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Oncomine™ Tumor Mutation Load Assay, manual library preparation

Catalog No. A37909
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A37909 24 samples
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Catalog No. A37909 Supplier Thermo Fisher Scientific Supplier No. A37909

Please call Customer Service at 1-800-234-7437 or send an email to help@thermofisher.com for assistance.

Specifications

Content And Storage • 1 Oncomine Tumor Mutation Load Panel, 24 reactions, store at -5 to -30°C.
• 1 Ion AmpliSeq Library Kit Plus, 24 reactions, store at -5 to -30°C.
Sequencing Type Genome & DNA Sequencing
For Use With (Equipment) Ion GeneStudio S5 System, Ion S5™ XL System
Libraries Targeted Sequencing Library
Product Line Oncomine
Product Type Tumor Mutation Load Assay
Quantity 24 samples
Shipping Condition Dry Ice
Throughput 24 Samples
Workflow Step Library Generation

Frequently Asked Questions (FAQs)

With the Oncomine Tumor Mutation Load Assay, do I need to adjust the PCR cycles based on sample type?

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.
In the Oncomine Tumor Mutation Load Assay, can I get the total variant calls (i.e., not just somatic)?

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.
In the Oncomine Tumor Mutation Load Assay, does the germline filter cover across different ancestry types?

Yes, germline filters cover across different ancestry types. They are not ethnicity-based, but are global population-based.
Can the Oncomine Tumor Mutation Load Assay (TML) workflow 2.0 be edited to run on other panels (such as Oncomine Comprehensive Assay v3)?

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).
Can I run the Oncomine Comprehensive Assay BAM file through the Ion Reporter workflow for the Oncomine Tumor Mutation Load (TML) Assay?

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.
What method do you recommend using for library quantitation with Oncomine Tumor Mutation Load Assay, manual library preparation vs Oncomine Tumor Mutation Load Assay, Chef-ready library preparation?

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.
How many barcoded libraries can I multiplex on Ion chips for calculation of Mutation Load/perform variant calling?

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.

What is the definition of Mutation Load?

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.
How many primer pools are present in the Oncomine Tumor Mutation Load Assay panel?

There are 2 pools of primers, each at 2X concentration.
Which barcode kit is compatible with the Oncomine Tumor Mutation Load Assay, manual library preparation?

Ion Xpress Barcode Adapters Kit (Cat. No. 4471250, 4474009, 4474518, 4474519, 4474520, 4474521, or 4474517) is compatible with the Oncomine Tumor Mutation Load Assay, manual library preparation. The barcode adapters need to be diluted as following: Barcode X at a final dilution of 1:4 for each adapter.
What is the starting material for the Oncomine Tumor Mutation Load Assay panel?

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.

How many libraries can be prepared with the Oncomine Tumor Mutation Load Assay, manual library preparation?

The Oncomine Tumor Mutation Load Assay, manual library preparation, includes panel primers and reagents sufficient for the preparation of 24 barcoded sample libraries from DNA.

What kits do you recommend using for nucleic acid isolation for the Oncomine Tumor Mutation Load Assay panel?

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).
What are the components of the Oncomine Tumor Mutation Load Assay, manual library preparation (Cat. No. A37909)?

The Oncomine Tumor Mutation Load Assay, manual library preparation consists of the Oncomine Tumor Mutation Load Assay - Manual primer panel (2 pools) and the Ion AmpliSeq Library Kit Plus. Sufficient panel primers and reagents are provided for the preparation of 24 barcoded sample libraries from DNA.

What is the Oncomine Tumor Mutation Load Assay?

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).