Failed Library Prep
|Input DNA contains an inhibitor||
Ensure DNA does not contain inhibitor.
Consider additional cleanup step.
|Failed step - Any of the enzymatic steps can fail if a critical reagent is omitted, or if the reagent has become inactive||
Confirm reagents were added for each step in the protocol.Ensure reagents have been stored at the appropriate temperature.
|Low Library Yield||
Input DNA is damaged
|Shear input DNA on a Covaris® instrument in 1X TE Buffer, and/ or use the NEBNext FFPE DNA Repair Mix (NEB #M6630) after shearing and prior to library prep.|
|Adaptor is denatured||
When diluting NEBNext adaptors, use 10 mM Tris HCl (pH 7.5-8.0) with 10 mM NaCl.Keep the adaptor on ice until use.
Mix samples well with 80-90% of the total volume in the well or tube by pipetting up and down. Keep the tip in the liquid to avoid the formation of bubbles.
For enzymatic steps, follow the manual recommendations (usually 10 mix cycles).Try to avoid losing sample in the pipette tip or on the source tube during transfer.
|SPRI beads have dried out before elution||
Add Elution Buffer and mix before the beads turn lighter brown and start cracking.For additional tips about SPRI beads view our video.
|Incomplete ethanol removal during SPRI bead wash||
Quickly spin the tube after the last ethanol wash at each SPRI bead step, keep the tube on the magnet and remove residual ethanol with a p10 tip.For additional tips about SPRI beads view our video.
|SPRI bead sample loss||
Mix slowly to avoid droplets clinging to the inside of the tip, which may not recombine with the sample before the tip is ejected. Dispense the last mix slowly into the sample tube so that the liquid stays together. Wait 1 second before pushing the pipette to the second stop, so that everything can be dispensed.
When removing the supernatant, take care not to remove any beads. Check your tip over a white piece of paper. If beads are visible, dispense everything back into the tube and allow beads to resettle.For additional tips about SPRI beads view our video.
|Sample storage after A-tailing||Avoid prolonged storage of sample before moving to ligation. If sample inputs are low, avoid overnight storage and move immediately from end prep to adaptor ligation.|
|Adaptor self-ligation (Adaptor dimer formation)||
Do not add adaptor to the ligation master mix. This can cause increased adaptor dimer formation.For best results, add the adaptor to the sample, mix and then add ligase master mix and ligation enhancer.
|Ligation incubation temperature is too warm||If ligation incubation occurs above 20˚C the DNA ends may breathe, which could reduce ligation efficiency.|
|Adaptor Dimer Formation (sharp 127 bp peak on Bioanalyzer)||To recover the samples, repeat the bead cleanup using a 0.9 x bead ratio.|
|Adaptor concentration too high||
Optimize adaptor dilution based on your sample input, quality and type using an adaptor titration experiment.Adaptor titration may need to be repeated if the source of the sample input changes (e.g., extraction method, tissue type, etc.).
|Adaptor self-ligation (Adaptor Dimer formation)||
Do not add adaptor to ligation master mix. This can cause increased adaptor dimer formation.For best results, add adaptor to sample, mix and then add ligase master mix and ligation enhancer. Mix again.
|Adaptor or primers remaining after PCR (e.g., visible on Bioanalyzer or similar instrument after PCR)||Excess adaptor or primer used or inefficient cleanup||Perform another 0.9 x SPRI cleanup|
|Overamplification (Once PCR primers are depleted, library fragments will become single stranded and/ or form heteroduplexes. These appear as high molecular weight fragments on a Bioanalyzer or similar instrument)||Too many PCR Cycles||
We recommend starting with the number of PCR cycles provided in the product manual. The ideal number of PCR cycles for your samples may vary.
Reduce the number of PCR cycles if you are seeing overamplification.Data quality may be compromised if overamplified libraries are sequenced.
|Not enough PCR primer||
Check primer concentration and ensure that you are adding the primer volume recommended in the manual.
Store primers at the correct temperature to prevent degradation.Data quality may be compromised overamplified libraries are sequenced.
|Too much input DNA||
The higher the input of template for the PCR, the sooner the primers will be depleted.NEBNext adaptor and PCR primers require a minimum of 3 PCR cycles. If you cannot further reduce the number of PCR cycles, consider a size selection step, or using only a fraction of the ligated library as input for PCR.
|Library not the correct size||PCR size bias||
During PCR, short fragments can amplify more efficiently than long ones. If you have sufficient input material, consider a size selection step after ligation to narrow the input size range.Reduce the number of PCR cycles.
FFPE DNA is crosslinked, which can inhibit amplification of longer PCR products.Crosslinks cannot be reversed, but less fragmentation before input into library prep may shift the library towards longer inserts.
Size selection ratios or bead volumes are based on sample volume and on the buffer that the sample is in.
When following a size selection protocol, be sure the sample is in the reagent or buffer specified in the previous step of the protocol.
Be sure to use accurate volumes.
Samples that are severely evaporated should be topped off with water to the expected volume.For additional tips about SPRI beads view our video.
Short library fragments cluster more efficiently than long ones, which may lead to a discrepancy between the average library size measured by fragment analyzer and the average insert size as determined by the sequencer.A more stringent size selection (e.g., using a gel cut or Pippin Prep) will remove the short fragments and increase the average insert size. Additional size selection may also result in lower library yields.