Best Practices

Best Practices

Consistency

  • To make sure there is consistency across samples, use a multichannel pipette where possible. Calibrate pipettes periodically.
  • To minimize sample loss from manual resuspension, use the recommended plates and plate shaker to mix samples.
  • Make sure the correct amplification plate is used.
  • Make sure to take aliquots as recommended after specific procedures as a quality check for the success of long amplification.

Avoid Cross-Contamination

  • Pipette carefully to avoid spillage.
  • Clean work surfaces thoroughly before and after the procedure.
  • Use only the recommended assay PCR plates required to physically mate with the custom Illumina plastic plates.

Avoid Potential DNA Contaminants

  • Incorrect DNA quantitation might result from DNA contamination caused by interference from superfluous nucleic acids in a sample (e.g., RNA, small nucleic acid fragments, nucleotides, single-stranded DNA), excess proteins, or other contaminating materials.
  • DNA quality might also affect the quantity of usable DNA in a sample. If the DNA is damaged (e.g., heavily nicked or containing extensive apurinic/apyrimidinic sites), then many of these fragments might fail during library preparation.
  • High molecular weight dsDNA derived from host genomes can also interfere with accurate quantitation. Bacterial artificial chromosomes (BACs) and other bacterially-derived plasmids usually contain a small percentage of the chromosomal DNA from the host cells, despite the best purification efforts. These sequences may ultimately give rise to unwanted clusters on a flow cell lane. However, this contamination can be accurately quantified by analyzing aligned reads generated during sequencing against known bacterial sequences and subtracting these out. High molecular weight contamination may also be estimated prior to library preparation using qPCR assays designed to target unique chromosomal markers.

Temperature Considerations

  • Keep libraries at temperatures ≤ 37°C, except where specifically noted.
  • Place reagents on ice after thawing at room temperature.
  • Avoid elevated temperatures, particularly in the steps preceding the adapter ligation, except where specifically noted.

Handling Liquids

  • Make sure you pipette the correct amount into each well.
  • Small differences in volumes (±0.5 µl) can sometimes give rise to very large differences in cluster numbers (~100,000).
  • Small volume pipetting can be a source of potential error in protocols that require generation of standard curves, such as PicoGreen assays or qPCR, or those that require small but precise volumes, such as the Agilent Bioanalyzer.
  • If small volumes are unavoidable, then due diligence should be taken to ensure that pipettes are correctly calibrated.
  • Make sure that pipettes are not used at the volume extremes of their performance specifications.
  • Take care with solutions of high molecular weight double-stranded DNA (dsDNA). These can be viscous and not evenly dispersed, resulting in aliquot measurements that are not representative of the true concentration of the solution.
  • Prepare reagents for multiple samples simultaneously to minimize pipetting errors, especially with small volume enzyme additions. Pipette once from the reagent tubes with a larger volume, rather than many times with 1 μl volumes. This method allows you to aliquot in a single pipetting movement to individual samples and standardize across multiple samples.
  • Dilute in low binding tubes.

Handling Master Mix Reagents

  • Prepare master mixes at time of use and mix them well.
  • Minimize freeze-thaw cycles. If you do not intend to consume the reagents in one use, dispense the reagent into aliquots after the initial thaw and refreeze the aliquots in order to avoid excessive freeze-thaw cycles. However, if you aliquot, you may not have enough reagents for the full number of reactions over multiple uses.
  • Add reagents in the order indicated.
  • Take care while adding ATL (A-Tailing Mix) and LIG (Ligation Mix) due to the viscosity of the reagents.

Handling Magnetic Beads

Follow appropriate handling methods when working with Sample Purification Beads (SPB):

  • Prior to use, allow the beads to come to room temperature.
  • Do not reuse beads. Always add fresh beads when performing the procedures.
  • Immediately prior to use, vortex the beads until they are well dispersed. The color of the liquid should appear homogeneous.
  • When pipetting the beads, pipette very slowly and dispense very slowly due to the viscosity of the solution.
  • The kit contains enough Sample Purification Beads to prepare the number of libraries supported by the kit. However, if you aliquot, you might not have enough beads to support the full number of reactions over multiple uses.
  • After adding the beads to the reaction, mix the solution gently and thoroughly by pipetting up and down 10 times, making sure the liquid comes in contact with the beads and that the beads are resuspended homogeneously.
  • Pipetting with the tips at the bottom of the well and not pipetting the entire volume of the solution helps prevent the solution from foaming. Excessive foaming leads to sample loss, because the foam is not transferred out of the plate efficiently.
  • Take care to minimize bead loss which can impact final yields.
  • Change the tips for each sample.
  • Let the mixed samples incubate at room temperature for the full duration specified in the protocol to ensure maximum recovery.
  • When removing and discarding supernatant from the wells, use a single channel or multichannel pipette and take care not to disturb the beads.
  • When aspirating the cleared solution from the reaction plate and wash step, it is important to keep the plate on the magnetic stand and to not disturb the separated magnetic beads. Aspirate slowly to prevent the beads from sliding down the sides of the wells and into the pipette tips.
  • To prevent the carryover of beads after elution, approximately 2.5 μl of supernatant are left when the eluates are removed from the bead pellet.
  • Prepare fresh 80% ethanol, as required, in the protocol. Ethanol tends to absorb water from the air, therefore, fresh 80% ethanol should be prepared for optimal results.
  • Be sure to remove all of the ethanol from the bottom of the wells, as it may contain residual contaminants.
  • Keep the reaction plate on the magnetic stand and let it air-dry at room temperature to prevent potential bead loss due to electrostatic forces. Allow for the complete evaporation of residual ethanol, as the presence of ethanol will impact the performance of the subsequent reactions. Illumina recommends at least 5 minutes drying time, but a longer drying time may be required. Remaining ethanol can be removed with a 10 μl pipette.
  • Avoid over drying the beads, which can impact final yields.
  • Use the Resuspension Buffer (RSB) for DNA elution.
  • For DNA elution, keep the plate in the magnetic stand and add the Resuspension Buffer to the beads for all samples before resuspending them to avoid the loss of dried pellets. Then remove the plate from the magnetic stand and resuspend the beads by repeatedly passing the Resuspension Buffer over the pellet until fully resuspended.
  • Do not scrape of the beads from the edge of the well using the pipette tip.
  • Resuspend the dried pellets using a single channel or multichannel pipette.
  • To maximize sample recovery during elution, incubate the DNA/bead mix for 2 minutes at room temperature before placing the samples onto the magnet.

Working with SP Beads for Size Selection

  • Size selection is done using various ratios of Sample Purification Beads (SPB) to sample. Improper bead usage or dilution will impact the final yield and selected insert size. Make sure the beads are resuspended and that appropriate bead dilutions with water are made depending on the target insert size.
  • The DNA of interest remains in the supernatant during the first size selection step after end repair using the diluted bead mixture. Make sure that you retain this supernatant and move forward with the correct solution.
  • Discard the remaining diluted bead mixture after the first bead size selection step. The second size selection step uses undiluted beads.
  • Add Resuspension Buffer to the beads while they are still on the magnetic stand. Once RSB is added, remove the plate from the magnet and resuspend the beads. This will ensure beads are not lost due to electrostatic forces and will result in higher yields.

Performing qPCR

  • Under or over quantitation may produce low yield and/or inaccurate results.
  • Use the validated qPCR systems.
  • Qualifying the SYBR Green is highly recommended. Excess concentration of SYBR green is a known inhibitor that can lead to inaccurate quantitation. For more information on how to calibrate SYBR Green, see the TruSeq Synthetic Long-Read DNA Library Prep Guide.

Equipment

  • Review the programming instructions for your thermal cycler.
  • Make sure that the thermal cycler is programmed using the heated lid function.
  • Calibrate the microplate shaker with a stroboscope and set it to 1600 rpm.
  • Follow the recommended PCR cycling parameters.