生物
大规模并行测序
转座酶
寡核苷酸
单细胞测序
DNA测序
计算生物学
杂交测序
巨量平行
霰弹枪测序
基因组文库
遗传学
计算机科学
DNA
DNA测序器
转座因子
基序列
基因
并行计算
基因组
外显子组测序
突变
作者
Simone Picelli,Åsa K. Björklund,Björn Reinius,Sven Sagasser,Gösta Winberg,Rickard Sandberg
出处
期刊:Genome Research
[Cold Spring Harbor Laboratory]
日期:2014-07-30
卷期号:24 (12): 2033-2040
被引量:790
标识
DOI:10.1101/gr.177881.114
摘要
Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large-scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from subpicogram amounts of cDNA. The comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, because naked Tn5 can be annealed to any oligonucleotide of choice, for example, molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enable innovation in sequencing-based applications.
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