c Houming Liu b and Youchun Xu a* a

Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084,

China. b

Laboratory of ShenZhen Third People’s Hospital, ShenZhen, GuangDong, 518112, China.

c

CapitalBio Technology, Beijing 101111, China.

#

These authors are co-first authors on this work.

*Correspondence should be addressed to Y.X. ([email protected]). Tel: (86)-10-62796071

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The genotyping of SNPs (single nucleotide polymorphisms) is a prerequisite for the analysis of many genetic diseases, including hereditary hearing-loss. However, the existed methods for SNP detection suffer from long detection period, tedious operation and a high risk of carryover contamination. To address these challenges, a microfluidic chip is constructed for rapid and efficient SNP genotyping by dividing the sample into many independent chambers for Kompetitive Allele Specific PCR in this study. Using this strategy, multiple detection can be easily accomplished and the challenge for the establishment of multiplex PCR is fundamentally overcame. The whole detection can be finished within 2 hours in a fully sealed manner with this method, which is quite simple compared to SNaPshot and MassArray. After the assessment of the basic performance, this chip was applied to screen 15 mutations, including SNPs and InDels (insertion-deletion markers), that can cover more than 80% cases of hereditary hearing-loss in China. Over 40 clinical samples were analyzed with this microfluidic chip for SNP genotyping, and the results are consistent with that obtained by Sanger sequencing, demonstrating its practicability and potential in the application of genetic diseases detection.

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Introduction： SNPs (single nucleotide polymorphisms) and InDels (insertion-deletion markers), the most common heritable variations in the human genome, have been extensively applied to the analysis of disease genetics, population genetics, forensic medicine, pharmacogenomics, and biodiversity as the third generation of genetic markers, and they will continuously play important roles in the development of life sciences.1-4 To facilitate relative researches, many methods were developed for SNPs/InDels detection, which can be generally divided into two types: 1) The gel-based methods with low throughput, including restriction enzyme fragment length polymorphic analysis (RFLP),5 single-strand conformational polymorphic analysis (SSCP),6 denaturing gradient gel electrophoresis (DGGE), conformation sensitive gel electrophoresis (CSGE),7 oligonucleotide link analysis (OLA), and allele-specific polymerase chain reaction analysis (ASP). These methods have the advantages of simple equipment requirements and low-costs, but limited by their tedious operations, long analysis periods and low throughputs.8-10 2) The high throughput methods follow the principle of allele-specific site hybridization (ASH), mass spectrometry (MS),11-12 denaturing high performance liquid chromatography (DHPLC),13 allele-specific site primer extension (ASPE), single base extension (SBCE) or target sequencing.14-16 For instance, the SNaPshotTM OpenArrayTM system

20-22

17-19

and TaqMan

have been used for high-throughput screening of SNPs,23-24 while the

GoldenGateTM platform can detect 1536 SNPs

25

in one time. These methods have significantly

improved throughputs, but highly rely on the costly instruments and need many samples to decrease cost on each sample. This limits their flexibility for small-scale analyses. Besides, these large-scale SNP genotyping methods need separate PCR step before detection, bringing the risk of carryover contaminate that may lead to false positive results.26-27 A common strategy to prevent carryover 3 / 26



contaminants is to perform the detection in separate rooms, minimize the steps of pipetting, and prevent the exposure of amplicons, which impede the popularization of molecular diagnosis. Combining microfluidic technology with SNP detection methodology offers a feasible strategy to overcome the issues including carryover contamination and flexibility for small-scale analyses, and many efforts have been made to construct microfluidic-based nucleic acid detection systems.28-31 For instance, the microfluidic TaqMan array card was used to identify ten drug resistance SNPs related to mycobacterium tuberculosis resistance to 9 main anti-tuberculosis drugs using sequence-specific probes and high-resolution melt analysis (HRM).23 However, HRM is the melt temperature variation analysis on a DNA fragment rather than on a single nucleotide site. Therefore, the length and base composition of pieces contributed somewhat to a reduction in repeatability.32-34 Recently, the integrated microfluidic chip for “sample-in-to-answer-out” detection have become the goals of many researchers, but the establishment of an ideal microfluidic system to automatically realize the whole procedures for nucleic acid extraction, amplification and detection is still a challenge.35-37 For instance, Choi et al.35 developed an integrated portable microarray gene analysis system which can facilitate rapid, accurate multiplex SNP genotyping to distinguish animal varieties by combining allele-specific PCR with a microarray chip. Although the integrated detection was accomplished in this study,35 the fabrication of this chip is complicated, limiting its disposable use due to high cost. Moreover, the establishment of a multiplex PCR system is a challenge owing to the intrinsic interference and competition of primer pairs. Overall, the need for rapid, accurate, contamination-free, cost-effective and flexible SNP detection of small-scale samples with low cost is still not satisfied. To approach this goal, a microfluidic chip was constructed to realize multiple SNP detections using Kompetitive Allele Specific PCR (KASP) for the first time. The chip possesses 112 reaction 4 / 26


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chambers and each reaction chamber was thermally sealed after sample distribution to eliminate the potential contamination of PCR products. The chip is made of polymer, and the volume of each reaction chamber is about 1.5 µL; thus the average cost of detection can be greatly reduced to as low as 0.1 dollars per SNP. To validate the practicability of our methods, the chip was applied to the detection of hearing-loss related mutations. Hearing-loss is one of the most common congenital disabilities, with the morbidity of 0.1%-0.3% in newborns.38-39 Approximately 50% of congenital hearing-loss is genetic, including 70% of cases of non-syndromic hearing-loss and around 30% of cases of syndromic hearing-loss.40 Currently, over 50 genetic hearing-loss genes have been cloned with over 100 relevant NHL sites identified (http://hereditaryhearingloss.org). Among these genes, the most common genes used for hearing-loss mutation screening are GJB2, SLC26A4, and several mitochondrial genes. In this study, 15 mutations in 4 genes were detected with our microfluidic chip in a convenient and fully sealed manner within 2 hours.

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Methods Preparation of template DNA and primers The DNA templates were extracted from blood and dried blood spots using a TIA Namp Blood DNA Kit (Beijing, China) and stored at -20°C until use. The primer pairs for SNPs/InDels detection were designed and synthesized by LGC (Shanghai, China). The PCR master-mix for KASP was also purchased from LGC. Chip fabrication The chip was designed by SolidWorks and inject molded with polypropylene (PP). As shown in Figure 1A, the size of the chip is 75 × 25 × 2 mm. There are 112 reaction chambers on a chip that are divided into four units, and each unit has an inlet/outlet for sample loading/venting. The infusing channel has a “sine” shape, and 28 reaction chambers are connected to the bottom of the sine-shaped infusing channel. Primer pairs (the final volume for each primer pair is 0.14 μL, including two forward primers (2.5 μM for each) and one reverse primer (0.5 μM) for SNPs/InDels detection, were pre-spotted into the reaction chambers of the chip manually or by an automatic Spotter (PersonalArrayer-16, CapitalBio, Beijing, China) at a humility of 55-70%. Then, the chip was sealed by a heat sealable PCR film (LGC) and stored at room temperature before using. The volume of each reaction chamber is about 1.5 µL while the volume of each “sine” shape is slightly bigger than it to ensure all the reaction chamber can be sufficiently filled after sample distribution. PCR program For on-chip PCR, the sample (40 μL) consisted of 8.6 μL of 1 × TE, 20 μL of 2 × KASP master mix, 11.4 μL of DNA template was loaded into the chip, and the chip was placed on a thermal cycler (CapitalBio) for PCR. The program for thermal cycling was set as follows: 37°C for 5 min; 95°C for 6 / 26


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15 min; 10 cycles of 95°C for 20 s, 61°C for 60 s (annealing 61°C to 55°C, decreasing 0.6°C per cycle); 29 cycles of 95°C for 20 s, 55°C for 60 s. Date analysis Once PCR finished, the chip was scanned with the LuxScan 10K-D fluorescence scanner (CapitalBio). The parameters of the scanner were determined and uniquely used after the calibration with the standard chip with fluorescence dyes provided by the manufacturer. The median intensities of the corresponding reaction chambers in FAM/HEX fluorescence channel were calculated by subtracting the median of background signal of the substrate of the chip. The genotyping criteria is as follow: 1) To exclude false positive signals generated by primer dimer, the median intensity of signal for an allele must be at least three times of that of the negative control before it can be considered as a positive signal. 2) The ratio value (median intensity in FAM channel / median intensity in HEX channel) to distinguish heterozygous and homozygous follows: when 0.5 < ratio < 2.0, the allele was judged to be heterozygous; when ratio ≤ 0.5 or ratio ≥ 2.0, the allele was homozygous. The selection of SNPs/InDels for hereditary hearing-loss detection The selection of SNPs/InDels for hereditary hearing-loss screening follows these principles: 1) The deafness genes were selected by consulting Haploview 4.2 and the HapMap website (http://hapmap.ncbi.nlm.nih.gov/) with more than one or two tags SNP located at each gene. 2) The minimum allele frequency of the chosen SNPs/InDels in Chinese population is higher than 0.1. 3) More than two research institutions have reported the selected SNP/InDel. Finally, 15 SNPs/InDels were selected from four deafness genes that reported in 21 Asian populations, as listed in Table S1.

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Chip performance evaluation For accuracy verification of the chip, forty samples were selected to detect 15 mutations and the results were compared with those generated by Sanger sequencing (Sangon, Shanghai, China). For sensitivity verification of the chip, fifteen positive samples were selected and the final amount of DNA template in the PCR mixture was separately diluted to 25, 20, 3 and 1 ng/µL. For stability verification of the chip, four samples were randomly selected and each sample was tested in quadruplicate.

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Results and discussion The detection process of the chip Compared to SNaPshot and MassArray, KASP has advantages in its simple and rapid detection procedure, but limited in the throughput. In this study, microfluidic technology is applied to overcome this drawback to build a KASP-based SNP genotyping method. Since only one mutation can be detected in a KASP reaction, the essential to realize high-throughput analysis on a chip is to divide the reaction mixture into many independent reaction units and each reaction unit should be fully sealed

to

eliminate

the

potential

cross-contamination.

To

accomplish

this

goal,

the

centrifugation-based sample distribution and thermal sealing are used. The whole detection procedure is illustrated in Fig. 1B-E, including sample distribution and sealing, PCR, fluorescence scanning, and extracted signal analysis. As shown in Fig. 1B, once the sample (template mixed with PCR master mix) was injected into the infusing channel (Fig. 1B1), the chip was placed on a motor and centrifuged to drive the liquid in the infusing channel into the corresponding reaction chamber below it (Fig. 1B2). After that, the chip was sealed by a hot metal block to physically isolate each reaction chamber (Fig. 1B3). This thermal sealing process can eliminate adverse effect induced by the evaporation and condensation of the liquid in the reaction chambers in the following PCR process (Fig. 1C). Once PCR finished, the chip was inserted into a fluorescence scanner and the fluorescence intensities of reaction chambers in FAM and HEX channels were obtained (Fig. 1D). These extracted fluorescence intensities were used to finally genotype the mutations (Fig. 1E). The whole process takes about 2 h in a fully sealed manner, which is superior to the current SNP genotyping methods, including MassArray and SNaPshot.

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Basic performance tests The chip constructed in this study has 112 reaction chambers, thus 112 mutations can be simultaneously detected on a chip at most in theory. To achieve accurate and uniform detection of these mutations, the consistent of the reaction conditions among different reaction chambers should be guaranteed. Two factors have been considered in our study. First, the uniformity of liquid distribution and amplification efficiency among different reaction chambers had been assessed by loading a complete PCR mixture with template, primer pair, and mater mix into the chip. After PCR, the fluorescence image of the chip was scanned, as shown in Fig. 2A. By extracting the fluorescence signal of each reaction chamber (Fig. 2B), we can generally conclude that the distribution and amplification efficiency of the chip is homogeneous because the CV (coefficient of variation) of fluorescence intensities in FAM/HEX channel of reaction chambers is less than 5%. Second, the tightness of reaction chamber after thermal sealing was evaluated by reverse centrifugation due to its importance in PCR process. As shown in Fig. 2C, we centrifuged the thermal-sealed chip from the reaction chamber to the infusing channel at 4000 rpm for 2 min, and no liquid leaking was observed. This result indicated our thermal sealing process can completely isolate the reaction chambers, preventing the fetal contamination issue that may existed for chip using. These two efforts make the accurate SNPs/InDels detection possible. Cross-contamination tests of the chip Since all the reaction chambers are connected by the infusing channels, the cross-contamination between adjacent reaction chambers was also evaluated even the physical isolation of the reaction chamber had been confirmed in Fig. 2C. We preloaded primer pair for the 1555A>G in the odd-numbered reaction chambers and the control primer pair in the even-numbered reaction chambers 10 / 26


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of the chip. Then, the sample with the SNP for 1555A>G (5 ng/µL) was mixed with master mix and loaded into the chip to finish the detection process. The fluorescence image of amplified chip was shown in Fig. 3A. The odd-numbered reaction chambers showed a strong signal compared the even-numbered reaction chambers (Fig. 3B). The extracted fluorescence intensities of odd- and even-numbered reaction chambers fit the criteria for positive and negative results, respectively. In addition, the amplicon in each reaction chamber was pipetted out by piercing the film of the chip and further examined by gel electrophoresis (Fig. 3C). Similar with Fig. 3A, strong signals were detected in the odd-numbered chambers, and no signals were found in the even-numbered chambers, demonstrating that no cross-contamination of primers occurred between adjacent reaction chambers during the reaction. The specificity, sensitivity and stability tests In this study, the chip-based SNP genotyping method was utilized in the detection of hereditary hearing-loss. Fifteen common mutations that covered over 80% of hereditary hearing-loss in China were chosen to construct a rapid screening tool for new born babies.41-43 The specificity and sensitivity of chip were carefully examined. For specificity tests, the chips were utilized to detect samples with pre-confirmed different mutations. The schematic diagram of primer pairs for all SNPs/InDels and the control on the chip is shown in Fig. 4A. Since 15 primer pairs were pre-spotted in the corresponding reaction chamber and samples with different SNPs were separately infused into the chip for detection, only the chambers containing corresponding primer pair would show positive signals in theory. As shown in Fig. 4B, the results were consistent with the expected signal configurations, indicating that the chip was able to accurately identify the samples with these 15 SNPs/InDels. The specific typing results of 15 11 / 26



SNPs/InDels are shown in Fig. S1. Moreover, the current chip design can be easily upgraded to detect more mutations, because all mutations were detected in separate reaction chambers without the intrinsic interference and competition of primer pairs. Actually, 23 mutations were chosen at the beginning (Fig. 4A), however, 8 mutations were eliminated because the difficulty in samples collection due to their ultra-low incidence rates. To assess the sensitivity, the samples with different concentrations were detected with the chip. The fluorescent images of the corresponding chambers for 15 SNPs/InDels were shown in Fig. 4C and the corresponding fluorescence intensities were extracted and plotted as Fig. S2. When the template concentration decreased to 1 ng/μL, some reaction chambers showed reduced fluorescence intensities that not reach the criteria for positive results (Fig. S2). Therefore, the limit of detections for these 15 SNPs/InDels can be preliminarily determined as 3 ng/μL. This LOD is enough for the detection of dried blood spot and finger-prick blood. The stability of the chip for the detection of these 15 SNPs/InDels was also evaluated. The corresponding primer pairs were pre-loaded on a chip, and each primer pair was repeated four times. Three chips were amplified with the same DNA template. As shown in Fig. S3, there was no significant difference in the fluorescence intensities between these chips, proving the stability and repeatability of the chip. SNP/InDel genotyping of real samples To verify the practicability of the chip, 40 clinical samples were analyzed by our chip and confirmed by Sanger sequencing. The on-chip results of these samples were listed in Table 1, which has a 100% consistency with the results obtained by Sanger sequencing. The results got from these two methods show different patterns. As shown in Fig. 5, the fluorescence images of the amplified 12 / 26


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chips for sample No. 30 and No. 6 can be easily distinguished. The reaction chambers pre-spotted with the primer pair for 235delC in the chip for detecting sample No. 30 show a significant different color (yellow) compared to other reaction chambers (red), indicating heterozygous allele for sample No. 30. Similarly, the amplified chip for the detection of sample No. 6 show a significant green color in the reaction chambers preloaded with primer pair for 1555A>G, indicating the homozygous allele for sample No. 6. These results are more intuitive for new users compared to the below sequencing results (Fig. 5). The comparison results for all positive samples are shown in Fig. S4. Our chip-based genotyping method is rapid, easy to use, and fully sealed, which is superior to Sanger sequencing in some way and more suitable for rapid screening of hereditary hearing-loss.

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Conclusion A microfluidic system is proposed for rapid and accurate SNP genotyping and applied for hereditary hearing-loss screening for the first time. About 96 SNPs (exclude control) can be simultaneously detected on a chip within 2 hours. Fifteen common mutations of hereditary hearing-loss related genes, which can cover more than 80% cases of patients in China. The results obtained by our system is 100% consistent with results obtained by Sanger sequencing in 40 real samples, proving its practicability. The chip constructed in this study has following merits: 1) The risk of contamination is utmost eliminated because the chip can be disposably used in a fully sealed manner. Compared to the commonly used methods, such as SNaPshot and MassArray, the PCR products in our chip cannot be exposed to the environment; therefore the requirement of the operating environment is remarkably reduced. 2) The chip is easy to operate and the whole analysis time is as less as 2 hours. 3) The chip is flexible for small-scale samples detection. Since each unit of the chip is separated and all reaction chambers are physically isolated in PCR, the number of mutations and samples can be easily adjusted to fit the needs of different applications. 4) The optimization of amplification system on our chip is quite easy compared to SNaPshot and MassArray because multiplex PCR isn’t needed. Overall, the microfluidic-based SNP genotyping system constructed in this study provides a rapid, convenient and reagent/time-saving tool for the analysis of hereditary deafness.

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Acknowledgements This study was supported by the National Natural Science Foundation of China (31870853), the Science and Technology Program of ShenZhen, China (JCYJ20170307095303424), the Special Support Funds of ShenZhen for Introduced High-Level Medical Team, China (SZSM201412005), and the Beijing Lab Foundation.

Supporting Information The scatter plots of 15 SNP/InDels; plotted images of sensitivity tests; results of the stability tests; Sanger sequencing results of positive samples; list of 15 SNPs/InDels; information of 40 clinical samples.

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Figures

Figure 1. Workflow of the chip for SNPs/InDels genotyping. (A) The diagram of the chip. (B) The process for (1) sample loading, (2) sample distribution and (3) the thermal sealing of reaction chambers (The diagrams on the left, the images of the chip on the right). (C) The chip was placed on a thermal cycler for PCR. (D) The scanned fluorescence image of the chip after PCR. (E) The discrimination plot of the fluorescence signals extracted from reaction chambers.

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Figure 2. Basic performance tests of the chip. (A) The scanned fluorescence image of the chip after PCR. (B) Analysis of the average fluorescence intensities of each row of reaction chambers in FAM and HEX channels. (C) The image of the chip after reverse centrifugation (Left). The local enlarged images of the chip before and after reverse centrifugation (Middle and Right, respectively).

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Figure 3. Cross-contamination tests between the adjacent reaction chambers after PCR. Primer pairs were preloaded on the chip alternatively in the same row. (A) The fluorescence image shows that only the reaction chambers preloaded with corresponding primer pair have significant signals. In this image, “+” and “-” represent reaction chambers preloaded with and without corresponding primer pair, respectively. (B) The extracted fluorescence intensities of the corresponding reaction chambers. (C) Gel electrophoresis of the amplicons in the reaction chambers in a row of the chip. M represents the DNA marker.

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Figure 4. The specificity and sensitivity tests of the chip. (A) The layout of primer pairs and control pre-loaded on the chip. (B) The fluorescence images of the reaction chambers for the detection of the pre-validated samples. (C) The detection results for the samples with each mutation in different template concentrations.

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Figure 5. Typical detection results using our chip (Left) and Sanger sequencing (Right), respectively. The genotyping results for sample No. 30 and No. 6 are GJB2 c. 235delC and MT RNR1 m. 1555A>G, respectively. The mutation site is highlighted with gray color in the result obtained from Sanger sequencing.

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Tables Table 1. The on-chip detection results of 40 real samples. Samples

Genotypes

Samples

genotypes

1

WT/1229C>T

21

WT/235delC

2

WT/C. 235delC

22

-

3

-

23

WT/919-2A>G

4

-

24

-

5

-

25

-

6

1555A>G/1555A>G

26

-

7

-

27

WT/1975G>C

8

-

28

-

9

-

29

-

10

-

30

WT/235delC

11

WT/35delG

31

WT/176_191del16

12

-

32

-

13

1494C>T/1494C>T

33

-

14

-

34

WT/2027T>A

15

WT/299_300delAT

35

-

16

WT/1174A>T

36

WT/9191-2A>G, WT/1707+5G>A

17

-

37

-

18

-

38

WT/538C>T

19

WT/1226G>A

39

-

20

-

40

WT/2168A>G

Abbreviations: “WT” means wild type; “-” means no mutation has been detected.

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A Microfluidic-Based SNP Genotyping Method for Hereditary Hearing

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