A method for reliable detection of genomic point mutations based on single-cell target-sequencing
Received date: 2020-04-26
Revised date: 2020-06-02
Online published: 2020-06-15
Supported by
Supported by Shanghai Jiao Tong University Scientific and Technological Innovation Funds No(2019TPA09);the National Natural Science Foundation of China No(81802806)
The analysis of genomic point mutations is one of the research strategies to explore the clonal evolution of tumor cells. At present, clonal evolution of tumor cells is mainly determined by bulk sampling and sequencing of different sections of the tumor. Since this approach analyzes a mixture of different cell types, it may not accurately represent the clonal evolution of specific tumor cell populations and likely miss low frequency mutations, especially when the sequencing depths are not sufficient. To address this issue, we have developed a strategy to analyze genomic point mutations from prostate basal cell carcinoma (BCC) tissues at single-cell resolution. Firstly, we optimized the single-cell whole genome amplification procedure with HepG2 cells. Then the single cells from BCC tissue were captured by a microfluidic chip of Fluidigm and processed for whole-genome amplification. Both SCUBE3 and MST1L genomic mutations were obtained by whole exome sequencing. Finally, we examined the genomic mutations through single-cell targeted amplification and Sanger sequencing. The established method successfully reconfirmed the mutations of SCUBE3 and MST1L in BCC at single cell level. The strategy established in this study could provide a useful tool for determining the clonal evolution of tumor cells based on genomic mutations at single-cell resolution.
Key words： tumor cell; mutation; clonal evolution; single-cell
Linan Zhao, Na Wang, Guoliang Yang, Xianbin Su, Zeguang Han . A method for reliable detection of genomic point mutations based on single-cell target-sequencing[J]. Hereditas(Beijing), 2020 , 42(7) : 703 -712 . DOI: 10.16288/j.yczz.20-046
| [1] | Terry S, Buart S, Chouaib S . Hypoxic stress-induced tumor and immune plasticity, suppression, and impact on tumor heterogeneity. Front Immunol, 2017,8:1625. |
| [2] | Junttila MR, de Sauvage FJ . Influence of tumour micro- environment heterogeneity on therapeutic response. Nature, 2013,501(7467):346-354. |
| [3] | Greaves M, Maley CC . Clonal evolution in cancer. Nature, 2012,481(7381):306-313. |
| [4] | Marusyk A, Almendro V, Polyak K . Intra-tumour heterogeneity: a looking glass for cancer? Nat Rev Cancer, 2012,12(5):323-334. |
| [5] | Gerlinger M, Rowan AJ, Horswell S, Math M, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S, Mcdonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA, Swanton C . Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med, 2012,366(10):883-892. |
| [6] | Brunner SF, Roberts ND, Wylie LA, Moore L, Aitken SJ, Davies SE, Sanders MA, Ellis P, Alder C, Hooks Y, Abascal F, Stratton MR, Martincorena I, Hoare M, Campbell PJ . Somatic mutations and clonal dynamics in healthy and cirrhotic human liver. Nature, 2019,574(7779):538-542. |
| [7] | Zong CH, Lu SJ, Chapman AR, Xie XS . Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science, 2012,338(6114):1622-1626. |
| [8] | Gawad C, Koh W, Quake SR . Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics. Proc Natl Acad Sci USA, 2014,111(50):17947-17952. |
| [9] | Hou Y, Song LT, Zhu P, Zhang B, Tao Y, Xu X, Li FQ, Wu K, Liang J, Shao D, Wu HJ, Ye XF, Ye C, Wu RH, Jian M, Chen Y, Xie W, Zhang RR, Chen L, Liu X, Yao XT, Zheng HC, Yu C, Li QB, Gong ZL, Mao M, Yang X, Yang L, Li JX, Wang W, Lu ZH, Gu N, Laurie G, Bolund L, Kristiansen K, Wang J, Yang HM, Li YR, Zhang XQ, Wang J . Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell, 2012,148(5):873-885. |
| [10] | Xu X, Hou Y, Yin XY, Bao L, Tang AF, Song TL, Li FQ, Tsang S, Wu K, Wu HJ, He WM, Zeng L, Xing MJ, Wu RH, Jiang H, Liu X, Cao DD, Guo GW, Hu XD, Gui YT, Li ZS, Xie WY, Sun XJ, Shi M, Cai ZM, Wang B, Zhong MM, Li JX, Lu ZH, Gu N, Zhang XQ, Goodman L, Bolund L, Wang J, Yang HM, Kristiansen K, Dean M, Li YR, Wang J . Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor. Cell, 2012,148(5):886-895. |
| [11] | Su XB, Long Q, Bo JJ, Shi Y, Zhao LN, Lin YX, Luo Q, Ghazanfar S, Zhang C, Liu Q, Wang L, He KY, He J, Cui XF, Yang JYH, Han ZG, Yang GL, Sha JJ . Mutational and transcriptomic landscapes of a rare human prostate basal cell carcinoma. Prostate, 2020,80(6):508-517. |
| [12] | Huang J, Deng Q, Wang Q, Li KY, Dai JH, Li N, Zhu ZD, Zhou B, Liu XY, Liu RF, Fei QL, Chen H, Cai B, Zhou BP, Xiao HS, Qin LX, Han ZG . Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma. Nat Genet, 2012,44(10):1117-1121. |
| [13] | Gunderson KL, Steemers FJ, Lee G, Mendoza LG, Chee MS . A genome-wide scalable SNP genotyping assay using microarray technology. Nat Genet, 2005,37(5):549-554. |
| [14] | Wang Y, Waters J, Leung ML, Unruh A, Roh W, Shi XQ, Chen K, Scheet P, Vattathil S, Liang H, Multani A, Zhang H, Zhao R, Michor F, Meric-Bernstam F, Navin NE . Clonal evolution in breast cancer revealed by single nucleus genome sequencing. Nature, 2014,512(7513):155-160. |
| [15] | Lasken RS . Genomic DNA amplification by the multiple displacement amplification (MDA) method. Biochem Soc Trans, 2009,37(Pt 2):450-453. |
| [16] | Gefrides LA, Powell MC, Donley MA, Kahn R . UV irradiation and autoclave treatment for elimination of contaminating DNA from laboratory consumables. Forensic Sci Int Genet, 2010,4(2):89-94. |
| [17] | Archer E, Allen H, Hopwood A, Rowlands D . Validation of a dual cycle ethylene oxide treatment technique to remove DNA from consumables used in forensic laboratories. Forensic Sci Int Genet, 2010,4(4):239-243. |
| [18] | Bolger AM, Lohse M, Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 2014,30(15):2114-2120. |
| [19] | Li H, Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009,25(14):1754-1760. |
| [20] | Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R . The Sequence Alignment/Map format and SAMtools. Bioinformatics, 2009,25(16):2078-2079. |
| [21] | Feng RM, Zong YN, Cao SM, Xu RH . Current cancer situation in China: good or bad news from the 2018 Global Cancer Statistics? Cancer Commun (Lond), 2019,39(1):22. |
| [22] | L?hnemann D, K?ster J, Szczurek E, Mccarthy DJ, Hicks SC, Robinson MD, Vallejos CA, Campbell KR, Beerenwinkel N, Mahfouz A, Pinello L, Skums P, Stamatakis A, Attolini CS, Aparicio S, Baaijens J, Balvert M, Barbanson B, Cappuccio A, Corleone G, Dutilh BE, Florescu M, Guryev V, Holmer R, Jahn K, Lobo TJ, Keizer EM, Khatri I, Kielbasa SM, Korbel JO, Kozlov AM, Kuo TH, Lelieveldt B, Mandoiu II, Marioni JC, Marschall T, Molder F, Niknejad A, Raczkowski L, Reinders M, Ridder J, Saliba AE, Somarakis A, Stegle O, Theis FJ, Yang H, Zelikovsky A, Mchardy AC, Raphael B J, Shah SP, Sch?nhuth A . Eleven grand challenges in single-cell data science. Genome Biol, 2020,21(1):31. |
| [23] | Gawad C, Koh W, Quake SR . Single-cell genome sequencing: current state of the science. Nat Rev Genet, 2016,17(3):175-188. |
| [24] | Huang L, Ma F, Chapman A, Lu SJ, Xie XS . Single-cell whole-genome amplification and sequencing: methodology and applications. Annu Rev Genomics Hum Genet, 2015,16:79-102. |
| [25] | Yao YX, La YF, Di R, Liu QY, Hu WP, Wang XY, Chu MX . Comparison of different single cell whole genome amplification methods and MALBAC applications in assisted reproduction. Hereditas(Beijing), 2018,40(8):620-631. |
| [25] | 姚雅馨, 喇永富, 狄冉, 刘秋月, 胡文萍, 王翔宇, 储明星 . 不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用. 遗传, 2018,40(8):620-631. |
| [26] | Woyke T, Sczyrba A, Lee J, Rinke C, Tighe D, Clingenpeel S, Malmstrom R, Stepanauskas R, Cheng JF . Decontamination of MDA reagents for single cell whole genome amplification. PLoS One, 2011,6(10):e26161. |
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