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.
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.
['Ishige|Takayuki|T|', 'Itoga|Sakae|S|', 'Kawasaki|Kenji|K|', 'Utsuno|Emi|E|', 'Beppu|Minako|M|', 'Sawai|Setsu|S|', 'Nishimura|Motoi|M|', 'Ichikawa|Tomohiko|T|', 'Nomura|Fumio|F|', 'Matsushita|Kazuyuki|K|']
D001483:Base Sequence D004247:DNA D010505:Familial Mediterranean Fever D015723:Gene Library D059014:High-Throughput Nucleotide Sequencing D006801:Humans D025202:Molecular Diagnostic Techniques D000071198:Pyrin D060888:Real-Time Polymerase Chain Reaction
Long-range PCR Massively parallel sequencing Post-PCR cleanup Library preparation