Low cycle number multiplex PCR : A novel strategy for the construction of amplicon libraries for next-generation sequencing
© 2024 Wiley‐VCH GmbH..
Multiplex PCR is a critical step when preparing amplicon library for next-generation sequencing. However, there are several challenges related to multiplex PCR including poor uniformity, nonspecific amplification, and primer-dimers. To address these issues, we propose a novel solution strategy that involves using a low cycle number (<10 cycles) in multiplex PCR and then employing carrier DNAs and magnetic beads for the selection of targeted products. This technique improves the amplicon uniformity while also reducing primer-dimers and PCR artifacts. To evaluate our technique, we initially utilized 120 DNA fragments from mouse genome containing single nucleotide polymorphism (SNP) sites. Sequencing results demonstrated that with only 7 cycles of multiplex PCR, 95.8% of the targeted SNP sites were mapped, with a coverage of at least 1×. The average sequencing depth of all amplicons was 1705.79 ± 1205.30×; 87% of them reached a coverage depth that exceeded 0.2-fold of the average sequencing depth. Our method had a greater uniformity (87%) when compared to Hi-Plex PCR (53.3%). Furthermore, we validated our strategy by randomly selecting 90 primer pairs twice from the initial set of 120 primer-pairs. Next, we used the same protocol to prepare amplicon libraries. The two groups had an average sequencing depth of 1013.30 ± 585.57× and 219.10 ± 158.27×, respectively; over 84% of the amplicons had a sequencing depth that exceeded 0.2-fold of average depth. These results suggest that the use of a low cycle number in multiplex PCR is a cost-effective and efficient approach for the preparation of amplicon libraries.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2024 |
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| Enthalten in: |
Electrophoresis - (2024) vom: 27. März |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Lu, Meng [VerfasserIn] |
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| Links: |
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| Themen: |
Amplicon library |
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| Anmerkungen: |
Date Revised 27.03.2024 published: Print-Electronic Citation Status Publisher |
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| doi: |
10.1002/elps.202300160 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Multiplex PCR is a critical step when preparing amplicon library for next-generation sequencing. However, there are several challenges related to multiplex PCR including poor uniformity, nonspecific amplification, and primer-dimers. To address these issues, we propose a novel solution strategy that involves using a low cycle number (<10 cycles) in multiplex PCR and then employing carrier DNAs and magnetic beads for the selection of targeted products. This technique improves the amplicon uniformity while also reducing primer-dimers and PCR artifacts. To evaluate our technique, we initially utilized 120 DNA fragments from mouse genome containing single nucleotide polymorphism (SNP) sites. Sequencing results demonstrated that with only 7 cycles of multiplex PCR, 95.8% of the targeted SNP sites were mapped, with a coverage of at least 1×. The average sequencing depth of all amplicons was 1705.79 ± 1205.30×; 87% of them reached a coverage depth that exceeded 0.2-fold of the average sequencing depth. Our method had a greater uniformity (87%) when compared to Hi-Plex PCR (53.3%). Furthermore, we validated our strategy by randomly selecting 90 primer pairs twice from the initial set of 120 primer-pairs. Next, we used the same protocol to prepare amplicon libraries. The two groups had an average sequencing depth of 1013.30 ± 585.57× and 219.10 ± 158.27×, respectively; over 84% of the amplicons had a sequencing depth that exceeded 0.2-fold of average depth. These results suggest that the use of a low cycle number in multiplex PCR is a cost-effective and efficient approach for the preparation of amplicon libraries | ||
| 650 | 4 | |a Journal Article | |
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| 650 | 4 | |a low number of cycles | |
| 650 | 4 | |a multiplex PCR | |
| 650 | 4 | |a next‐generation sequencing | |
| 700 | 1 | |a Sun, Xiuxiu |e verfasserin |4 aut | |
| 700 | 1 | |a Zhao, Yuxin |e verfasserin |4 aut | |
| 700 | 1 | |a Zheng, Linlin |e verfasserin |4 aut | |
| 700 | 1 | |a Lin, Junjie |e verfasserin |4 aut | |
| 700 | 1 | |a Tang, Chen |e verfasserin |4 aut | |
| 700 | 1 | |a Chao, Kaiyue |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Ye |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Kai |e verfasserin |4 aut | |
| 700 | 1 | |a Zhou, Yuxun |e verfasserin |4 aut | |
| 700 | 1 | |a Xiao, Junhua |e verfasserin |4 aut | |
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