Cell3™ Target: Custom NGS Panels

Enabling targeted resequencing for prenatal testing

Cell3™ Target is a novel enrichment system developed for prenatal diagnosis converting cell-free DNA
or genomic DNA into libraries for next generation sequencing.

 

Validated for cell-free and genomic DNA

NGS enrichment developed for and validated on cell-free DNA as well as gDNA for prenatal applications like non-invasive prenatal diagnosis

Confidently detect fetal fraction in maternal blood

QC validation by NGS guarantees uniformity of coverage which combined with error suppression technology ensures confident detection of even the lowest fetal fraction

 

Simple and rapid design process

Our online probe design tool means you can design and optimise a panel in minutes and our novel rapid manufacture process means you will receive your panel within 4 weeks of ordering.

Optimised for large and small panels

Cell3 Target enrichment has been optimised to deliver efficient target capture and unrivalled on-target performance regardless of panel size from 1 gene to 1,000.

Non-invasive prenatal testing

Cell-free DNA (cfDNA) are degraded DNA fragments released into the bloodstream through a natural process of cell death. During pregnancy, the mother’s blood contains cfDNA from her own tissue and from the fetus (cell-free fetal DNA (cffDNA)) via the placenta.  Sequence analysis of cfDNA from a sample of maternal blood can therefore provide genetic information about the fetus and inform clinical decisions without the need for invasive techniques like amniocentesis. However, as only between 2 and 20% of the total cell-free DNA in maternal blood is fetal1,2 (the fetal fraction), its analysis requires ultra-sensitive sequencing methods capable of accurately detecting genetic variants in samples with even the lowest of fetal fractions.

Targeted NGS/resequencing panels for NIPD or prenatal testing

Targeted sequencing, through the use of custom designed NGS panels, offers a cost-effective method for the ultra-sensitive sequencing required for non-invasive prenatal genetics. Without the data burden of whole-genome sequencing (WGS) or whole-exome sequencing (WES) it allows researchers to achieve very high depths of coverage providing the increased sensitivity and accuracy needed for the analysis of low abundance cell free fetal DNA (cffDNA).

Cell3™ Target is a novel target enrichment system developed by Nonacus for converting cell-free DNA or genomic DNA into libraries for next generation sequencing that has been validated to ensure confident detection and analysis for even the lowest of fetal fractions.

Reliable target enrichment for cffDNA and gDNA

We understand that our customers have different questions to answer so Cell3 Target has been developed for and validated on cffDNA and gDNA for a range of prenatal applications. Whether you are using amniocentesis or non-invasive prenatal testing (NIPT), you know you will be able to use the same method and panel design.

Liquid biopsy sample, DNA from a blood sample
COVID-19 Whole Genome Sequencing Service

Quick and convenient workflow for cfDNA

Cell3 Target offers quick, Covaris-free, enzymatic shearing for genomic DNA samples and is available without enzymatic fragmentation reagents for cfDNA.

  • Quick and easy workflow takes less than 10 hours, with less than 2 hours hands-on time.
  • Supports manual or automated preparation of 1 – 96 samples in a single batch
  • 384 patient/sample indexes ensure that customer can use Cell3™ Target on the smallest to the largest output sequencers.
  • Suitable for use with 10ng cfDNA or genomic DNA

Ultra-sensitive NGS enrichment for NIPD

Cell3™ Target enrichments incorporate error suppression technology including unique molecular indexes (UMIs) and unique dual indexes (UDI’s) which remove of both PCR and sequencing errors and index hopping events. This error suppression technique, combined with our excellent uniformity of coverage, allows you to confidently and accurately call variants in even the lowest of fetal fractions. (and from as little as 10ng cfDNA or gDNA input). We provide advice and provision of ready to go analysis scripts for error removal using UMI’s.

Umi diagram

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

Screenshot of Nonacus Probe design tool homepage

Successful custom panel design for targeted NGS

Good design is key to a successful NGS panel ensuring adequate coverage and robust variant calling.  It’s not always easy to achieve, especially first time, but our Probe Design tool, makes it simple and easy allowing you to optimise panels in minutes. Create designs instantly from scratch using uploaded BED files, gene lists or genomic coordinates, customise catalogue products or add your own content onto one of our fixed panels.

• Easily merge, compare and share panels

• Get coverage data instantly and request a quote

• Receive your fully validated* custom panel within 4 weeks of order.

 

Log into the Probe Design Tool at My Nonacus  to start on a design and get a quote.

*All panels are NGS validated to ensure uniformity of coverage meets our QC requirements across all targeted regions

Optimised sequencing performance for NIPD

The baits used in Cell3 Target are designed to deliver excellent uniformity of coverage. By improving uniformity of coverage and reducing the number of low coverage exons, our Cell3 Target enrichment optimises sequencing efficiency and sample capacity per sequencing run ultimately reducing your sequencing costs.

Nonacus-Uniformity-of-coverage-ST1
sequence-metrics-summary

Figure 2. Coverage uniformity and % target capture for a 40 Kb panel designed using Cell3 Target technology and Company (A) High Sensitivity enrichment kit. Targeted enrichment performed on genomic DNA samples and sequenced on the Illumina MiSeq

Improved on-target for small and large panels

Most capture target enrichment technologies suffer from a significant percentage of off target sequencing reads when targeting small regions of the genome. Cell3 Target has been developed so that whether you wish to sequence 1 or 1,000 genes you will find lower off target and more uniform coverage when compared with alternative capture technologies.

 

References

  1. Barrett, A, Zimmerman BG, Wang D, Holloway A, Chitty L. Implementing prenatal diagnosis based on cell-free fetal DNA: Accurate identification of factors affecting fetal DNA yield. PLoS One. 2011;6(10):e25202.
  2. Nigam A, Saxena P, Prakash A, Acharya A.Detection of fetal nucleic acid in maternal plasma: A novel noninvasive prenatal diagnostic technique. J Interntl Med Sci Acad. 2012; 25(3): 119-120.

Publications

van Campen, J., Silcock, L., Yau, S., Daniel, Y., Ahn, J.W., Ogilvie, C., Mann, K. and Oteng‐Ntim, E. (2020), A novel non‐invasive prenatal sickle cell disease test for all at‐risk pregnancies Br J Haematol, 190: 119-124. https://doi.org/10.1111/bjh.16529

Product Support

If you have a query relating to any of our products, please take a look at our FAQs or complete our contact me form and one of our team 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.

Learn more about our products for prenatal testing

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The first direct from plasma prenatal diagnosis kit for fetal RhD genotyping.

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ExomeCG

Clinically enhanced exome capture for whole exome sequencing and copy number analysis in a single test.

every sample is precious

Cell3™ Preserver

A whole blood stabilisation tube for the collection of blood in cell free DNA applications.

cell free DNA extraction

Cell3™ Extract

A cell free DNA extraction kit that is a fast, accurate and flexible solution to obtain cfDNA directly from plasma.