Best Practices

Best Practices

Separate Laboratory Areas

  • A pre-PCR area is recommended for setting up the PCR1 reaction (no amplified DNA or DNA libraries).
  • Two post-PCR areas are recommended. One area free of DNA libraries for setting up PCR2. Another area for libraries only, with the magnetic stand and microplate shaker that is used for SPRI purification, bead-based-normalization, and pooling. This area is also used for adding library pools to the MiSeq ForenSeq reagent cartridge.

Ensuring Consistency

  • To ensure consistency across samples, use a multichannel pipette where possible. Calibrate pipettes periodically.

Avoid Cross-Contamination

  • Always use fresh pipette tips between samples and between dispensing index primers.
  • Mix samples with a multichannel pipette and centrifuge the plate when indicated. Do not vortex the plates.
  • Pipette carefully to avoid spillage.
  • Clean pipettes and change gloves between handling different adapter stocks.
  • Clean work surfaces thoroughly before and after the procedure.

Handling Magnetic Beads

  • Allow the beads to come to room temperature before use.
  • Vortex the beads until they are well dispersed immediately before use. The color of the liquid should appear homogeneous.
  • Take care to minimize bead loss, which can affect 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 aspirating the clear solution from the reaction plate and wash step, it is important to keep the plate on the magnetic stand and 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.
  • Always prepare fresh 80% ethanol. Ethanol tends to absorb water from the air, therefore, fresh 80% ethanol should be prepared fresh for optimal results.
  • Be sure to remove all of the ethanol from the bottom of the wells, as it can 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 impacts the performance of the subsequent reactions. Illumina recommends at least 15 minutes drying time, but a longer drying time may be required.
  • Resuspend the dried pellets using a single channel or multichannel pipette.
  • When removing and discarding supernatant from the wells, use a single channel or multichannel pipette and take care not to disturb the beads.
  • To maximize DNA recovery during elution, incubate the DNA/bead mix for two minutes at room temperature before placing the samples onto the magnet.

Choosing Indexed Adapters

  • For 16 or fewer samples, Illumina recommends adding the adapters individually to each sample. It is best to use a unique pair for low input samples. 
  • For eight or fewer samples, each sample will have a unique pair of index sequences.
  • Example: Indexed Adapter Combinations for 8 or Fewer Samples

    Sample

    Index 1

    Index 2

    1

    A501

    R701

    2

    A501

    R702

    3

    A501

    R703

    4

    A501

    R704

    5

    A501

    R705

    6

    A501

    R706

    7

    A501

    R707

    8

    A501

    R708

  • For 9–16 samples, make sure that reference samples and positive controls have unique pairs of adapters
  • Example: Indexed Adapter Combination for 9–16 Samples

    Sample

    Type

    Index 1

    Index 2

    1

    Reagent Blank

    A501

    R701

    2

    Assay Blank A502

    R702

    3

    2800M Positive Control

    A503

    R703

    4

    Reference

    A504

    R704

    5

    Reference

    A505

    R705

    6

    Reference

    A506

    R707

    7

    Extraced Sample 1

    A507

    R708

    8

    Extraced Sample 2 A508

    R709

    9 Extraced Sample 3
    A501 R710
    10 Extraced Sample 4
    A502 R711
    11 Extraced Sample 5 A507 R712
    12 Extraced Sample 6 A508 R711
    13 Extraced Sample 7 A507 R709
    14 Extraced Sample 8 A508 R710
    15 Extraced Sample 9 A501 R708
    16 Extraced Sample 10 A502 R709
  • For more than 16 samples, Illumina recommends using the ForenSeq Index Adapter Fixture to array the adapter tubes and add the adapters with a multichannel pipette
  • Extra caps are included in the kit to help prevent adapter cross-contamination. Discard caps when tubes are opened and seal with a fresh cap when storing remaining adapter.

Numbers of Samples Sequenced

  • If only single source reference or database samples are run in an experiment, up to 96 samples can be sequenced together as long as they each have a different index combination.
  • If casework or evidentiary samples are run in an experiment, up to 32 samples can be sequenced together as long as they each have a different index combination.
  • If a mixture of samples using both DNA Primer Mix A and B are used, the limit should be 32 samples sequenced together.