Evaluation of analytical factors associated with targeted MEFV gene sequencing using long-range PCR/massively parallel sequencing of whole blood DNA for molecular diagnosis of Familial Mediterranean fever.
Clin Chim Acta
; 495: 562-569, 2019 Aug.
Article
em En
| MEDLINE
| ID: mdl-31173732
ABSTRACT
BACKGROUND:
Long-range PCR (LR-PCR) is used to enrich the target regions of the genome. This study aimed to establish the pipeline of targeted gene sequencing using LR-PCR and massively parallel sequencing (MPS).METHODS:
The 14-kb-long MEFV gene, including the entire coding exons, was selected as a target gene and amplified using LR-PCR. The evaluated analytical factors were as follows LR-PCR conditions, three types of post-PCR cleanup methods, and two types of MPS library preparation methods.RESULTS:
With regard to LR-PCR conditions, Tks Gflex DNA polymerase at 7-min (30-s/kb) annealing/extension with 100-ng genomic DNA input had the highest yield. Regarding post-PCR purification methods, the magnetic beads-based method had high recovery and purity. In the MPS library preparation methods, the ligation-based method had a higher base coverage in the target (94.58%), uniformity of base coverage (99.95%), and target bases with no strand bias (97.40%). The exonic variants determined by Sanger sequencing were detected by both ligation- and transposon-based methods.CONCLUSIONS:
Various analytical factors were evaluated, and the pipeline of targeted gene sequencing using LR-PCR and MPS was established. These data can enable the optimization of targeted gene sequencing using LR-PCR and MPS in the clinical laboratory.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Febre Familiar do Mediterrâneo
/
DNA
/
Técnicas de Diagnóstico Molecular
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Sequenciamento de Nucleotídeos em Larga Escala
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Pirina
Tipo de estudo:
Diagnostic_studies
/
Risk_factors_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article