A method for near full-length amplification and sequencing for six hepatitis C virus genotypes.

BACKGROUND: Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient. RESULTS: In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90%, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1-3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies. CONCLUSIONS: The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications.

Perimetry while moving the eyes: implications for the variability of visual field defects.

BACKGROUND: In standard perimetry, subjects fixate so that saccades are reduced and testing precision is increased. However, because vision in daily life requires eye movements, it is appropriate to assess visual fields during eye movement. METHODS: Perimetry was carried out in 8 healthy subjects and in 16 patients with visual field defects under conditions of a stable and moving fixation spot. Eye movements were simultaneously recorded with an eye tracker. Outcome measures included stimulus detection, variability of visual field border, and saccade amplitudes. RESULTS: Perimetric performance during stable fixation was comparable to that during eye movement. All subjects showed 92%-96% correct detections of the fixation controls and a stable and comparable blind spot position in the stable and moving fixation spot conditions. The eye tracker revealed that 97% of the time the eyes were positioned within +/-1 from fixation. CONCLUSIONS: Visual fields obtained by perimetry while moving the eyes is comparable to standard perimetry in which a stable fixation spot minimizes eye movements.