Cell3™ Target: Pan-Cancer (524) TMB/MSI Panel

Comprehensive NGS analysis of cancer mutations, TMB and MSI

A panel targeting 524 oncogenes that allows you to profile and stratify all common cancers and predict response to immunotherapy.

One workflow for multiple biomarkers

Detect all variant types including SNVs, CNVs and indels as well as TMB and MSI across 524 oncogenes in a single NGS enrichment.

Run ctDNA and FFPE samples

Validated on ctDNA and FFPE samples, as well as gDNA, giving you the option of profiling either primary, metastatic or liquid biopsies.

Measure tumor genomic instability

Confidently extrapolate tumor mutation burden (TMB) from panel sequencing data for immunotherapy response prediction.

An alternative to whole exome sequencing

Simplified analysis and reduced costs make this targeted panel an attractive alternative to tumor whole exome sequencing (WES) for routine use.

Profile solid tumors with Cell3 Target Pan-Cancer Panel

If you have limited time and limited sample, targeted sequencing offers the best approach to profile common cancer genes via a comprehensive set of biomarkers in a single panel.

The Cell3™ Target: Pan-Cancer (524) TMB/MSI panel is a next generation sequencing (NGS) panel that covers common driver mutations including SNVs, CNVs and indels in 524 oncogenes and supports the analysis of immuno-oncology biomarkers like tumor mutation burden (TMB) and microsatellite instability (MSI).

This targeted enrichment panel has been validated on a range of sample types from circulating tumor DNA (ctDNA) to formalin-fixed paraffin-embedded (FFPE) DNA meaning you can process all of your oncology samples – regardless of sample type or tumour origin – in a single, simple workflow.

Comprehensive content for clinical cancer research

The Pan-Cancer (524) TMB/MSI panel covers 63 genes from NCCN/FDA cancer treatment guidelines, 116 cancer driver genes and 345 genes in vital cancer signalling pathways. The design, whilst exon focused, covers key intronic and promoter regions and contains a selection of CNV probes to support copy number calling across the genome. It is a comprehensive panel that allows you to accurately identify and profile variants associated with cancer and stratify all common cancers in a single workflow.

Table 1. Pan-Cancer (524) TMB/MSI Panel gene content.

Profiling cancer to predict response to immunotherapy

Analysis of TMB and MSI using NGS panels

Immune checkpoint inhibitors have shown great potential as treatments across a number of cancers. However, as not all patients will respond to immunotherapies, positive biomarkers are needed to help match patients with the appropriate treatment.  Tumor genomic instability has been shown to correlate positively with immunotherapy response for which there are two known biomarkers: TMB and MSI.

Targeted NGS sequencing offers a cost-effective way to measure TMB and MSI, but the size of a panel can influence the precision of TMB measurement1. Too small and the measurement is imprecise (and therefore clinically suboptimal for patient stratification and response prediction) but too large and it is not cost effective for routine use. At 1.58Mb, the Nonacus Pan-Cancer (524) TMB/MSI panel, delivers accurate TMB estimation, cost effectively.

Validated on FFPE and ctDNA samples

Running a clinical cancer laboratory can require the analysis of a range of sample types from blood samples (liquid biopsies) to solid tumors (fresh or frozen).

Our Pan-Cancer TMB/MSI Panel has been validated on a broad range of samples including formalin-fixed, paraffin-embedded (FFPE), fresh frozen, genomic and circulating tumor DNA (ctDNA).

That means you can process all of your oncology samples – regardless of sample type or tumour origin– in a single, simple workflow.

ffpe-sample
Umi diagram

Figure 1. Using UMI’s to identify and quantify individual DNA molecules during library preparation increases sensitivity

Detect low-level variants with pan-cancer panels

The Cell3™ Target technology behind the Pan-Cancer panel incorporates error suppression technology, which includes unique molecular indexes (UMIs) and unique dual indexes (UDIs), to remove both PCR and sequencing errors and index hopping events. This allows confident and sensitive calling of mutations down to 0.1% VAF from as little as 10ng ctDNA input.

Maximise sequencing efficiency

By increasing the yield per sample, Cell3™ Target libraries allow you to run more samples per flow cell, which increases your efficiency and reduces your cost per sample.

Pan-Cancer-Panel-Table2

Table 2. Number of samples per flow cell to achieve 500x mean depth of coverage based on 2 x 100bp library and maximum quoted sequencer output for Nonacus Cell3™ Target Pan-Cancer (524) TMB/MSI Panel.

Screenshot of Nonacus Probe design tool homepage

Customise content for NGS cancer panels

Designed to be flexible, our Cell3 Target Pan-Cancer panel allows you to add extra content specific to your project. Whether this is additional content or increased coverage of existing content, our Probe Design Tool makes this a simple and easy process to implement. And our rapid production turnaround means you will receive a fully NGS-validated custom exome within 4 weeks.

  • Additional content with high enrichment uniformity
  • Increased coverage of specific genes covered by Cell3™ Target Pan Cancer Panel
  • Optimization of spike-in ratio

Log into the Probe Design Tool at My Nonacus to start the design process and get a quote or drop us an email at info@nonacus.com

Ordering Information

Product: Cell3™Target Pan-Cancer (524), Tumor Mutational Burden/MSI Panel, (16 samples)
Catalogue No.: C3299 (options A/B*)

Product: Cell3™Target Pan-Cancer (524), Tumor Mutational Burden/MSI Panel, (96 samples)
Catalogue No.: C3300 (options A/B/C*)

*Cell3™ Target panels are available in the following options:

A = non-fragmentation e.g. cffDNA/ctDNA,
B = fragmentation e.g. gDNA or FFPE,
C = Both Fragmentation and Non-Fragmentation (half of each)

Product Support

If you have any questions about any of our products, including access to the BED files and example data sets, please fill in the support request form here and we will get back to you as soon as possible.

Frequently asked questions

1. Cell3™ Target Library preparation: methods, equipment & reagents

For best results, we recommend that you extract cfDNA and quantify just before you start your library prep. However, sometimes there is a need to extract and store cfDNA at an earlier time. In this case we advise you to store extracted cfDNA in a low-bind tube at -20˚C and quantify your samples after thawing when you are ready to start the library preparation.

We don’t recommend mixing Cell3™ Target enrichment probes with libraries prepared from other library prep kits because, we cannot guarantee the results. However, if you choose to do this you should be aware that some compatibility issues may arise. For example, the blockers in our kits are designed for our library prep kits and are for hybridisation-based kits with 8nt or lower indexing. Therefore, depending on the kit you used, you may see a high percentage of off target reads.

Our elution buffer consists of 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA; therefore, this low level of EDTA should not affect most downstream applications, including NGS library prep. It  is perfectly fine to use water in place of the supplied buffer to elute your cfDNA. If doing so, please ensure that the pH is >6.0.  However, if you are storing your samples the buffer should be used. In addition, it is advisable to only extract cfDNA at the point where you plan to proceed with the library preparation.

We have successfully run the protocol using cyclers that have a set max volume of 50 or 100ul.

A heated lid that has a max of 99˚C should work fine. However, we would not recommend adjusting the denaturation temperature to 95 ˚C in the library amplification step and recommend keeping this at 98 ˚C.

We recommend the hybridisation to run between 4 and 16 hours. However, we regularly run the hybridization overnight. For example, start the reaction at 4/5pm and then perform probe capture at 9 or 10am the following day. We have tested longer hybridisation times only up to 24 hours. If you want to break in between, you can store the dried down library pool with COT-1 DNA and universal blockers overnight at 4˚C if you want to delay starting the hybridisation reaction.

A. Often, we find that the beads dry quicker than 5 minutes, so it is advisable to check at 3 mins. Ensure that they are not shiny and try not to wait until they are cracking and overdried as beads are more difficult to resuspend and you may recover less of your sample (see Fig.1 below for an example)

beads

Figure 1. example of dried clean up beads showing matt appearance. Well E shows a slight sheen on the beads just before completely drying.

Section 2C Captured library amplification, of the Cell3 Target protocol gives general guidelines on the suggested number of post capture PCR cycles. For very small targets the number of cycles would need to be increased. For example, 0.004Mb may need an additional 2 cycles (total 18) to ensure there is enough library for sequencing. Therefore, please note that for very small targets you may need to optimise the number of post capture PCR cycles.

The correct type of Dyna Beads is critical to the success of the capture. Only M-270 Streptavidin beads are suitable and will give the correct post capture yield. Any other beads are likely to be a different diameter and concentration and have shown to give a significant decrease in final yield.

As you have plenty of DNA for your WGS, and therefore not using the UMIs, we recommend performing a PCR free method to eliminate PCR bias. To do this you would need a minimum of 100ng of DNA to input and if your FFPE DNA has very low DIN scores (e.g. less than 3) you would need to increase your DNA input by 5-10 fold (see pg. 11 in Cell3 Target protocol).

A.    The following are the steps for running a PCR free method for Cell3 Target library prep:

  1. Fragmentation/End repair/A-tailing (use 1 ul for tapestation to verify size)
  2. Ligation of adapters
  3. Bead clean-up
  4. Quantitation by qPCR (not Qubit and TapeStation)

When conducting a PCR free library prep, the QC at the end (following adapter ligation) will not give accurate Qubit readings (they will appear lower than what they should be) and the Tapestation will not show fragments in the right range. This is due to the ligation of Y-shaped adapters. The best way to QC is using qPCR. If you want to confirm the average fragment size, you could use 1 ul of the fragmentation reaction (post incubation) and run that on the tapestation. The average fragment length + adapters (144bp) will give the final library average fragment length.

Quantity of probe set is 18 ul for a 4x reaction kit for the cancer 50 panel. This allows you to prepare 4 x hybridisation pools with one probe set.

Library pooling guidelines for all custom and off the shelf products are listed on pg. 27 of the protocol. For the Cancer 50 panel up to 16 libraries can be pooled into 1 hybridisation reaction and you use 4 ul of probe set per reaction. For the exome panel, up to 8 libraries can be pooled. By all means, you can pool less than this number. However, you will need to ensure that the concentration of the combined pool reaches a total of 1000ng.

2. Cell3™ Target Library preparation: Quality controls (QC) & troubleshooting

A. The TapeStation profile and yield of extracted cfDNA will vary depending on the input sample. The following descriptions and images give examples of these:

a) cfDNA extracted from cancer patients will be a mixture of cfDNA from the normal process of apoptosis and circulating tumour DNA (ctDNA) shed from the patient’s tumour. The amount of ctDNA shed into the blood varies with tumour type, stage and whether the patient has received treatment. There is a size difference observed in fragment length between cfDNA derived from the tumour and the patient’s normal background cfDNA. The normal cfDNA is generally around 166 bp (160-180bp peak), which corresponds to the length of DNA wrapped around the nucleosome and is likely to be the result of normal apoptosis. Peaks in multiples of 160-180 bp are often observed. The ctDNA portion in the sample is generally a smaller peak and reveal a shorter fragment length of approx. 145bp.

b) cfDNA extracted from patients with inflammatory conditions, infections, late stage cancer or transplant rejection may reveal larger peaks of cfDNA on the TapeStation analysis. You may see several peaks of differing sizes as multiples of 166bp (e.g. 332bp, 498bp, 664bp…).

profile-DNA

Fig.2. Example of cell free DNA profile for patient with an inflammatory condition

A. In all cases it is important to accurately determine the DNA concentration in your sample, especially when using <100 ng of DNA as input. To do this we recommend using a fluorometric method (such as the Qubit assay, Invitrogen).  Additionally, you may want to perform different QC steps on your input material according to your material type.

a) FFPE derived DNA may be compromised due to formalin fixation so we recommend performing a quality check by running your samples on a genomic screen tape (TapeStation) to determine the DIN (DNA integrity) score. This allows you to optimise the DNA input adding more material with lower DIN scores.

b) DNA derived from plasma may be contaminated with gDNA and the extent of this can be determined using a High Sensitivity D1000 Screen Tape on the TapeStation. Contamination with gDNA can arise during the processing of plasma due to haemolysis of white blood cells. A peak at >1500 bp is evidence of sample contamination with gDNA. You may also want to run this check to determine that the TapeStation profile reveals fragments of the expected size range (see Fig. 2 above).

enzymatic

Figure.3. Fragment size distribution of unsuccessful library prepared with 10 ng of input high molecular weight genomic DNA. The presence of a tail in the long fragment size range suggests that the sample was not entirely sheared during enzymatic fragmentation. The small 160 bp peak (indicated by the arrow) represents the presence of a small amount of adapter-dimers.

There are several possible causes for this. Accurate quantification of material and dilution upfront is a primary concern if you have an under fragmented library. Additionally, please ensure that the correct time and temperature have been used for fragmentation. If you are not using 10-100ng and this is your first use of Cell3™Target with this sample type, we recommend first trying 3 different times to find the best option for your sample type and input quantity.

Have you got EDTA in your DNA extraction elution buffer? EDTA affects the enzymatic shearing and causes poor/under fragmentation. DNA stored in EDTA buffers should be cleaned up with bead or column clean up prior to use. Ideally non EDTA elution buffers such as EB buffer (10mM Tris HCL pH 7.5-8) should be used.

Did you prepare the Enzymatic shearing reaction on ice and mix adequately before incubation? Mixing should be quite vigorous by pipetting or vortexing.

 

A. A small amount of adapter-dimers seen at the pre-capture library preparation QC (as seen in fig.3 Q16 above) should not be carried over to the final library pool. If your libraries show a significant adapter-dimer peak (see fig. 4 below) this may be because you have input less than 50ng of DNA into the initial reaction without diluting the adapters to 1.5 µM (1:10 dilution). Always ensure you have accurately determined the concentration in your samples immediately prior to library preparation (see Q.1). You can attempt to reduce the adapter-dimer peak by performing a 0.9X (bead to sample ratio) clean-up step. However, this will result in sample loss and you will need to perform additional PCR cycles to ensure you have sufficient material to continue.

adapter-dimers

Figure 4. Library preparation from cfDNA showing significant adapter-dimer peak at 152bp

3. Sequencing Cell3™ Target libraries

A. Yes. Provided all libraries are Cell3™ Target libraries and they all have different sample indexes then a mix of Enzymatically sheared and Covaris sheared is fine.

A. Sequencing coverage can be estimated by using the Illumina Sequencing Coverage Calculator, which can be found at

https://support.illumina.com/downloads/sequencing_coverage_calculator.html

Below is an example for 2x coverage for WGS:

genome-table

A. This is possible; however, it is relevant to note that the read length you set may alter from your usual parameters. cfDNA is on average 166bp in length therefore, we usually recommend 2x75bp read length. For FFPE DNA fragments are often longer, and we would recommend a 2x150 bp read length. If you run at the longer read length you will sequence into adapter sequences and need to remove these during analysis. Alternatively, you can sequence all your libraries at the shorter read length of 2x75bp, however, this will impact the achievable depth of coverage for your FFPE DNA libraries.

4. Cell3™ Target: ordering & purchasing

A mixture of fragmentation and non-fragmentation kits can be purchased. For example, 48 fragmentation, 48 non-frag or 16 sample of one and 32 of the other so that both FFPE and cfDNA can be prepared.

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Profiling cancer to predict response to immunotherapy