title: Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications.
abstract: Ultra-deep sequencing (UDS) of amplicons is a major application for next-generation sequencing technologies, even more so for the 454 Genome Sequencer FLX. Especially for this application, errors that might be introduced during any of the sample processing or data analysis steps should be avoided or at least recognized, as they might lead to aberrant sequence variant calling. Since 454 pyrosequencing relies on PCR-driven target amplification, it is key to differentiate errors introduced during the amplification step from genuine minority variants. Thereto, optimal primer design is imperative because primer selection, primer dimer formation, and nonspecific binding may all affect the quality and outcome of amplicon-based deep sequencing. Also, other intrinsic PCR characteristics including amplification drift and the formation of secondary structures may influence sequencing data quality. We illustrate these phenomena using real life case studies and propose experimental and analytical evidence-based solutions for effective practice. Furthermore, because accuracy of the DNA polymerase is vital for reliable UDS results, a comparative analysis of error profiles from seven different DNA polymerases was performed and experimentally assessed in parallel by 454 sequencing. Finally, intra and interrun variability evaluation of the 454 sequencing protocol revealed highly reproducible results in amplicon-based UDS.
authors: Tibotec-Virco Virology, Department of Translational Genomics & Genetics, Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, Beerse, Belgium.
authors: Ina
authors: I
authors: Vandenbroucke
authors: Herwig
authors: H
authors: Van Marck
authors: Peter
authors: P
authors: Verhasselt
authors: Kim
authors: K
authors: Thys
authors: Wendy
authors: W
authors: Mostmans
authors: Stéphanie
authors: S
authors: Dumont
authors: Veerle
authors: V
authors: Van Eygen
authors: Katrien
authors: K
authors: Coen
authors: Marianne
authors: M
authors: Tuefferd
authors: Jeroen
authors: J
authors: Aerssens
mesh_terms: D001483:Base Sequence; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D012015:Reference Standards; D017422:Sequence Analysis, DNA / Q000379:methods*