NGS Library Preparation Support—Troubleshooting

In addition to input RNA quality and accurate quantification, the clean-up and size selection steps are critical to generating a successful RNA-Seq library.

• Be sure to mix the nucleic acid binding beads well before dispensing, and follow the workflow and incubation times as closely as possible.
• Use fresh ethanol and pre-wet pipette tips prior to transferring ethanol, as the volume is critical for size selection.
• Remove residual ethanol before elution using a small-volume pipette. Do not over-dry or under-dry the beads. 

The ~ 70 bp or ~90 bp peak is likely standard or barcoded adapter dimers, respectively. Adapter dimers may form during the adapter ligation step and are usually removed during the size selection process. The adapter dimers will amplify on the Ion Torrent™ Ion Sphere™ particles during template preparation and decrease the overall throughput of usable sequencing reads; thus, we highly recommend removing the adapter dimers by performing an additional clean-up step prior to template preparation.

We recommend using the qPCR result, as quantification by qPCR is generally more accurate than quantification using the Bioanalyzer instrument.

No, the Ion Library Quantitation Kit for qPCR library quantification is unable to differentiate amplifiable primer-dimers from library fragments. We recommend assessing the library size distribution, including checking for the presence of adapter dimers, using the Bioanalyzer instrument. Libraries containing adapter dimers will have a sharp peak at ~70 bp for non-barcoded libraries or ~90 bp for barcoded libraries.

Make sure that you are using the recommended seals and compression pads (depending on the thermal cycler being used). If you are able to avoid it, we recommend against using Rows A and H (depending on the thermocycler, there can be more evaporation in these outer wells).

Here are some suggestions:

• Double check how the DNA was originally quantitated. We recommend using TaqMan™ RNase P Detection Reagents Kit for quantifying amplifiable DNA.

• If you are still getting low yield using 50-100 ng input, add cycles to the initial amplification. You could try to add 1-3 cycles. It is better to add additional cycles to the target amplification rather than to the 2nd amplification step. Keep in mind that it is important to limit the number of cycles done during the final amplification as you do not want to amplify fragments exponentially as this will introduce bias toward smaller fragments in the amplicon pool.

• Evenness of coverage is affected by bias in “AMP” cycles. Additionally, overamplification will put the sample concentration above the dynamic range of detection for the High Sensitivity BioAnalyzer Chip. It is better to repeat the amplification reaction to generate sufficient product that to overamplify and dilute.