title: NGS of HCV 6 weeks post LTX2
design: Viral RNA was purified from 200 l patient serum using the NucleoSpin Virus kit (Macherey-Nagel). cDNA based on the extracted RNA was generated using the SuperScript IV First-Strand Synthesis kit (Invitrogen) with HCV genotype specific primers. To amplify the DNA, nested PCR was performed using the SuperFi II DNA polymerase kit (Invitrogen) according to the manufacturers instructions. A first 50 l PCR reaction with 25% of the cDNA as template was run for 30 cycles. 5 l of this PCR product was used as template for the second PCR running again for 30 cycles. The desired amplicon was purified via gel electrophoresis with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). NGS was performed using the MiSeq v2 (2x250bp) platform (Illumina) with an average sequencing depth of ~4000 reads (Microsynth AG).