What's wrong with my NGS?

• Re-purify samples using proteinase

• Ensure that your DNA extraction protocol includes RNase

• Re-purify samples by ethanol precipitation

• Ensure correct DNA input concentration by Qubit^® (or similar fluorometer) reading immediately before starting the protocol

• Treat samples with SureSeq™ FFPE Repair Mix*

• Measure DNA concentration and dilute samples to ~ 70 ng/μl in nuclease-free water prior to shearing or fragmentation

Suboptimal DNA fragmentation

Covaris^®:

• Check settings in SureSeq Handbook and/or equipment manual to ensure correct shearing conditions are used
• Perform recommended maintenance and calibration per equipment manual

NEBNext^® dsDNA Fragmentase^®:

• Ensure samples are kept on ice prior to addition of the master mix
• Ensure samples are diluted in nuclease-free water, not an EDTA-containing buffer
• Ensure sample is at 65-75 ng/μl. If samples are >75 ng/μl dilute them to this concentration
• Be sure to vortex master mix prior to use and after adding to sample
• Ensure to stop the reaction with EDTA timely and consistently for all samples
• Proceed immediately to post-fragmentation purification

DNA degradation or FFPE samples

• Check quality of the samples prior to fragmentation using TapeStation^® (or similar), to determine optimal shearing conditions
• Ensure correct volume of AMPure^® beads is used for post-fragmentation clean-up (1.8x bead to sample ratio)

• Adaptor ligation is temperature sensitive, prepare reaction on ice and ensure reaction is at 20°C without heated lid

Inefficient PCR reaction

• Keep samples and reagents on ice when preparing the PCR reaction
• Ensure proper mixing of reagents and sample by vortex mixing

Suboptimal number of PCR cycles

• Follow guidelines in SureSeq Handbook to optimise number of PCR cycles
• Repeat PCR reaction on remaining ligation product (if available)

Suboptimal elution

• Ensure beads are not over-dried at any point
• The bead pellet has dried sufficiently when the appearance of the surface changes from shiny to matt. The bead pellet is over-dried when there are multiple cracks and it begins to flake
• Prepare fresh 70% ethanol daily for bead clean-up procedures and avoid evaporation

• Should not affect downstream processing, but leave 2 μl in tube to reduce bead carry-over

Hybridisation and/or wash was not efficient and resulted in high amount of off-target DNA

• Repeat the hybridisation and wash steps with remaining pre-capture library (if available)
• Ensure temperatures of buffers are maintained throughout wash procedure by using a thermal cycler (or similar) to heat the buffers

Suboptimal input amount for hybridisation

• Verify hybridisation input calculations, this should be ~ 1000 ng

• Check for evaporation. It is recommended to test consumables and equipment beforehand to avoid evaporation
• Perform routine maintenance of thermal cycler

Stringency of post-hybridisation washes too low

• Maintain the correct temperature during hybridisation and washing procedures; verify using a calibrated thermometer

Hybridisation reaction exposed to room temperature

• Work in close proximity to the thermocycler
• Complete the washing procedure in one step, without stopping

• Increase input sample concentration, optimal amount is 1000 ng
• Minimise the number of PCR cycles to reduce duplication rates

• Optimise number of samples for sequencer model and flow cell capacity
• If using Fragmentase, the optimal number of samples per sequencing run may be slightly lower compared to Covaris shearing
• If degraded DNA, e.g. extracted from FFPE samples, performs poorly the batch size may need to be reduced