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Linear amplification for deep sequencing
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DOI：10.1038/nprot.2011.345.png)
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摘要
En 中文
Linear amplification for deep sequencing (LADS) is an amplification method that produces representative libraries for Illumina next-generation sequencing within 2 d. The method relies on attaching two different sequencing adapters to blunt-end repaired and A-tailed DNA fragments, wherein one of the adapters is extended with the sequence for the T7 RNA polymerase promoter. Ligated and size-selected DNA fragments are transcribed in vitro with high RNA yields. Subsequent cDNA synthesis is initiated from a primer complementary to the first adapter, ensuring that the library will only contain full-length fragments with two distinct adapters. Contrary to the severely biased representation of AT- or GC-rich fragments in standard PCR-amplified libraries, the sequence coverage in T7-amplified libraries is indistinguishable from that of nonamplified libraries. Moreover, in contrast to amplification-free methods, LADS can generate sequencing libraries from a few nanograms of DNA, which is essential for all applications in which the starting material is limited.
Keyword:
T7 RNA-POLYMERASE
PLASMODIUM-FALCIPARUM
GENOME SEQUENCE
DNA
SEQ
PLATFORM
CELL
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