We had a run with a very low on-target rate (0 – 20%) and with many low coverage regions. What could be the reason for this result?
On-target rates are expected to be around 70 – 80 %. Low on-target rates are caused by problems in the hybridization and capture step and are also reflected in a high number of low coverage regions. On-target rates below 10 % have been observed when one of the following experimental errors occurred:
- Probes were not added to the hybridization reaction, resulting in libraries without target enrichment. We suggest checking the remaining volumes in the probe tube, to make sure that the correct volume has been removed.
- The hybridization reaction was not placed at 95°C before adding the probes and starting the hybridization. In this scenario, the double stranded DNA is not denatured, hence, no binding between probes and DNA can occur.
- The hybridization reaction cooled down after DNA denaturation. If the reaction is placed at room temperature for too long, the DNA strands re-anneal, and the probes cannot access the DNA. This can also occur when the same thermal cycler is used for both denaturation and hybridization. In this case, the thermal cycler might take too long to cool down from 95°C to 65°C, and during this time the DNA stands anneal again. We recommend using to different blocks for denaturation and hybridization.
- Cot DNA was not added to the hybridization reaction. Cot I DNA is used to block non-specific hybridization of repetitive sequences. We suggest checking the remaining volume in the tube, to make sure that the correct volume was removed.
- Universal blockers were not added to the hybridization reaction. The universal blocking oligos prevent non-specific hybridization between adapter sequences, thereby enhancing specificity of the capture. We suggest checking the remaining volumes in the probe tube, to make sure that the correct volume was removed.