單細胞測序

單細胞測序（英語：Single cell sequencing）採取優化的下一代 DNA測序技術（NGS）檢測單細胞的序列，可以獲得特定微環境下的細胞序列差異以方便研究其功能差異等^[1]。對個體細胞的DNA測序可以幫助我們了解例如在癌症中的小範圍細胞的變異；對其進行RNA測序可以幫助我們了解和鑑別不同的細胞類型與其表現的基因，對研究發育生物學等有較大裨益。^[2]

需要考慮的問題

分離單細胞

在全基因組擴增和測序前，有很多方法可以分離細胞個體，其中流式細胞術（FACS）較為常用。個體細胞可以用顯微操作來收集，^[3]^[4]這樣較為快捷而且成本較低，但是比較有技術難度，而且顯微鏡下對細胞的錯誤分類容易影響實驗結果。雷射捕獲顯微切割（英語：Laser-capture microdissection）技術（LCM）亦可以用來收集單細胞。但是儘管雷射捕獲顯微切割可以保留樣本細胞在組織中的空間位置，但是捕獲單細胞的時候容易連帶周圍細胞的物質。^[5]^[6]^[7] 高通量的細胞個體分離還可以使用微流控技術。微流控技術和流式細胞技術都能準確而自動地分離無偏樣本。但是，兩種方法都要求首先將細胞從其為環境中脫離，因此可能在RNA表達分析中造成對轉錄數據的干擾。^[8]^[9]

細胞分析數量

單細胞RNA測序

單細胞RNA測序也稱scRNA-Seq。通常而言，一般的組織細胞RNA-seq實驗會產生1000萬個讀數，如果一個基因的頻率超過50RPKM，即每百萬讀數中每千個鹼基中超過50個，這一基因即被認為有表達。泊松分佈下，1kb長的基因有超過500個讀數和4%的最小變異係數，即CV值；哺乳動物細胞通產含有200,000個mRNA，因此至少要用50個單細胞一起才能到達最小CV值；考慮到逆轉錄的效率和環境干擾等因素，為得到準確的表達和細胞種類的識別，需要使用的細胞數量實際要更多。^[5]

參見

參考資料

^ Eberwine J, Sul JY, Bartfai T, Kim J. The promise of single-cell sequencing. Nature Methods. January 2014, 11 (1): 25–7. PMID 24524134. doi:10.1038/nmeth.2769.
^ Pennisi E. Chronicling embryos, cell by cell, gene by gene. Science. April 2018, 360 (6387): 367. PMID 29700246. doi:10.1126/science.360.6387.367.
^ Zong C, Lu S, Chapman AR, Xie XS. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science. December 2012, 338 (6114): 1622–6. Bibcode:2012Sci...338.1622Z. PMC 3600412 . PMID 23258894. doi:10.1126/science.1229164.
^ Kurimoto K, Yabuta Y, Ohinata Y, Saitou M. Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nature Protocols. 2007, 2 (3): 739–52. PMID 17406636. doi:10.1038/nprot.2007.79.
^ ^5.0 ^5.1 "Shapiro E, Biezuner T, Linnarsson S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nature Reviews. Genetics. September 2013, 14 (9): 618–30. PMID 23897237. doi:10.1038/nrg3542. "
^ Yachida S, Jones S, Bozic I, Antal T, Leary R, Fu B, Kamiyama M, Hruban RH, Eshleman JR, Nowak MA, Velculescu VE, Kinzler KW, Vogelstein B, Iacobuzio-Donahue CA. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature. October 2010, 467 (7319): 1114–7. Bibcode:2010Natur.467.1114Y. PMC 3148940 . PMID 20981102. doi:10.1038/nature09515.
^ Frumkin D, Wasserstrom A, Itzkovitz S, Harmelin A, Rechavi G, Shapiro E. Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues. BMC Biotechnology. February 2008, 8 (17): 17. PMC 2266725 . PMID 18284708. doi:10.1186/1472-6750-8-17.
^ Dalerba P, Kalisky T, Sahoo D, Rajendran PS, Rothenberg ME, Leyrat AA, Sim S, Okamoto J, Johnston DM, Qian D, Zabala M, Bueno J, Neff NF, Wang J, Shelton AA, Visser B, Hisamori S, Shimono Y, van de Wetering M, Clevers H, Clarke MF, Quake SR. Single-cell dissection of transcriptional heterogeneity in human colon tumors. Nature Biotechnology. November 2011, 29 (12): 1120–7. PMC 3237928 . PMID 22081019. doi:10.1038/nbt.2038.
^ White AK, VanInsberghe M, Petriv OI, Hamidi M, Sikorski D, Marra MA, Piret J, Aparicio S, Hansen CL. High-throughput microfluidic single-cell RT-qPCR. Proceedings of the National Academy of Sciences of the United States of America. August 2011, 108 (34): 13999–4004. Bibcode:2011PNAS..10813999W. PMC 3161570 . PMID 21808033. doi:10.1073/pnas.1019446108.

[Eberwine-1] Eberwine J, Sul JY, Bartfai T, Kim J. The promise of single-cell sequencing. Nature Methods. January 2014, 11 (1): 25–7. PMID 24524134. doi:10.1038/nmeth.2769.

[ReferenceA-2] Pennisi E. Chronicling embryos, cell by cell, gene by gene. Science. April 2018, 360 (6387): 367. PMID 29700246. doi:10.1126/science.360.6387.367.

[3] Zong C, Lu S, Chapman AR, Xie XS. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science. December 2012, 338 (6114): 1622–6. Bibcode:2012Sci...338.1622Z. PMC 3600412 . PMID 23258894. doi:10.1126/science.1229164.

[4] Kurimoto K, Yabuta Y, Ohinata Y, Saitou M. Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nature Protocols. 2007, 2 (3): 739–52. PMID 17406636. doi:10.1038/nprot.2007.79.

[Shapiro-5] 5.0 ^5.1 "Shapiro E, Biezuner T, Linnarsson S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nature Reviews. Genetics. September 2013, 14 (9): 618–30. PMID 23897237. doi:10.1038/nrg3542. "

[6] Yachida S, Jones S, Bozic I, Antal T, Leary R, Fu B, Kamiyama M, Hruban RH, Eshleman JR, Nowak MA, Velculescu VE, Kinzler KW, Vogelstein B, Iacobuzio-Donahue CA. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature. October 2010, 467 (7319): 1114–7. Bibcode:2010Natur.467.1114Y. PMC 3148940 . PMID 20981102. doi:10.1038/nature09515.

[7] Frumkin D, Wasserstrom A, Itzkovitz S, Harmelin A, Rechavi G, Shapiro E. Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues. BMC Biotechnology. February 2008, 8 (17): 17. PMC 2266725 . PMID 18284708. doi:10.1186/1472-6750-8-17.

[8] Dalerba P, Kalisky T, Sahoo D, Rajendran PS, Rothenberg ME, Leyrat AA, Sim S, Okamoto J, Johnston DM, Qian D, Zabala M, Bueno J, Neff NF, Wang J, Shelton AA, Visser B, Hisamori S, Shimono Y, van de Wetering M, Clevers H, Clarke MF, Quake SR. Single-cell dissection of transcriptional heterogeneity in human colon tumors. Nature Biotechnology. November 2011, 29 (12): 1120–7. PMC 3237928 . PMID 22081019. doi:10.1038/nbt.2038.

[9] White AK, VanInsberghe M, Petriv OI, Hamidi M, Sikorski D, Marra MA, Piret J, Aparicio S, Hansen CL. High-throughput microfluidic single-cell RT-qPCR. Proceedings of the National Academy of Sciences of the United States of America. August 2011, 108 (34): 13999–4004. Bibcode:2011PNAS..10813999W. PMC 3161570 . PMID 21808033. doi:10.1073/pnas.1019446108.

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