Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing.
BMC Genomics
; 19(1): 30, 2018 01 08.
Article
em En
| MEDLINE
| ID: mdl-29310587
ABSTRACT
BACKGROUND:
Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk.RESULTS:
Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative.CONCLUSIONS:
For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications.Palavras-chave
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Base de dados:
MEDLINE
Assunto principal:
Análise de Sequência de DNA
/
Biologia Computacional
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Sequenciamento de Nucleotídeos em Larga Escala
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article