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2017 ; 18
(1
): 440
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Application of nonsense-mediated primer exclusion (NOPE) for preparation of
unique molecular barcoded libraries
#MMPMID28583065
Shagin DA
; Turchaninova MA
; Shagina IA
; Shugay M
; Zaretsky AR
; Zueva OI
; Bolotin DA
; Lukyanov S
; Chudakov DM
BMC Genomics
2017[Jun]; 18
(1
): 440
PMID28583065
show ga
BACKGROUND: Recently we proposed efficient method to exclude undesirable primers
at any stage of amplification reaction, here termed NOPE (NOnsense-mediated
Primer Exclusion). According to this method, added oligonucleotide overlapping
with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously
provides a template for its elongation. This elongation disrupts specificity of
unwanted primer, preventing its further participation in PCR. The suggested
approach allows to rationally manage the course of PCR reactions in order to
facilitate analysis of complex DNA mixtures as well as to perform multistage PCR
bypassing intermediate purification steps. RESULTS: Here we apply NOPE method to
DNA library preparation for the high-throughput sequencing (HTS) with the
PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE
oligo efficiently neutralizes UMI-containing oligonucleotides after introduction
of UMI into sample DNA molecules, thus allowing to proceed with further
amplification steps without purification and associated loss of starting
material. At the same time, NOPE oligo does not affect the efficiency of target
PCR amplification. CONCLUSION: We describe a simple, robust and cheap
modification of UMI-labeled HTS libraries preparation procedure, that allows to
bypass purification step and thus to preserve starting material which may be
limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of
isolated cells of interest. Furthermore, demonstrated simplicity and robustness
of NOPE method should make it popular in various PCR protocols.
free
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