TruSight Tumor 170 Kit FAQs

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  • General


  • TruSight Tumor 170 includes library prep; enrichment and sequencing reagents; and software to generate variant calls from DNA and RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue.

    Not currently. Consult your Illumina sales representative to determine future availability.

    During library prep and enrichment, the DNA and RNA samples go through the same workflow following sample shearing (DNA) and cDNA synthesis (RNA). The effect is that one user with one plate can process the DNA and RNA samples in parallel. The DNA and RNA libraries can then be sequenced on the same sequencing run.

    An experienced TruSight Tumor 170 assay operator can generate variant calls from extracted nucleic acids in approximately 72 hours.

    Illumina recommends performing the TruSight Tumor 170 assay workflow according to the following schedule:

    • Day 1: cDNA Synthesis, DNA Shearing, Library Preparation, and begin Overnight (First) Hybridization.
    • Day 2: Enrichment, Enriched Library QC Check (AccuClear), Bead-Based Normalization of Enriched Libraries, and Loading of Libraries onto Sequencing Platform (NextSeq 500, NextSeq 550, or HiSeq 2500).
    • Day 3: Kick off analysis in BaseSpace and review res

    Approximately seven hours for an experienced operator.

    The library prep kits support 24 samples (48 libraries—24 from DNA and 24 from RNA). The library prep kit can be ordered alone (OP-101-1004) or as a bundle with 3 NextSeq High Output v2 (300 cycles) kits (OP-101-1003).

    The reagents provided in this kit do not support the preparation of more than 24 DNA libraries and 24 RNA libraries.

  • Input


  • Extraction reagents are not included in the TruSight Tumor 170 kit. The QIAGEN AllPrep DNA/RNA FFPE Kit (Cat. # 80234) has demonstrated high yield of nucleic acids in comparison to other extraction methods for this assay. The QIAGEN AllPrep DNA/RNA FFPE Kit includes a DNase I digestion step during RNA extraction. You can select other commercially available extraction kits at your discretion.

    Illumina recommends adding a minimum of 40 ng of DNA or RNA. For samples that are highly degraded, assay performance may be improved by using a maximum of 120 ng of DNA and 85 ng of RNA.

    Illumina recommends adding a minimum of 40 ng of DNA or RNA. For samples that are highly degraded, assay performance may be improved by using a maximum of 120 ng of DNA and 85 ng of RNA.

    Results can be obtained with less than 40 ng, though performance may be lower or result in reduced sensitivity.

    DNA sample quality can be assessed using the Illumina FFPE qPCR kit. Illumina recommends a DNA sample quality cut-off ΔCq value of ≤ 5 to obtain optimal results. RNA sample quality can be assessed using the Agilent Bioanalyzer. Illumina recommends a sample quality cut-off DV200 score of ≥ 20 to obtain optimal results.

  • Protocol


  • Illumina recommends that you prepare and sequence at least three samples at a time.

    The reagents of the TST 170 kit can go through up to four freeze-thaw cycles. The exception is OPD1 and OPR1 reagents, which can go through up to eight freeze-thaw cycles.

    Illumina recommends mechanical shearing of the DNA using the Covaris ultrasonicator.

    Currently, Illumina supports the LE220, LE220R, E220, E220R, and E220 evolution. 

    If you are using a Covaris model that is not supported, Illumina recommends that you use the Covaris E220 settings, found in the E220 User Manual, as a starting point.

    See the question "What are the settings for the S220 and M220 Covaris models?" for additional settings. Contact Covaris customer support for further assistance. For other ultrasonicator models not supported by Illumina, you can verify settings on their ultrasonicator to achieve the desired fragment size.

    If you are using the Covaris S220 or M220 models, fragment the gDNA using the following settings. 

    S220 Instrument

    Holder

    S-Series Holder microTUBE-50 Screw-Cap (PN 500492)

    Consumable

    microTUBE-50 Screw-Cap (PN 520166)

    Sample volume

    55 µl

    PIP

    100W

    DF

    30%

    cpb

    1000

    Time

    150 seconds

    M220 Instrument

    Holder

    M220 Holder XTU (PN 500414)

    Insert M220 Holder XTU Insert microTUBE 50 µl (PN 500488)

    Consumable

    microTUBE-50 Screw-Cap (PN 520166)

    Sample volume

    55 µl

    PIP

    75W

    DF

    15%

    Time

    360 seconds

    cpb

    1000

      NOTE: It is critical that water is in contact with Insert when using microTUBE-50. Visually inspect even if the Water Level check button is green in SonoLab.
    • Adjust settings as needed to achieve gDNA that has been fragmented to 90–250 bp (with a peak at ~125 bp).
    • [Optional] Fragment size distribution of sheared samples may be assessed using the Agilent DNA 1000 Kit with the Agilent Bioanalyzer 2100.

    You should expect an average fragment size of approximately 120 bp for high-quality DNA. However, because post-shearing fragment size is a function of both the Covaris settings and the fragment size of the starting material, the average post-shearing fragment size is expected to be lower for degraded FFPE DNA.

    The kit contains Unique Index Primer Mixes (UPXX) and combinatorial Index Primer Mixes (CPXX) (XX = index primer mix number). RNA libraries require the use of UPXX index primer mixes. Assigning CPXX index primer mixes to RNA libraries may result in decreased performance. DNA libraries can be run with either CPXX or UPXX index primer mixes.

    Yes. Illumina recommends using UPXX index primers for low-plex runs. One or more combinations of the following sets should be used for low-plex runs: UP01-03, UP04-06, UP07-09, UP10-12, or UP13-15.

    Random hexamers are used for priming during cDNA synthesis.

    All beads required for the protocol are supplied in the kit.

    Performance of the assay has been demonstrated using a Hybex incubator with midi heat block inserts. Illumina recommends that you follow the protocol as written.

    Clean up procedures have been optimized with the stand specified, the Thermo Fisher 96-well magnetic stand (#AM10027). Comparable performance is not guaranteed when using other magnets.

    Reagents have color-coded caps to identify the associated sample type. Blue caps identify reagents used only with DNA. Red caps identify reagents used only with RNA.

    Illumina has included the bead-based normalization reagents for convenience; you can perform manual normalization if preferred.

    There are several safe stopping points listed in the reference guide after the following steps.

    Pre-enrichment:

    • cDNA Clean Up for RNA samples or after Fragment DNA for DNA samples
    • Index PCR

    Post-Enrichment:

    • Perform Second Capture
    • Clean Up Amplified Enriched Library
    • Normalize Libraries
  • Library Evaluation


  • Illumina has found that the amount of library that goes into the enrichment assay does not affect performance. Removing the QC check step also removes the requirement to perform a DNA cleanup step, potentially increasing yield while reducing hands-on and turnaround time.

    Quantify DNA libraries to make sure that there is sufficient yield by using a fluorometric quantification for double stranded DNA such as AccuClear Ultra High Sensitivity dsDNA Quantitation Kit. The protocol is optimized for ≥ 3 ng/uL of each library for efficient bead-based normalization.