What is this peak?
The presence of a peak at ~ 200 bp in QuantSeq libraries is an indication of mis-hybridization.
Mis-hybridization in this case (the mis-priming of the oligo(dT) primers) often happens as a result of temperature deviations during reverse transcription.
How do I prevent mis-hybridization?
To prevent mis-hybridization, it is particularly important during the First Strand cDNA Synthesis that the
RNA / FS1 mix is stably kept at 42°C, after the denaturation step (steps 2, 3, and 4 of the protocol, in the user guide), and that the FS2 / E1 mastermix is also pre-warmed and kept at this same temperature (42°C) until is added to the samples (step 3).
If the alternative protocol for low input/degraded samples is used, and step 2 is skipped, the
F1 / F2 / E1 mastermix should be pre-warmed at 42°C before being added to the RNA samples (step 3) and the thermocycler should also be pre-heated at 42°C before the samples are transferred on it (step 4).
ATTENTION: The centrifugation steps should always be carried out at room temperature (never at 4°C)!
TIP: Raising the reaction temperature to 50 °C for reverse transcription could also help prevent mis-priming.
How does the peak look like?
Figure: Example of a QuantSeq-FWD library prepared from 50ng of UHR RNA and showing signs of mis-hybridization (peak at ~200 bp, black arrow).
What does this mean for my results?
The presence of mis-hybridization products in the final libraries can impact your results.
Mis-hybridization products correlate with a high proportion of ribosomal RNA (rRNA) reads in the sequencing output. This happens because rRNAs contain some A-rich regions that could be targeted during oligo(dT) mis-priming and amplified in your final libraries.
Other A-rich regions (present in various RNA molecules) could also be amplified in a similar way.
It is not possible to estimate the proportion of mis-hybridization products present in a library, before sequencing it.
The increased reads mapping to rRNAs and other mis-primed regions will reduce the proportion of reads of interest in the sequencing output.