Substitution and insertion/deletion rates and their sources using 454 pyrosequencing.

<p>A) The molecular full-length HIV-1 clone pYK-JRCSF was used to generate three different samples for the determination of error rates and error sources during the different steps of sample preparation. The blue (left) pathway indicates the procedure NGS, i.e., no amplification step was performed before emulsion PCR and pyrosequencing. The green (right) pathway shows the procedure PCR-NGS, i.e., the target was amplified once prior to 454 emulsion PCR/pyrosequencing. The orange (middle) pathway depicts the commonly used procedure RT-2PCR-NGS to reverse transcribe, amplify and sequence HIV-1 RNA genomes reflecting the errors that will occur using patients’ plasma samples to analyze HIV-1 haplotypes. Detailed description of each step is given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074249#s4" target="_blank">materials and methods</a> section. B) Error rates per positions are shown for forward reads (left) and reverse reads (right). For each duplicate, one example is shown (always sample a as presented <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074249#pone-0074249-t001" target="_blank">table 1</a>).