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View additional product information for Oncomine™ Tumor Mutation Load Assay, Chef-ready library preparation - FAQs (A37910)
14 product FAQs found
No, the Oncomine Tumor Mutation Load Assay is optimized for use with FFPE tissue. The in-house development of the assay specifically focused on three tissue types: melanoma, lung, and colon.
Yes, total variant calls are quantified and reported by the Oncomine Tumor Mutation Load Assay workflow and a .vcf file of all called variants is available to download in the user interface.
Yes, germline filters cover across different ancestry types. They are not ethnicity-based, but are global population-based.
Yes, by following the instructions in the appendix of the User guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0015885_OncomineComprehensiveAssay_v3_UG.pdf).
Yes, Oncomine Comprehensive Assay BAM files can be used with the Oncomine TML workflow. Targets that are not in Oncomine TML workflow will just be discarded during the analysis.
With the Oncomine Tumor Mutation Load Assay, manual library preparation, you can quantify the libraries using a Qubit fluorometer or the Ion Library TaqMan Quantitation Kit. The expected manual library yield is >50 pM.
With the Oncomine Tumor Mutation Load Assay, Chef-ready library preparation, the expected automated library pool yield is 100 pM (with range of 50-200 pM). On the Ion Chef Instrument, the Ion Library Equalizer Kit is used to normalize the library to 100 pM.
IonCode Barcode Adapters 1-384 Kit (Cat. No. A29751) is compatible with the Oncomine Tumor Mutation Load Assay, Chef-ready library preparation. The set includes 4 PCR plates containing a total of 384 unique barcodes:
- IonCode 0101-0196 in 96 Well PCR Plate (red)
- IonCode 0201-0296 in 96 Well PCR Plate (yellow)
- IonCode 0301-0396 in 96 Well PCR Plate (green)
- IonCode 0401-0496 in 96 Well PCR Plate (blue)
The Oncomine Tumor Mutation Load Assay, Chef-ready library preparation consists of the Oncomine Tumor Mutation Load Assay - Chef-ready primer panel (2 pools), at 2X concentration pre-measured in barcoded Primer Pool tubes, ready to load into an Ion AmpliSeq Chef Reagents DL8 cartridge. In addition, the kit contains the Ion AmpliSeq Library Kit Plus. Sufficient panel primers and reagents are provided for the preparation of 32 barcoded sample libraries from DNA.
Calculation of the Mutation Load alone does not require as many reads as when performing variant calling. To calculate the Mutation Load alone, we recommend combining up to a maximum of 8 libraries on an Ion 540 Chip, or up to a maximum of 16 libraries on an Ion 550 Chip.
To perform variant calling along with calculation of the Mutation Load, we recommend multiplex sequencing of no more than 4 libraries on an Ion 540 Chip and no more than 6 libraries on an Ion 550 Chip, to achieve sufficient read depth for variant calls at a ≥5% allele frequency.
Mutation Load, also known as Tumor Mutational Burden (TMB), as determined by the Oncomine Tumor Mutation Load Assay, is defined as the number of nonsynonymous variants (missense and nonsense single-nucleotide variants (SNVs)), plus insertion and deletion variants (INDELs) detected per megabase (Mb) of exonic sequence. The Ion Reporter Software TMB algorithm filters out germline mutations using the Mutation Load Calculation Filter Chain when calculating the Mutation Load result.
There are 2 pools of primers, each at 2X concentration.
The starting material is formalin-fixed paraffin-embedded (FFPE) tumor samples. We recommend using 10 ng of DNA per primer pool. As there are 2 pools, total input is 20 ng of DNA. For each target amplification reaction, use 300-30,000 copies of DNA (10 ng of mammalian gDNA) from normal or FFPE tissue. Increasing the amount of DNA results in higher-quality libraries, especially when DNA quality or quantity is unknown. We recommend using 1 ng of gDNA(300 copies) only with high-quality, accurately quantified samples.
For sequential extraction of RNA and DNA from formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue samples, we recommend using the RecoverAll Multi-Sample RNA/DNA Isolation Workflow (Cat. Nos. A26069, A26135).
For extraction of total nucleic acid from formalin- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue samples, we recommend using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Cat. No. AM1975).
The Oncomine Tumor Mutation Load Assay is a targeted next-generation sequencing (NGS) assay that is designed for tumor profiling by annotating cancer driver variants and provides an accurate assessment of mutation load (mutations/Mb). The assay detects and annotates low-frequency somatic variants (SNPs and INDELs) from 409 genes, spanning ˜1.7 Mb of genomic space, encompassing 1.2 Mb of exonic sequence. The assay is designed to facilitate successful selection and identification of samples most likely to derive responses in cancer immunotherapy research.
We offer the Oncomine Tumor Mutation Load Assay, manual library preparation (Cat. No. A37909) and the Oncomine Tumor Mutation Load Assay, Chef-ready library preparation (Cat. No. A37910).