Commercially available reverse transcription enzyme and temperature comparison in high throughput sequencing of bacterial ribosomal 16S rRNA.. Reverse transcription comparison

We compare three reverse transcription enzymes (ImProm-II, SuperScriptIV and TGIRT), commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized reverse transcription conditions. The comparison have been performed on soil samples collected under a desert shrub of genus Hammada in the Negev Desert, near Midreshet Ben Gurion. We have observed up to 6 fold differences in bacterial class abundance in our comparisons. A temperature induced bias can be partially explained by a G-C content of the affected groups, thus pointing towards a need for higher reaction temperatures. However another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus might require the use of one enzyme for the sake of cross-study comparison.